Giant magnetoresistance-based detection of analytes

Abstract

In implementations described herein, processes, methods, and techniques are described for the detection of analytes in a sample using multiplexing methods for giant magnetoresistance sensors.

Description

6356.002W01GIANT MAGNETORESISTANCE-BASED DETECTION OF ANALYTESCLAIM TO PRIORITY

[0001] This patent application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 688,133, filed August 28, 2024, which is incorporated by reference herein in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in ST26 format and hereby incorporated by reference in its entirety. Said ST26 file, created on August 27, 2025, is named 6356002W01 .xml and is 55,608 bytes in size.BACKGROUND

[0003] The detection of biological conditions that underlie disease can be a first step in identifying the proper treatment for the disease. Diagnostic tests can be performed to identify a biological condition that results in symptoms within a subject. For example, samples can be obtained from subjects and analyzed to determine the possibility of a biological condition being present in the subjects. The samples can include tissue samples or the samples can include a biological fluid, such as whole blood, plasma, and the like. The samples can be analyzed to identify the presence of one or more biological markers that indicate the presence of a biological condition within a subject.

[0004] For example, analytes can be extracted from the samples and the presence, absence, or amount of the analytes extracted from the samples can indicate whether a biological condition is present within subjects and / or the severity of the biological condition. The analytes can include biological molecules and / or genetic material. To illustrate, various proteins can be biomarkers for diseases, such as Alzheimer’s disease, cardiovascular disease, diseases of the joints (e.g., rheumatoid arthritis), kidney disease, liver disease, among others. Additionally, the detection of genetic mutations in genomic material, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), can indicate the presence of a number of diseases, such as many forms of cancer. The presence of genomic material in samples can also be used to identify6356.002W01 the presence of cardiovascular disease, organ transplant rejection, autoimmune diseases, and viral infections.

[0005] There is a need for improved systems and methods for improved analyte detection through the analysis of samples obtained from subjects that include cell-free DNA. It is an object of the disclosure to provide methods that have improved capability to classify a sample as being derived from a subject having a biological condition.BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain implementations, and together with the written description, serve to explain certain principles of the methods, computer readable media, and systems disclosed herein. The description provided herein is better understood when read in conjunction with the accompanying drawings which are included by way of example and not by way of limitation. It will be understood that like reference numerals identify like components throughout the drawings, unless the context indicates otherwise. It will also be understood that some or all of the figures may be schematic representations for purposes of illustration and do not necessarily depict the actual relative sizes or locations of the elements shown.

[0007] Figure 1 is a diagrammatic representation of an example process that identifies nucleic acids that correspond to target sequences that are indicative of an underlying biological condition being present in a subject, according to one or more example implementations.

[0008] Figure 2 is a diagrammatic representation of an example process of delivering a number of solutions across a sensor array to cause nucleic acids that correspond to target sequences to bind to a surface of the sensor array, according to one or more example implementations.

[0009] Figure 3 is a diagrammatic representation of a flow diagram of a process to capture nucleic acids having target sequences and analyzing quantitative measures related to the nucleic acids to determine the presence or absence of a biological condition within a subject, according to one or more example implementations.

[0010] Figure 4 is a representation of a signals derived from samples in relation to detection of Aspergillus fumigatus within the samples.

[0011] Figure 5 is a representation of a signals derived from samples in relation to detection of Candida albicans within the samples.6356.002W01

[0012] Figure 6 is a representation of a signals derived from samples in relation to detection of Histoplasma capsulatum within the samples.

[0013] Figure 7 is a representation of a signals derived from samples in relation to detection of Candida krusei within the samples.

[0014] Figure 8 is a table showing electrical signals produced with respect to a number of fungi in relation to the number of copies of nucleic acids corresponding to the fungi present in a sample.SUMMARY

[0015] Methods of detecting analytes can include providing an apparatus that includes (i) a plurality of giant magnetoresistance (GMR) sensors and (ii) a plurality of chambers for performing amplification of nucleic acids and causing a plurality of probes to be attached to the plurality of GMR sensors. The plurality of probes correspond to a plurality of fungal species. In addition, the methods can include causing a first solution to be supplied to a first chamber of the plurality of chambers with the first solution including a first set of primers that correspond to a first group of fungi included in the plurality of fungal species. The methods can also include causing a second solution to be supplied to a second chamber of the plurality of chambers with the second solution including a second set of primers that correspond to a second group of fungi included in the plurality of fungal species and are different from the first group of fungi. Further, the methods can include causing a first sample portion to be supplied to the first chamber and a second sample portion to be supplied to the second chamber with the first sample portion including first nucleic acids and the second sample portion including second nucleic acids. A first amplification process can be performed in the first chamber to produce a first amplification product that includes first double stranded amplicons and a second amplification process can be performed in the second chamber to produce a second amplification product that includes second double stranded amplicons, wherein the second amplification process is performed concurrently with respect to the first amplification process. The first amplification product and the second amplification product can be supplied to a chamber that includes the plurality of GMR sensors and the method can include determining a change in electrical signals produced by at least one GMR sensor of the plurality of6356.002W01GMR sensors based on a first number nucleic acids derived from the first double stranded amplicons coupled to one or more first probes attached to the at least one GMR sensor or a second number derived from the second double stranded amplicons coupled to one or more second probes attached to the at least one GMR sensor. The one or more first probes can correspond to the first group of fungi and the one or more second probes can correspond to the second group of fungi.

[0016] Methods of detecting analytes can comprise extracting nucleic acids from a sample and performing a first amplification process in relation to a first number of the nucleic acids to produce first amplicons. The first amplification process can be performed using primers that are complementary with first genomic regions of a first group of fungi. The method can also comprise performing a second amplification process in relation to a second number of the nucleic acids to produce second amplicons. The second amplification process can be performed using primers that are complementary with second genomic regions of a second group of fungi different from the first group of fungi. The first group of fungi and the second group of fungi can include two of the following groups of fungi: (1 ) Aspergillus flavus, Candida albicans, Candida auris, Scedosporium spp and Candida glabrata', (2) Aspergillus fumigatus, Aspergillus niger, Candida krusei, and Cryptococcus., (3) Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, and Fusarium solanr', and (4) Candida tropicalis, Coccidioides, Histoplasma capulatun, Mucorales, Blastomyces, or Pneumocystis jirovecii. Further, the method can comprise determining an electrical signal that corresponds to at least one of a number of single stranded first amplicons or a number of single stranded second amplicons bound to a surface of a sensor.

[0017] Additionally, methods of detecting analytes can comprise forming a surface of a sensor. The surface of the sensor can include a polymeric coating and a number of probe sequences. Individual probe sequences can include a first section that includes a linker sequence having from 2 to 15 consecutive thymine or adenine molecules and a 5’ amino group coupled to the polymeric coating. Individual probe sequences can also include a second section having a nucleotide sequence that is complementary to a genomic region of a fungi selected from Aspergillus flavus, Candida albicans, Candida auris, Candida glabrata, Aspergillus fumigatus, Aspergillus niger, Candida krusei, Cryptococcus, Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, Fusarium solani, Candida tropicalis, Coccidioides, Histoplasma capulatun, Scedosporium spp, Mucorales, Blastomyces, or Pneumocystis jirovecir', or6356.002W01 one or more control sequences. The methods can also include determining an electrical signal that corresponds to a number of single stranded nucleic acids bound to the surface of the sensor. The single stranded nucleic acid molecules can be derived from a sample.

[0018] Further, methods of detecting analytes can comprise extracting nucleic acids from a sample, the nucleic acids comprising cell-free deoxyribonucleic acid (cfDNA) molecules and providing the nucleic acids and a plurality of primer sets to one or more containers. The plurality of primer sets can include at least a first primer set and a second primer set selected from two of the following groups of primers: first primers that are complementary with first genomic regions of a first group of fungi including Aspergillus flavus, Candida albicans, Candida auris, Scedosporium spp, and Candida glabrata and one or more first controls sequences; second primers that are complementary with second genomic regions of a second group of fungi including Aspergillus fumigatus, Aspergillus niger, Candida krusei, and Cryptococcus and one or more second controls sequences; third primers that are complementary with third genomic regions of a third group of fungi including Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, and Fusarium solani and one or more third control sequences; or fourth primers that are complementary with fourth genomic regions of a fourth group of fungi including Candida tropicalis, Coccidioides, Histoplasma capulatun, Mucorales, Blastomyces, or Pneumocystis jirovecii and one or more fourth control sequences. The methods can also include performing amplification of a portion of the nucleic acids using the plurality of primer sets to produce an amplification product. The amplification product can include double stranded amplicons of the portion of the nucleic acids complementary to at least two of the first group of fungi, the second group of fungi, the third group of fungi, or the fourth group of fungi corresponding to the first primer set and the second primer set. Individual double stranded amplicons can include a first strand having a 5’ biotin and a second strand having a 5’ phosphate group. Further, the methods can include producing a group of single stranded nucleic acids corresponding to the first strands of the double stranded amplicons having the 5’ biotin and forming a surface of a sensor. The surface of the sensor can include a polymeric coating and a number of probe sequences. Individual probe sequences can include a first section having a nucleotide sequence that is complementary to a genomic region included in the two of the first genomic regions, the second genomic regions, the third genomic regions, or6356.002W01 the fourth genomic regions that correspond to the first primer set and the second primer set. Individual probe sequences can also include a second section that includes a linker sequence having at least five nucleotides and a 5’ amino group coupled to the polymeric coating. Further, the methods can include contacting the surface of the sensor with the group of single stranded nucleic acids to produce a modified surface of the sensor that includes a number of captured single stranded nucleic acids. Individual captured single stranded nucleic acids can be bound to an individual probe sequence. The methods can also include contacting the surface of the sensor with a solution including superparamagnetic streptavidin magnetic particles to produce a number of modified single stranded nucleic acids bound to the modified surface of the sensor. Individual modified single stranded nucleic acids having a streptavidin particle bound to the 5’ biotin. In addition, the methods can include determining an electrical signal that corresponds to a number of the modified single stranded nucleic acids bound to the surface of the sensor.

[0019] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.DEFINITIONS

[0020] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms may be set forth through the specification. If a definition of a term set forth below is inconsistent with a definition in an application or patent that is incorporated by reference, the definition set forth in this application should be used to understand the meaning of the term.

[0021] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons of ordinary skill in the art upon reading this disclosure and so forth.

[0022] It is also to be understood that the terminology used herein is for the purpose of describing particular implementations only, and is not intended to be limiting. Further, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which6356.002W01 this disclosure pertains. In describing and claiming the methods, computer readable media, and systems, the following terminology, and grammatical variants thereof, will be used in accordance with the definitions set forth below.[QQ23]About: As used herein, “about” or “approximately” as applied to one or more values or elements of interest, refers to a value or element that is similar to a stated reference value or element. In certain implementations, the term “about” or “approximately” refers to a range of values or elements that falls within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11 %, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value or element unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value or element).

[0024] Administer. As used herein, “administer” or “administering” a therapeutic agent (e.g., an immunological therapeutic agent) to a subject means to give, apply or bring the composition into contact with the subject. Administration can be accomplished by any of a number of routes, including, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal and intradermal.

[0025] Amplify: As used herein, “amplify” or “amplification” in the context of nucleic acids refers to the production of multiple copies of a polynucleotide, or a portion of the polynucleotide, starting from a small amount of the polynucleotide (e.g., a single polynucleotide molecule), where the amplification products or amplicons are generally detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes.

[0026] Cell-Free Nucleic Acid: As used herein, “cell-free nucleic acid” refers to nucleic acids not contained within or otherwise bound to a cell or, in some implementations, nucleic acids remaining in a sample following the removal of intact cells. Cell-free nucleic acids can include, for example, all non-encapsulated nucleic acids sourced from a bodily fluid (e.g., blood, plasma, serum, urine, cerebrospinal fluid (CSF), etc.) from a subject. Cell-free nucleic acids include DNA (cfDNA), RNA (cfRNA), and hybrids thereof, including genomic DNA, mitochondrial DNA, circulating DNA, siRNA, miRNA, circulating RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), long non-coding RNA (long ncRNA), and / or fragments of any of these. Cell-free nucleic acids can be double-stranded, singlestranded, or a hybrid thereof. A cell-free nucleic acid can be released into bodily fluid6356.002W01 through secretion or cell death processes, e.g., cellular necrosis, apoptosis, or the like. Some cell-free nucleic acids are released into bodily fluid from cancer cells, e.g., circulating tumor DNA (ctDNA). Others are released from healthy cells. CtDNA can be non-encapsulated tumor-derived fragmented DNA. A cell-free nucleic acid can have one or more epigenetic modifications, for example, a cell-free nucleic acid can be acetylated, 5-methylated, ubiquitylated, phosphorylated, sumoylated, ribosylated, and / or citrullinated.

[0027] Ce / / u / ar Nucleic Acids: As used herein, “cellular nucleic acids” means nucleic acids that are disposed within one or more cells at least at the point a sample is taken or collected from a subject, even if those nucleic acids are subsequently removed as part of a given analytical process.

[0028] Confidence Interval: As used herein, “confidence interval” means a range of values so defined that there is a specified probability that the value of a given parameter lies within that range of values.

[0029] Control Region: As used herein, “control region” refers to a genomic region that is located in a human reference sequence.

[0030] Deoxyribonucleic Acid or Ribonucleic Acid: As used herein, “deoxyribonucleic acid” or “DNA” refers to a natural or modified nucleotide which has a hydrogen group at the 2'-position of the sugar moiety. DNA can include a chain of nucleotides comprising four types of nucleotide bases: adenine (A), thymine (T), cytosine (C), and guanine (G). As used herein, “ribonucleic acid” or “RNA” refers to a natural or modified nucleotide which has a hydroxyl group at the 2'-position of the sugar moiety. RNA can include a chain of nucleotides comprising four types of nucleotides: A, uracil (U), G, and C. As used herein, the term “nucleotide” refers to a natural nucleotide or a modified nucleotide. Certain pairs of nucleotides specifically bind to one another in a complementary fashion (called complementary base pairing). In DNA, adenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine (G). In RNA, adenine (A) pairs with uracil (U) and cytosine (C) pairs with guanine (G). When a first nucleic acid strand binds to a second nucleic acid strand made up of nucleotides that are complementary to those in the first strand, the two strands bind to form a double strand. As used herein, “nucleic acid sequencing data”, “nucleic acid sequencing information”, “sequence information”, “sequence representation”, “nucleic acid sequence”, “nucleotide sequence”, “genomic sequence”, “genetic sequence”, “fragment sequence”, “sequencing read”, or “nucleic acid sequencing read” denotes6356.002W01 any information or data that is indicative of the order and identity of the nucleotide bases (e.g., adenine, guanine, cytosine, and thymine or uracil) in a molecule (e.g., a whole genome, whole transcriptome, exome, oligonucleotide, polynucleotide, or fragment) of a nucleic acid such as DNA or RNA. It should be understood that the present teachings contemplate sequence information obtained using all available varieties of techniques, platforms or technologies, including, but not limited to: capillary electrophoresis, microarrays, ligation-based systems, polymerase-based systems, hybridization-based systems, direct or indirect nucleotide identification systems, pyrosequencing, ion- or pH-based detection systems, and electronic signature-based systems.

[0031] L / m / t of Detection (LoD): As used herein, “limit of detection” means the smallest amount of a substance (e.g., a nucleic acid) in a sample that can be measured by a given assay or analytical approach.

[0032] Polynucleotide: As used herein, “polynucleotide”, “nucleic acid”, “nucleic acid molecule”, “polynucleotide molecule”, or “oligonucleotide” refers to a linear polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by internucleosidic linkages. A polynucleotide can comprise at least three nucleosides. Oligonucleotides often range in size from a few monomeric units, e.g., 3-4, to hundreds of monomeric units. Whenever a polynucleotide is represented by a sequence of letters, such as “ATGCCTG,” it will be understood that the nucleotides are in 5’ 0 3’ order from left to right and that in the case of DNA, “A” denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotes deoxyguanosine, and “T” denotes deoxythymidine, unless otherwise noted. The letters A, C, G, and T may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.

[0033] Probe: As used herein, “probe” refers to a polynucleotide comprising a functionality. The functionality can be a detectable label (fluorescent), a binding moiety (biotin), or a solid support (a magnetically attractable particle or a chip). Probes can include single-stranded DNA / RNA polynucleotides or double stranded DNA polynucleotides that hybridize to target nucleic acid sequences. Sequence capture using probes generally depends, in part, on the number of consecutive nucleotides in at least a portion of the target nucleic acid sequence that is complementary (or nearly complementary) to the sequence of the probe.6356.002W01

[0034] Quantitative Measures. As used herein, “quantitative measures” refers to an absolute or relative measure. A quantitative measure can be, without limitation, a number, a statistical measurement (e.g., frequency, mean, median, standard deviation, or quantile), or a degree or a relative quantity (e.g., high, medium, and low). A quantitative measure can be a ratio of two quantitative measures. A quantitative measure can be a linear combination of quantitative measures. A quantitative measure may be a normalized measure.

[0035] Reference Sequence: As used herein, “reference sequence” refers to a known sequence used for purposes of comparison with experimentally determined sequences. For example, a known sequence can be an entire genome, a chromosome, or any segment thereof. A reference sequence can include at least about 20, at least about 50, at least about 100, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, at least about 500, at least about 1000, or more nucleotides. A reference sequence can align with a single contiguous sequence of a genome or chromosome or can include noncontiguous segments that align with different regions of a genome or chromosome. Example reference sequences, include, for example, human genome reference sequences, such as, hG19 and hG38.

[0036] Sample: As used herein, “sample” means anything capable of being analyzed by the methods and / or systems disclosed herein.

[0037] Specifically binds: As used herein, “specifically binds” in the context of a probe or other oligonucleotide and a target sequence means that under appropriate hybridization conditions, the oligonucleotide or probe hybridizes to its target sequence, or replicates thereof, to form a stable probe:target hybrid, while at the same time formation of stable probe: non-target hybrids is minimized. Thus, a probe hybridizes to a target sequence or replicate thereof to a sufficiently greater extent than to a nontarget sequence, to enable capture or detection of the target sequence. Appropriate hybridization conditions can be predicted based on sequence composition, or can be determined by using various testing methods (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nded. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) at §§ 1.90-1.91 , 7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly §§ 9.50-9.51 , 11.12-11.13, 11 .45-11 .47 and 11 .55-11 .57, incorporated by reference herein).6356.002W01

[0038] Subject. As used herein, “subject” refers to an animal, such as a mammalian species (e.g., human) or avian (e.g., bird) species, or other organism, such as a plant. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals (e.g., production cattle, dairy cattle, poultry, horses, pigs, and the like), sport animals, and companion animals (e.g., pets or support animals). A subject can be a healthy individual, an individual that has or is suspected of having a disease or a predisposition to the disease, or an individual that is in need of therapy or suspected of needing therapy. The terms “individual” or “patient” are intended to be interchangeable with “subject.”

[0039] Target Region: As used herein, “target region” refers to a genomic locus targeted for identification and / or capture, for example, by using probes (e.g., through sequence complementarity). A “target region set" or “set of target regions” refers to a plurality of genomic loci targeted for identification and / or capture, for exa ple, by using a set of probes (e.g., through sequence complementarity). In addition, a target region can be defined by a pair of primer binding sites. Further, a target region can be defined by a predetermined beginning genomic locus and a predetermined ending genomic locus. The target region can include from about 10 nucleotides to about 250 nucleotides, from about 25 nucleotides to about 200 nucleotides, or from about 50 nucleotides to about 150 nucleotides.

[0040] Threshold: As used herein, “threshold” refers to a predetermined value used to characterize experimentally determined values of the same parameter for different samples depending on their relation to the threshold.DETAILED DESCRIPTION

[0041] Invasive fungal infections (IFIs) pose a significant threat, particularly among immunocompromised and critically ill patients. Unlike superficial fungal infections, IFIs are severe conditions associated with serious, deep, deep-seated, disseminated, and systemic fungal infections. Despite advances in Clinical Microbiology, the diagnosis of IFIs remains challenging, often resulting in delayed or missed detection, leading to significant morbidity and mortality. According to CDC surveillance data, the in-hospital all-cause (crude) mortality rate for patients with candidemia exceeds 25%, while invasive aspergillosis (IA) in immunocompromised individuals exhibits an exceptionally high mortality rate ranging from 40% to 90%. Conventional diagnostic methods, such as culture and serology, suffer from limitations including prolonged6356.002W01 turnaround times and low sensitivity. Furthermore, numerous cryptic fungal species cannot be isolated and cultured on standard fungal culture media, thereby evading conventional detection methods. For invasive candidiasis, blood culturing is regarded as the gold standard; however, the extended turnaround time (up to five days for yeasts and up to four weeks for molds) could elevate the patient's risk due to delays in determining the required treatment strategy.

[0042] Consequently, there is a need for the development and implementation of novel diagnostics to enhance the timely and accurate detection of IFIs. In recent years, molecular-based diagnostics have seen significant advancements across various clinical diagnostic areas, including the detection of fungal infections, by identifying minute quantities of target DNA or RNA. Molecular diagnostic assays offer several advantages over traditional methods, including rapid detection, high sensitivity, detection of cryptic species, multiplexing, independence from culturing requirements, and the ability to identify specific fungal pathogens at the species level. Arastehfar and team identified nine cryptic species of Candida using one-step multiplex PCR. Polymerase chain reaction (PCR)-based assays targeting fungal nucleic acids can be used in clinical settings, enabling detection of fungal pathogens directly from clinical specimens. It is extensively demonstrated by some commercial assays available for fungal identification. However, these assays exhibit restricted multiplexing capabilities, allowing them to detect only a limited range of fungal genera and species.

[0043] High-multiplex assays offer several benefits. By simultaneously detecting a wide range of fungal genera / species within a single sample, they expedite diagnoses, reducing the turnaround time significantly. Rapid identification can lead to prompt initiation of effective treatment and improved clinical outcomes. By consolidating multiple tests into a single assay, high-multiplex testing can be cost-effective. High throughput capacity ensures efficient utilization of resources and laboratory infrastructure. Additionally, the potential to identify co-infections in a single analysis offers additional guidance to the treating clinician. Ultimately, the integration of high- multiplex assays into syndromic testing protocols represents a significant diagnostic advance.

[0044] Fungal cell-free DNA (cfDNA) detection in blood is a novel testing modality for the noninvasive diagnosis of IFI. Cell-free DNA fungal detection offers the potential for early and sensitive diagnosis of fungal infections, as it may identify fungal DNA even before symptoms appear. It also involves less invasive sampling compared to tissue6356.002W01 biopsy and bronchoalveolar lavage sampling. The Karius Test, a laboratory-developed test from Karius, Inc., has been commercially available for inpatients in the United States since 2017. Being analytically and clinically validated, this cfDNA-based diagnostic test can identify bacteria, mycobacteria, DNA viruses, fungi, and protozoa in the bloodstream at the species level. This further underscores the significant value of blood cell-free DNA for detecting fungal infections.

[0045] However, fungal detection techniques that use fluorescence-based technologies can have drawbacks. For example, the Karius Test implements next generation sequencing that uses fluorescent tags to identify nucleotides in the cell- free DNA sequence. These technologies can be inaccurate due to a lack of specificity in detecting fungal species. To illustrate, fungal detection assays that implement fluorescence technologies can suffer from an amount of noise in the data derived from these assays that makes it difficult to distinguish signal related to the presence of fungi from the noise present in the data. Further, fluorescence-based techniques can have low sensitivity because the measurements obtained from these systems can be easily influenced by contaminants present in the samples.

[0046] Giant magnetoresistance (GMR) is a technology that could be applied in diagnostics. GMR involves a drastic change in electrical resistance which occurs in the presence of a magnetic field, particularly in thin multilayer structures composed of ferromagnetic and non-magnetic layers. The exceptionally sensitive GMR sensor enables the detection of minute levels of biological markers, rendering it highly efficient for medical diagnostics, especially in detecting trace amounts of cfDNA. In terms of cost and automation, GMR technology is notable for its potential to consolidate all essential components onto a single cartridge, which diminishes the need for numerous distinct procedures.

[0047] Minute changes in magnetic fields can be detected by GMR. However, the presence of contaminants or non-fungal material in a sample does not impact the magnetic field and the amount of noise present in data generated using a GMR-based process is less than the noise present in data generated by existing techniques. Thus, by utilizing GMR sensors, diagnostic devices can achieve high sensitivity in detecting DNA in biological samples. Furthermore, GMR-based diagnostic tools offer advantages such as rapid detection, high precision, and the ability to miniaturize devices for medical applications. Herein, a high-multiplex molecular diagnostic assay coupled with GMR Lab-on-a-Chip technology is described for the detection of blood6356.002W01 fungal cfDNA. In at least some implementations, the GMR-based analyte detection devices can have improved specificity and sensitivity over electromagnetic radiation based devices when more than a few analytes are being detected together because of limitations on dividing the electromagnetic radiation spectrum into more than a few segments, such as 4, 5, or 6 segments. In these situations, the electromagnetic radiation spectrum segments used to identify multiple analytes can begin to overlap and decrease the specificity of these optical based analyte detection devices.

[0048] Figure 1 is a diagrammatic representation of an example process 100 to analyze nucleic acid molecules present in samples to determine the presence of one or more analytes with respect to subjects, in accordance with one or more example implementations. The process 100 can include the development of a primer pool 102. The primer pool 102 can include a number of primers 104. Primers are oligonucleotides that are complementary to target genomic sequences included in nucleic acids that correspond to the presence of one or more organisms within subjects. The organisms can include one or more fungi, one or more bacteria, or one or more viruses.

[0049] In one or more illustrative examples, the process 100 can be implemented to detect at least one of Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Blastomyces, Candida albicans, Candida auris, Candida glabrata, Candida krusei, Candida pa rapsilosis, Candida tropicalis, Coccidioides spp., Cryptococcus neoformans, Cryptococcus gattii, Cunninghamella bertholletiae, Fusarium verticillioides, Fusarium solani, Histoplasma capsulatum, lomentospora prolificans, Pneumocystis jirovecii, Rhizomucor miehei, Rhizopus microsporus, Rhizopus Oryzae, Blastomyces dermatitidis, Mucorales, Mucor circinelloides, MucorR, MucorF, Scedosporium spp., Scedosporium apiospermum, Candida lusitaniae (CL), or Candida dubliniensis.

[0050] In one or more additional examples, the process 100 can be implemented to detect at least one of treatment resistant bacteria or antimicrobial bacteria strains. In various examples, the process 100 can be implemented to detect at least one of Acinetobacter calcoaceticus-baumannii complex, Bacteroides fragilis, Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, Clostridioides (Clostridium) difficile (toxin A / B), Enterobacterales, Enterobacter cloacae complex, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Haemophilus influenzae, Klebsiella aerogenes, Klebsiella oxytoca, Klebsiella pneumoniae group,6356.002W01Legionella pneumophila, Listeria monocytogenes, Moraxella catarrhalis, Mycoplasma pneumoniae, Proteus spp., Salmonella spp., Serratia marcescens, Haemophilus influenzae, Neisseria meningitidis, Pseudomonas aeruginosa, Salmonella, Staphylococcus spp., Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus lugdunensis, Stenotrophomonas malto ph Hi a, Streptococcus spp., Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, orTuberculosis.

[0051] In one or more further examples, the process 100 can be implemented to detect at least one of Adenovirus, Coronavirus 229E, Coronavirus HKU, Coronavirus NL63, Coronavirus OC43, Cytomegalovirus (CMV), Human Metapneumovirus, Human Rhinovirus / Enterovirus, Influenza A virus, Influenza B virus, Parainfluenza virus 1 , Parainfluenza virus 2, Parainfluenza virus 3, Parainfluenza virus 4, Respiratory syncytial virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Mpox, Herpes Simplex Virus type 1 (HSV1 ), Herpes Simplex Virus type 2 (HSV2), Vesicular Stomatitis Virus (VZV), Human Papillomavirus (HPV), Human Immunodeficiency virus (HIV), Hepatitis C virus (HCV), or Hepatitis B virus (HBV).

[0052] The process 100 can include providing a primer pool 102. The primer pool 102 can include primers 104 that correspond to genomic regions of a number of analytes of interest. For example, the primer pool 102 can include primers 104 that correspond to genomic sequences of a number of fungi. In one or more additional examples, the primer pool 102 can include primers 104 that correspond to genomic sequences of a number of bacteria. In one or more further examples, the primer pool 102 can include primers 104 that correspond to genomic sequences of a number of viruses. In still other examples, the primer pool 102 can include primers 104 that correspond to reference sequences, such as genomic sequences that correspond to a human genome.

[0053] In various examples, the process 100 can include determining a number of primer sets 106. Individual primer sets 106 can correspond to a number of groups of analytes of interest. To illustrate, the individual primer sets 106 can correspond to genomic sequences of a number of fungi. In addition, the individual primer sets 106 can correspond to genomic sequences of a number of bacteria. In still other examples, the individual primer sets 106 can correspond to genomic sequences of a number of viruses.6356.002W01

[0054] In one or more illustrative examples, the primers 104 included in the individual primer sets 106 can include genomic sequences that have less than a threshold amount of homology with respect to one another. For example, individual primers 104 included in individual primer sets 106 can have no greater than about 50% homology with respect to one another, no greater than about 45% homology with respect to one another, no greater than about 40% homology with respect to one another, no greater than about 35% homology with respect to one another, no greater than about 30% homology with respect to one another, no greater than about 25% homology with respect to one another, no greater than about 20% homology with respect to one another, no greater than about 15% homology with respect to one another, no greater than about 10% homology with respect to one another, or no greater than about 5% homology with respect to one another. In one or more additional illustrative examples, the genomic sequences of individual primers 104 included in individual primer sets 106 can be unique with respect to a target analyte. In one or more further illustrative examples, the genomic sequences of individual primers 104 included in individual primer sets 106 can include a nucleotide sequence from 40 base pairs to 120 base pairs, from 50 base pairs to 110 base pairs, from 60 base pairs to 100 base pairs, or from 70 base pairs to 90 base pairs that is unique to a target analyte. Individual primer sets 106 can correspond include a number of primer pairs with each primer pair including a forward primer and a reverse primer.

[0055] In one or more examples, the primers 104 included in the primer sets 106 can have at least 10 base pairs, at least 12 base pairs, at least 14 base pairs, at least 16 base pairs, at least 18 base pairs, at least 20 base pairs, at least 22 base pairs, or at least 24 base pairs. Additionally, the primers 104 included in the primer sets 106 can have no greater than 40 bas pairs, no greater than 38 base pairs, no greater than 36 base pairs, no greater than 34 base pairs, no greater than 32 base pairs, no greater than 30 base pairs, no greater than 28 base pairs, or no greater than 26 base pairs. In various examples, the primers 104 included in the primer sets 106 can have from 10 base pairs to 40 base pairs, from 10 base pairs to 35 base pairs, from 10 base pairs to 30 base pairs, from 10 base pairs to 25 base pairs, from 10 base pairs to 20 base pairs, from 15 base pairs to 40 base pairs, from 15 base pairs to 35 base pairs, from 15 base pairs to 30 base pairs, from 15 base pairs to 25 base pairs, from 20 base pairs to 40 base pairs, from 20 base pairs to 35 base pairs, from 20 base pairs to 306356.002W01 base pairs, from 25 base pairs to 40 base pairs, from 25 base pairs to 35 base pairs, or from 30 base pairs to 40 base pairs.

[0056] In addition, one primer from each primer pair can be modified. For example, a first primer of each primer pair can have a phosphate group ligated to a 5’ end of the primer and can be referred to herein as a phosphorylated strand. Further, a second primer of each primer pair can have a biotin molecule ligated to a 5’ end of the primer and can be referred to herein as a biotinylated strand.

[0057] The primer sets 106 can include at least a first set of primers 108 and a second set of primers 110. In one or more additional examples, the primer sets 106 can include a greater number of primer sets. For example, the primer sets 106 can include three sets of primers, four sets of primers, five sets of primers, six sets of primers, seven sets of primers, eight sets of primers, nine sets of primers, ten sets of primers, eleven sets of primers, twelve sets of primers, thirteen sets of primers, fourteen sets of primers, fifteen sets of primers, or more. In one or more illustrative examples, at least a portion of the primer sets 106 can have a same number of primer pairs. In one or more additional illustrative examples, one or more of the primer sets 106 can have a different number of primer pairs. The individual primer sets 106 can include different combinations of primer pairs selected from the primer pool 102. In at least some illustrative examples, the individual primer sets 106 can include a unique combination of primer pairs with respect to one another. In one or more further illustrative examples, one or more of the primer sets 106 can have a same combination of primer pairs as at least one other primer set 106.

[0058] In various examples, the first set of primers 108 can include a first combination of primer pairs selected from the primer pool 102 and the second set of primers 110 can include a second combination of primer pairs selected from the primer pool 102. In one or more illustrative examples, the first set of primers 108 can include a first combination of primer pairs that correspond to a first number of target sequences of first fungi and the second set of primers 110 can include a second combination of primer pairs that correspond to a second number of target sequences of second fungi. In one or more additional illustrative examples, the first set of primers 108 can include a first combination of primer pairs that correspond to a first number of target sequences of first bacteria and the second set of primers 110 can include a second combination of primer pairs that correspond to a second number of target sequences of second bacteria. In one or more further examples, the first set of primers 108 can include a6356.002W01 first combination of primer pairs that correspond to a first number of target sequences of first viruses and the second set of primers 110 can include a second combination of primer pairs that correspond to a second number of target sequences of second viruses. In at least some examples, the first set of primers 108 and the second set of primers 110 can include one or more primer pairs that correspond to control sequences. In various examples, the control sequences can correspond to sequences of a human reference sequence.

[0059] The first set of primers 108 and the second set of primers 110 can include 2 primer pairs, 3 primer pairs, 4 primer pairs, 5 primer pairs, 6 primer pairs, 7 primer pairs, 8 primer pairs, 9 primer pairs, 10 primer pairs, 11 primer pairs, 12 primer pairs, 13 primer pairs, 14 primers pairs, 15 primer pairs, or more. In one or more illustrative examples, the first set of primers 108 can include a first number of primer pairs that include target sequences selected from Aspergillus flavus, Candida albicans, Candida auris, Candida glabrata , Aspergillus fumigatus, Aspergillus niger, Candida krusei, Cryptococcus, Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, Fusarium solani, Candida tropicalis, Coccidioides, Histoplasma capulatun, Scedosporium spp, Mucorales, Blastomyces, and Pneumocystis jirovecii. Additionally, the second set of primers 110 can include a second number of primer pairs that include target sequences selected from Aspergillus flavus, Candida albicans, Candida auris, Candida glabrata, Scedosporium spp, Aspergillus fumigatus, Aspergillus niger, Candida krusei, Cryptococcus, Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, Fusarium solani, Candida tropicalis, Coccidioides, Histoplasma capulatun, Pneumocystis jirovecii, Mucorales, and Blastomyces. At least one primer pair of the first set of primers 108 can correspond to target sequences of a fungi that is different from target sequences of at least one primer pair of the second set of primers 110.

[0060] In one or more additional illustrative examples, the primer sets 106 can include three sets of primers with each of the three primer sets including primer pairs corresponding to a different grouping of target sequences selected from Aspergillus flavus, Candida albicans, Candida auris, Candida glabrata, Scedosporium spp, Aspergillus fumigatus, Aspergillus niger, Candida krusei, Cryptococcus, Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, Fusarium solani, Candida tropicalis, Coccidioides, Histoplasma capulatun, Mucorales, Blastomyces, and Pneumocystis jirovecii. In one or more further illustrative examples, the primer sets6356.002W01106 can include four sets of primers with each of the four primer sets including primer pairs corresponding to a different grouping of target sequences selected from Aspergillus flavus, Candida albicans, Candida auris, Candida glabrata, Scedosporium spp, Aspergillus fumigatus, Aspergillus niger, Candida krusei, Cryptococcus, Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, Fusarium solani, Candida tropicalis, Coccidioides, Histoplasma capulatun, Mucorales, Blastomyces, and Pneumocystis jirovecii. In still other illustrative examples, the primer sets 106 can include five sets of primers with each of the five primer sets including primer pairs corresponding to a different grouping of target sequences selected from Aspergillus flavus, Candida albicans, Candida auris, Candida glabrata, Scedosporium spp, Aspergillus fumigatus, Aspergillus niger, Candida krusei, Cryptococcus, Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, Fusarium solani, Candida tropicalis, Coccidioides, Histoplasma capulatun, Mucorales, Blastomyces, and Pneumocystis jirovecii. In various additional illustrative examples, the primer sets 106 can include six sets of primers with each of the six primer sets including primer pairs corresponding to a different grouping of target sequences selected from Aspergillus flavus, Candida albicans, Candida auris, Candida glabrata, Scedosporium spp, Aspergillus fumigatus, Aspergillus niger, Candida krusei, Cryptococcus, Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, Fusarium solani, Candida tropicalis, Coccidioides, Histoplasma capulatun, Mucorales, Blastomyces, and Pneumocystis jirovecii. In at least some illustrative examples, the primer sets 106 can include a first set of primer pairs that are complementary with first genomic regions of a first group of fungi including Aspergillus flavus, Candida albicans, Candida auris, Candida glabrata, and Scedosporium spp. In addition, the primer sets 106 can include a second set of primer pairs that are complementary with second genomic regions of a second group of fungi including Aspergillus fumigatus, Aspergillus niger, Candida krusei, and Cryptococcus. Further, the primer sets 106 can include a third set of primer pairs that are complementary with third genomic regions of a third group of fungi including Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, and Fusarium solani. The primer sets 106 can also include a fourth set of primer pairs that are complementary with fourth genomic regions of a fourth group of fungi including Candida tropicalis, Coccidioides, Histoplasma capulatun, Mucorales, Blastomyces, or Pneumocystis jirovecii.

[0061] Example primers can be found below in Table 1 :6356.002W01Table 1. Forward Primers and Reverse Primers for Fungal Species6356.002W01

[0062] A concentration of individual primers corresponding to target sequences in a mixture of primers included in an individual primer set 106 can be at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least 110 mM, at least 120 mM, at least 130 mM, at least 140 mM, at least 150 mM, at least 160 mM, at least 170 mM, or at least 180 mM. Additionally, a concentration of individual primers in a mixture of primers corresponding to target sequences included in an individual primer set 106 can be no greater than 300 mM, no greater than 290 mM, no greater than 280 mM, no greater than 270 mM, no greater than 260 mM, no greater than 250 mM, no greater than 240 mM, no greater than 230 mM, no greater than 220 mM, no greater than 210 mM, no greater than 200 mM, or no greater than 190 mM. In one or more illustrative examples, a concentration of individual primers corresponding to a target sequence in a mixture of primers included in an individual primer set 106 can be from 50 mM to 300 mM, from 50 mM to 280 mM. from 50 mM to 260 mM, from 50 mM to 240 mM, from 50 mM to 220 mM, from 50 mM to 200 mM, from 50 mM to 180 mM, from 50 mM to 160 mM, from 50 mM to 140 mM, from 50 mM to 120 mM, from 50 mM to 100 mM, from 50 mM to 80 mM, from 80 mM to 300 mM, from 80 mM to 280 mM. from 80 mM to 260 mM, from 80 mM to 240 mM, from 80 mM to 220 mM, from 80 mM to 200 mM, from 80 mM to 180 mM, from 80 mM to 160 mM, from 80 mM to 140 mM, from 80 mM to 140 mM, from 80 mM to 120 mM, from 100 mM to 300 mM, from 100 mM to 280 mM. from 100 mM to 260 mM, from 100 mM to6356.002W01240 mM, from 100 mM to 220 mM, from 100 mM to 200 mM, from 100 mM to 180 mM, from 100 mM to 160 mM, from 100 mM to 140 mM, from 100 mM to 140 mM, from 120 mM to 300 mM, 120 mM to 280 mM. from 120 mM to 260 mM, from 120 mM to 240 mM, from 120 mM to 220 mM, from 120 mM to 200 mM, from 120 mM to 180 mM, from 120 mM to 160 mM, from 150 mM to 300 mM, 150 mM to 280 mM. from 150 mM to 260 mM, from 150 mM to 240 mM, from 150 mM to 220 mM, from 150 mM to 200 mM, from 150 mM to 180 mM, from 180 mM to 300 mM, 180 mM to 280 mM. from 180 mM to 260 mM, from 180 mM to 240 mM, from 180 mM to 220 mM, from 200 mM to 300 mM, 200 mM to 280 mM. from 200 mM to 260 mM, from 200 mM to 240 mM, from 220 mM to 300 mM, from 220 mM to 280 mM, from 220 mM to 260 mM, from 240 mM to 300 mM, from 240 mM to 280 mM, or from 260 mM to 300 mM.

[0063] In still additional examples, a concentration of individual primers corresponding to a control sequence in a mixture of primers included in an individual primer set 106 can be from 1 mM to 30 mM, from 1 mM to 28 mM. from 1 mM to 26 mM, from 1 mM to 24 mM, from 1 mM to 22 mM, from 1 mM to 20 mM, from 1 mM to 18 mM, from 1 mM to 16 mM, from 1 mM to 14 mM, from 1 mM to 120 mM, from 1 mM to 10 mM, from 1 mM to 8 mM, from 1 mM to 6 mM, from 1 mM to 4 mM, from 5 mM to 30 mM, from 5 mM to 28 mM. from 5 mM to 26 mM, from 5 mM to 24 mM, from 5 mM to 22 mM, from 5 mM to 20 mM, from 5 mM to 18 mM, from 5 mM to 16 mM, from 5 mM to 14 mM, from 5 mM to 14 mM, from 5 mM to 110 mM, from 10 mM to 30 mM, from 10 mM to 28 mM. from 10 mM to 26 mM, from 10 mM to 24 mM, from 10 mM to 22 mM, from 10 mM to 20 mM, from 10 mM to 18 mM, from 10 mM to 16 mM, from 10 mM to 14 mM, from 15 mM to 30 mM, 15 mM to 28 mM. from 15 mM to 26 mM, from 15 mM to 24 mM, from 15 mM to 22 mM, from 15 mM to 20 mM, from 20 mM to 30 mM, from 20 mM to 28 mM, from 20 mM to 26 mM, 20 mM to 24 mM. or from 25 mM to 30 mM.

[0064] The process 100 can include obtaining one or more samples 112 from a patient 114. The one or more samples 112 can include one or more biological fluids obtained from the patient 114. In one or more examples, the one or more samples 112 can include at least one of serum, plasma, blood, urine, cerebrospinal fluid, sputum, abdominal fluid, interstitial fluid, saliva, peritoneal fluid, pleural fluid, oral fluid, nasal fluid, semen, vaginal secretions, synovial fluid, pericardial fluid, amniotic fluid, lesion aspiration, bronchoalveolar lavage, genital swab, oropharyngeal swab, ano-rectal swab, nasal swab, nasopharyngeal swab, tissue biopsy, stool, bone marrow, sweat, or tears.6356.002W01

[0065] The analyte may be a nucleic acid derived from an organism. In some embodiments, the nucleic acid is a target nucleic acid derived from the organism that has been amplified to form an amplicon. In some embodiments, the organism is a plant, a mammal, a microbial species, or a virus. In several embodiments, the analyte may be derived from a microbial pathogen. In such embodiments, the biological sample may include cells and / or cell debris from the host mammalian subject as well as one or more microbial pathogen cells.

[0066] A number of nucleic acids 116 can be derived from the one or more samples 112. In various examples, the number of nucleic acids 116 can be extracted from the one or more samples 112. The extraction of the nucleic acids 116 from the one or more samples 112 can include the lysis of cells in the one or more samples 112. In one or more examples, the cells included in the samples 112 can by lysed using at least one of one or more physical cell lysis techniques, one or more chemical cell lysis techniques, or one or more enzymatic cell lysis techniques.

[0067] The one or more physical cell lysis techniques can include breaking cell walls by physically contacting the cell walls with one or more materials. In various examples, the one or more physical cell lysis techniques can include contacting the one or more samples 112 with an instrument or tool that breaks the cell walls. In one or more additional examples, the one or more physical cell lysis techniques can include contacting the one or more samples 112 with beads within one or more containers to disrupt the cell walls. The beads can be comprised of at least one of a ceramic material, a metallic material, or a polymeric material. In one or more illustrative examples, the beads can be comprised of stainless steel, zirconium oxide, borosilicate glass, silica, silicon carbide, or one or more combinations thereof. In one or more additional illustrative examples, the beads can be comprised of a magnetic material. The one or more physical cell lysis techniques can be performed in tubes, mills, homogenizers, vortexers, plates, grinders, sonicators, and the like. Commercially available beads can include magnetic beads offered by ZymoBiomics.

[0068] The one or more chemical cell lysis techniques can include contacting the one or more samples 112 with one or more solutions that include compounds that can disrupt cellular walls. In one or more examples, the one or more chemical cell lysis techniques can include contacting the one or more samples 112 with a solution that includes one or more detergents. In one or more additional examples, the one or more chemical cell lysis techniques can include contacting the one or more samples 1126356.002W01 with a solution that includes one or more chaotropes. In various examples, the one or more chemical cell lysis techniques can include contacting the one or more samples 112 with a solution that includes one or more non-ionic surfactants. In one or more illustrative examples, the one or more chemical cell lysis techniques sodium dodecyl sulphate (SDS), can include contacting the one or more samples 112 with a solution that includes 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1 -propanesulfonate (CHAPSO),4-octylphenol polyethoxylate (Triton-X 20, 50, 100), Tween, SDS, nonyl phenoxypolyethoxylethanol (NP-40), Cetyltrimethylammonium bromide (CTAB), or one or more combinations thereof.

[0069] Various techniques to lyse cell membranes in order to extract the nucleic acids 116 from cells included in the one or more samples are described in Shehadul Islam M, Aryasomayajula A, Selvaganapathy PR. A Review on Macroscale and Microscale Cell Lysis Methods. Micromachines (Basel). 2017 Mar 8;8(3):83. doi: 10.3390 / mi8030083. PMCID: PMC6190294; Klimek-Ochab M, Brzezihska-Rodak M, Zymahczyk-Duda E, Lejczak B, Kafarski P. Comparative study of fungal cell disruption-scope and limitations of the methods. Folia Microbiol (Praha). 2011 Sep;56(5):469-75. doi: 10.1007 / s12223-011 -0069-2. Epub 2011 Sep 8. PMID: 21901292; PMCID: PMC3189342; Hasim S, Coleman J J. Targeting the fungal cell wall: current therapies and implications for development of alternative antifungal agents. Future Med Chem. 2019 Apr;11 (8):869-883. doi: 10.4155 / fmc-2018-0465. Epub 2019 Apr 17. PMID: 30994368; PMCID: PMC6543504, and Sebastian Scharf, Anna Bartels, Mustafa Kondakci, Klaus Pfeffer, Birgit Henrich, Rainer Haas, Introduction of a bead beating step improves fungal DNA extraction from selected patient specimens, International Journal of Medical Microbiology, Volume 310, Issue 6, 2020, 151443, ISSN 1438-4221 , which are each incorporated by reference herein.

[0070] Column-based extraction is a method that employs selective binding of nucleic acid to a solid matrix such as silica that is packed in a column. After lysis, apply cell lysate to a column in the presence of a high salt buffer allowing nucleic acid and nucleic acid binding proteins to adsorb to the matrix. Other cellular components and proteins do not bind to the matrix and pass through the column as the lysate is applied. Complete the sample application, and later washing, and elution steps by centrifugation, pressure, or vacuum. After sample application, nucleic acid binding proteins may stay bound to nucleic acid and remain in the column. Pass a buffer6356.002W01 containing a low amount of chaotropic salt through the column to remove these proteins. Apply a wash buffer to the same column to wash out contaminants in order to improve purity of the eluted nucleic acid. Finally, apply an appropriate elution buffer of water to release the nucleic acid from the solid matrix into a collecting container.

[0071] Lysate can be produced by the one or more processes applied to break the cell membranes. One or more purification operations can be performed to remove waste products from the lysate. In one or more examples, the debris included in the lysate can include proteins, lipids, saccharides, and the like. In at least some examples, the purification of the lysate can take place using at least one of centrifugation, filtration, phase separation, precipitation, one or more affinity-based methods, or one or more bead-based methods.

[0072] After the removal of waste products from the lysate, nucleic acids can be isolated from the residual lysate. In one or more examples, DNA included in the residual lysate can be isolated using matrices that bind the DNA while allowing the remainder of the residual lysate to remain unbound to the matrices. In one or more examples, DNA can be separated from the residual lysate using silica binding material. The silica binding material can comprise a filter membrane, silica crystal, raw silica, immobilized silica material, or silica-coated magnetic particles. In one or more illustrative examples, the filter membrane comprising silica binding material can be included in a purification column. In one or more additional illustrative examples, silicon-coated magnetic particles can be used to capture DNA in solution in the presence of a magnetic field. The silica binding material can be combined with one or more chaotropic salts. In one or more illustrative examples, the one or more chaotropic salts can include guanidine hydrochloride, The one or more chaotropic salts can facilitate the binding of the nucleic acid molecules to silica. In still other examples, DNA can be bound to silica materials in the presence of kosmotripic salts.

[0073] Binding of DNA to silica surfaces can be performed under conditions described in Vandeventer PE, Lin JS, Zwang TJ, Nadim A, Johal MS, Niemz A. Multiphasic DNA adsorption to silica surfaces under varying buffer, pH, and ionic strength conditions. J Phys Chem B. 2012 May 17;116(19):5661 -70. doi: 10.1021 / jp3017776. Epub 2012 May 8. PMID: 22537288; PMCID: PMC3766398.

[0074] Additionally, DNA can be separated from the residual lysate using cellulose materials. The DNA can bind to a cellulose matrix in the presence of solutions having relatively high concentrations of one or more salts and alcohols. Further, DNA can be6356.002W01 separated from the residual lysate using ion-exchange techniques. For example, negatively charged phosphate groups in solutions having relatively low salt concentrations can bind to a matrix of positively charged particles. Unwanted particles, such as proteins and RNA, can be removed from the positively charged particles using salt solutions.

[0075] After binding of DNA to the binding material, one or more rinsing or washing operations can be performed. In various examples, the one or more rinsing or washing operations can include solutions with components that cause the removal of contaminants, such as proteins, polysaccharides and, in at least some cases unwanted RNA. For example, the solutions in the rinsing or washing operations can include a mixture of one or more salts in ethanol. Wash buffers including alcohols can be used to remove contaminants bound to the particle matrices and aid in the binding of DNA to the positively charged particles. In at least some scenarios, the one or more rinsing or washing operations can include a ribonuclease. In one or more examples, the ribonuclease can include RNase A.

[0076] Extracted DNA can be eluted using one or more solvents. The one or more solvents can include water or a buffer solution including tris(hydroxymethyl)aminomethane (Tris) and ethylenediaminetetraacetic acid (EDTA).

[0077] In still other examples, the DNA can be isolated from the residual lysate by alcohol precipitation. To illustrate, the residual lysate can be contacted with one or more solutions having a relatively high salt concentration that cause proteins and other contaminants to form a precipitate that can be filtered from the DNA molecules. The filtering of the contaminant precipitate can be achieved using centrifugation. One or more alcohol solutions can then be used to cause the DNA to form a precipitate. The DNA precipitate can be pelletized and isolated from the alcohol and salt solutions using centrifugation. In various examples, the pelletized DNA can be subjected to one or more additional washes, such as an ethanol wash. The pelletized DNA can be suspended in solution once again using a Tris-EDTA buffer or water.

[0078] The extracted nucleic acids 118 can be combined with the primer sets 106 in a nucleic acid amplification process 120. The nucleic acid amplification process 120 can produce an amplification product 122 that includes a number of copies of a nucleic acid subset 124. The nucleic acid subset 124 can include nucleic acids 118 that have been bound to a primer included in the primer sets 106. In this way, the amplification product 122 includes original nucleic acids included in the extracted nucleic acids 1186356.002W01 as well as copies of the extracted nucleic acids 118 that include target sequences of analytes of interest.

[0079] The nucleic acid amplification process 120 can take place in a container. In one or more examples, the container can include a channel that is included in a microfluidics device. In one or more additional examples, the container can include a well. In one or more further examples, the container can include a tube. In various examples, the nucleic acid amplification process 120 can be performed in multiple containers. In at least some examples, the nucleic acid amplification process 120 can take place in a number of containers that corresponds to the number of primer sets. For example, the nucleic acid amplification process 120 can be performed in a first container in relation to the first set of primers 108 and in a second container in relation to the second set of primers 110. In still other examples, the nucleic acid amplification process 120 can be performed using multiple primer sets in a single container. To illustrate, the nucleic acid amplification process 120 can be performed using the first set of primers 108 and the second set of primers 110 in a single container.

[0080] In one or more examples, the nucleic acid amplification process 120 can be performed using lyophilized components. In at least some examples, the lyophilized components can be included in a bead. In one or more illustrative examples, the lyophilized components used in the nucleic acid amplification process 120 can be reconstituted using an aqueous solution. In one or more additional illustrative examples, the lyophilized components used in the nucleic acid amplification process 120 can be reconstituted using a solution in which the extracted nucleic acids 118 are present and / or using a solution in which the primer sets 106 are present. The extracted nucleic acids 118 and the primer sets 106 can be present in aqueous solutions. The extracted nucleic acids 118 and the primer sets 106 can also be present in one or more buffer solutions. For example, the extracted nucleic acids 118 and the primer sets 106 can be present in a Tris-EDTA buffer solution.

[0081] In various examples, the lyophilized components used in the nucleic acid amplification process 120 can include a polymerase and nucleotides. In one or more illustrative examples, the lyophilized components used in the nucleic acid amplification process 120 can include a Taq DNA polymerase. The nucleotides included in the lyophilized components can comprise deoxynucleotide triphosphates (dNTPs). For example, the lyophilized components used in the nucleic acid amplification process 120 can include deoxyadenosine triphosphate (dATP); deoxythymidine triphosphate6356.002W01(dTTP); deoxycytosine triphosphate (dCTP); and deoxyguanosine triphosphate, dGTP. In one or more examples, the dNTPs included in the lyophilized components can include deoxyuridine triphosphate (dllTP). In at least some examples, the individual dNTPs can be present in substantially equimolar amounts. In one or more illustrative examples, the lyophilized components used in the nucleic acid amplification process 120 can include a buffer solution comprising water, 2-hydroxypropyl-[3- cyclodextrin (HPBCD), and mannitol. A commercial example of components that can be used in the nucleic acid amplification process 120 is a Taqman multiplex master mix available from Applied Biosystems.

[0082] In one or more examples, a volume of lyophilized components used in the nucleic acid amplification process 120 can be at least about 5 microliters pL, at least about 6 pL, at, at least about 7 pL at least about 8 pL, at least about 9 pL, at least about 10 pL, at least about 11 pL, at least about 12 pL, at least about 13 pL, at least about 14 pL, at least about 15 pL, at least about 16 pL, at least about 17 pL, at least about 18 pL, at least about 19 pL, or at least about 20 pL. In one or more additional examples, a volume of lyophilized components used in the nucleic acid amplification process 120 can be no greater than about 35 pL, no greater than about 34 pL, no greater than about 33 pL, no greater than about 32 pL, no greater than about 31 pL, no greater than about 30 pL, no greater than about 29 pL, no greater than about 28 pL, no greater than about 27 pL, no greater than about 26 pL, no greater than about 25 pL, no greater than about 24 pL, no greater than about 23 pL, no greater than about 22 pL, or no greater than about 21 pL. In one or more further examples, a volume of lyophilized components used in the nucleic acid amplification process 120 can be from about 5 pL to about 35 pL, from about 5 pL to about 30 pL, from about 5 pL to about 25 pL, from about 5 pL to about 20 pL, from about 5 pL to about 15 pL, from about 5 pL to about 10 pL, from about 10 pL to about 35 pL, from about 10 pL to about 30 pL, from about 10 pL to about 25 pL, from about 10 pL to about 20 pL, from about 10 to about 15 pL, from about 15 pL to about 35 pL, from about 15 pL to about 30 pL, from about 15 pL to about 25 pL, from about 15 pL to about 20 pL, from about 20 pL to about 35 pL, from about 25 pL to about 35 pL, from about 25 pL to about 30 pL, or from about 30 pL to about 35 pL.

[0083] In still other examples, the lyophilized components used in the nucleic acid amplification process 120 can be included in a bead having a volume from about 5 pL to about 35 pL, from about 10 pL to about 30 pL, from about 15 pL to about 25 pL,6356.002W01 from about 5 pL to about 20 pL, from about 10 pL to about 20 pL, from about 20 pL to about 30 pL or from about 20 pL to about 35 pL. In one or more examples, the bead can include a component comprised of a HPBCD solution having from about 20% by weight to about 40% by weight HPBCD, from about 25% by weight to about 35% by weight HPBCD, or from about 20% by weight to about 30% by weight HPBCD. Additionally, the bead can include a component comprised of a mannitol solution having from about 20% by weight to about 40% by weight mannitol, from about 25% by weight to about 35% by weight mannitol, or from about 20% by weight to about 30% by weight mannitol.

[0084] In various examples, the bead can include from about 20% by volume to about 40% by volume of the HPBCD solution, from about 20% by volume to about 35% by volume of the HPBCD solution, from about 20% by volume to about 30% by volume of the HPBCD solution, from about 20% by volume to about 25% by volume of the HPBCD solution, from about 25% by volume to about 40% by volume of the HPBCD solution, from about 25% by volume to about 35% by volume of the HPBCD solution, from about 25% by volume to about 30% by volume of the HPBCD solution, from about 30% by volume to about 40% by volume of the HPBCD solution, or from about 35% by volume to about 40% by volume of the HPBCD solution.

[0085] In addition, the bead can include from about 20% by volume to about 40% by volume of the mannitol solution, from about 20% by volume to about 35% by volume of the mannitol solution, from about 20% by volume to about 30% by volume of the mannitol solution, from about 20% by volume to about 25% by volume of the mannitol solution, from about 25% by volume to about 40% by volume of the mannitol solution, from about 25% by volume to about 35% by volume of the mannitol solution, from about 25% by volume to about 30% by volume of the mannitol solution, from about 30% by volume to about 40% by volume of the mannitol solution, or from about 35% by volume to about 40% by volume of the mannitol solution.

[0086] Further, the bead can include from about 20% by volume to about 50% by volume of the Taq polymerase solution, from about 20% by volume to about 45% by volume of the Taq polymerase solution, from about 20% by volume to about 40% by volume of the Taq polymerase solution, from about 20% by volume to about 35% by volume of the Taq polymerase solution, from about 20% by volume to about 30% by volume of the Taq polymerase solution, from about 20% by volume to about 25% by volume of the Taq polymerase solution, from about 25% by volume to about 50% by6356.002W01 volume of the Taq polymerase solution, from about 25% by volume to about 45% by volume of the Taq polymerase solution, from about 25% by volume to about 40% by volume of the Taq polymerase solution, from about 25% by volume to about 35% by volume of the Taq polymerase solution, from about 25% by volume to about 30% by volume of the Taq polymerase solution, from about 30% by volume to about 50% by volume of the Taq polymerase solution, from about 30% by volume to about 45% by volume of the Taq polymerase solution, from about 30% by volume to about 40% by volume of the Taq polymerase solution, from about 35% by volume to about 50% by volume of the Taq polymerase solution, from about 35% by volume to about 45% by volume of the Taq polymerase solution, from about 35% by volume to about 40% by volume of the Taq polymerase solution, from about 40% by volume to about 50% by volume of the Taq polymerase solution, from about 40% by volume to about 45% by volume of the Taq polymerase solution, or from about 45% by volume to about 50% by volume of the Taq polymerase solution.

[0087] In still other examples, the bead can include from about 15% by volume to about 30% by volume of the dNTPs solution, from about 15% by volume to about 25% by volume of the dNTPs solution, from about 15% by volume to about 20% by volume of the dNTPs solution, from about 20% by volume to about 30% by volume of the dNTPs solution, from about 20% by volume to about 25% by volume of the dNTPs solution, or from about 25% by volume to about 30% by volume of the dNTPs solution.

[0088] In one or more examples, at least one of the mannitol solution, the HPBCD solution, the Taq polymerase solution, and the dNTPs solution can include an amount of water. In at least some examples, the bead can include an amount of water that is in addition to the amount of water included in at least one of the mannitol solution, the HPBCD solution, the Taq polymerase solution, and the dNTPs solution. For example, the bead can include from about 2% by volume to about 15% by volume of additional water, from about 2% by volume to about 12% by volume of additional water, from about 2% by volume to about 10% by volume of additional water, from about 2% by volume to about 8% by volume of additional water, from about 2% by volume to about 6% volume of additional water, from about 2% by volume to about 4% by volume of additional water, from about 5% by volume to about 15% by volume of additional water, from about 5% by volume to about 12% by volume of additional water, from about 5% by volume to about 10% by volume of additional water, from about 5% by volume to about 8% by volume of additional water, from about 8% by volume to about 15% by6356.002W01 volume of additional water, from about 8% by volume to about 12% by volume of additional water, from about 8% by volume to about 10% by volume of additional water, from about 10% by volume to about 15% by volume of additional water, from about 10% by volume to about 12% by volume of additional water, or from about 12% by volume to about 15% by volume of additional water.

[0089] In scenarios where the lyophilized components of the nucleic acid amplification process 120 are provided as a bead, in one or more examples, a single bead including the lyophilized components can be provided to individual containers in which at least a portion of the nucleic acid amplification process 120 is performed. To illustrate, the nucleic acid amplification process 120 can include providing a first bead including the lyophilized components, the first set of primers 108, and a first portion of the extracted nucleic acids 118 to a first container and providing a second bead including the lyophilized components, the second primer set 110, and a second portion of the extracted nucleic acids 118 to a second container, where at least a portion of the nucleic acid amplification process 120 is performed in the first container and the second container. In one or more additional examples, multiple beads including the lyophilized components can be provided to individual containers in which at least a portion of the nucleic acid amplification process 120 is performed. In one or more illustrative examples, a first plurality of beads including the lyophilized components, the first set of primers 108, and a first portion of the extracted nucleic acids 118 can be provided to a first container and a second plurality of beads including the lyophilized components, the second set of primers 110, and a second portion of the extracted nucleic acids 118 can be provided to a second container, where at least a portion of the nucleic acid amplification process 120 is performed in the first container and the second container. In one or more additional illustrative examples, a plurality of beads including the lyophilized components, the first set of primers 108, the second set of primers 110, and at least a portion of the extracted nucleic acids 118 can be provided to a container in which at least a portion of the nucleic acid amplification process 120 is performed.

[0090] The nucleic acid amplification process 120 can include heating a mixture that includes the primer sets 106, the extracted nucleic acids 118, and polymerase chain reaction (PCR) components. In the mixture, the extracted nucleic acids 118 can include double stranded DNA. In these situations, the mixture can be heated to temperatures that cause denaturing of the double stranded nucleic acids 118. In this6356.002W01 way, single stranded DNA molecules can be produced from at least a portion of the extracted nucleic acids 118. In one or more examples, the nucleic acid amplification mixture can be heated to temperatures of at least about 75 °C, at least about 78 °C, at least about 80 °C, at least about 82 °C, at least about 85 °C, at least about 88 °C, or at least about 90 °C. In one or more additional examples, the nucleic acid amplification mixture can be heated to temperatures no greater than about 99 °C, no greater than about 98 °C, no greater than about 97 °C, no greater than about 96 °C, no greater than about 95 °C, no greater than about 94 °C, no greater than about 93 °C, no greater than about 92 °C, or no greater than about 91 °C. In one or more illustrative examples, the PCR amplification mixture can be heated to temperatures from about 75 °C to about 99 °C, from about 78 °C to about 97 °C, from about 80 °C to about 95 °C, from about 82 °C, to about 93 °C, from about 80 °C to about 90 °C, from about 75 °C to about 85 °C, from about 78 °C to about 88 °C, or from about 85 °C to about 95 °C.

[0091] In various examples, the PCR amplification mixture can be heated at temperatures to cause double stranded DNA of the extracted nucleic acids 118 to denature for a duration of at least about 0.5 minutes, at least about 0.8 minutes, at least about 1 minute, at least about 1.2 minutes, at least about 1.5 minutes, at least about 1.8 minutes, at least about 2 minutes, at least about 2.2 minutes, at least about 2.5 minutes, at least about 2.8 minutes, at least about 3 minutes, at least about 3.2 minutes, at least about 3.5 minutes, at least about 3.8 minutes, or at least about 4 minutes. Additionally, the PCR amplification mixture can be heated at temperatures to cause double stranded DNA of the extracted nucleic acids 118 to denature for a duration of no greater than about 8 minutes, no greater than about 7.8 minutes, no greater than about 7.5 minutes, no greater than about 7.2 minutes, no greater than about 7 minutes, no greater than about 6.8 minutes, no greater than about 6.5 minutes, no greater than about 6.2 minutes, no greater than about 6 minutes, no greater than about 5.8 minutes, no greater than about 5.5 minutes, no greater than about 5.2 minutes, no greater than about 5 minutes, no greater than about 4.8 minutes, no greater than about 4.5 minutes, or no greater than about 4.2 minutes. In one or more illustrative examples, the PCR amplification mixture can be heated at temperatures to cause double stranded DNA of the extracted nucleic acids 118 to denature for durations from about 0.5 minutes to about 8 minutes, from about 1 minute to about 7 minutes, from about 2 minutes to about 6 minutes, from about 0.5 minutes to about 56356.002W01 minutes, from about 0.5 minutes about 2 minutes, from about 1 minute to about 5 minutes, from about 1 minute to about 4 minutes, from about 2 minutes to about 8 minutes, from about 2 minutes to about 4 minutes, from about 3 minutes to about 8 minutes, from about 3 minutes to about 5 minutes, from about 4 minutes to about 8 minutes, from about 4 minutes to about 6 minutes, or from about 5 minutes to about 8 minutes.

[0092] After denaturing double stranded DNA molecules included in the extracted nucleic acids 118 to produce a number of single stranded DNA molecules, an annealing operation can be performed to cause primers included in the primer sets 106 to bind to the single stranded DNA molecules derived from the extracted nucleic acids 118. The annealing of the primers to the single stranded DNA molecules can take place at temperatures lower than the temperatures used to denature double stranded DNA molecules included in the extracted nucleic acids 118. For example, a solution including the single stranded DNA molecules can undergo a cooling process. The annealing of single stranded DNA molecules to primers of the primer sets 106 can take place at temperatures of at least about 40 °C, at least about 42 °C, at least about 45 °C, at least about 48 °C, at least about 50 °C, at least about 52 °C, at least about 55 °C, at least about 58 °C, or at least about 60 °C. In one or more additional examples, the annealing of single stranded DNA molecules to primers of the primer sets 106 can take place at temperatures no greater than about 70 °C, no greater than about 69 °C, no greater than about 68 °C, no greater than about 67 °C, no greater than about 66 °C, no greater than about 65 °C, no greater than about 64 °C, no greater than about 63 °C, no greater than about 62 °C, or no greater than about 61 °C. In one or more illustrative examples, the annealing of single stranded DNA molecules to primers of the primer sets 106 can take place at temperatures from about 40 °C to about 70 °C, from about 40 °C to about 65 °C, from about 40 °C to about 60 °C, from about 40 °C, to about 50 °C, from about 45 °C to about 65 °C, from about 45 °C to about 60 °C, from about 45 °C to about 55 °C, from about 50 °C to about 65 °C, or from about 50 °C to about 60 °C.

[0093] In one or more examples, the process of annealing single stranded DNA molecules to primers of the primer sets 106 can take place for a duration of at least about 0.5 minutes, at least about 0.6 minutes, at least about 0.7 minutes, at least about 0.8 minutes, at least about 0.9 minutes, at least about 1 minute, at least about 1.1 minutes, at least about 1.2 minutes, at least about 1.3 minutes, at least about 1.46356.002W01 minutes, or at least about 1.5 minutes. Additionally, the process of annealing single stranded DNA molecules to primers of the primer sets 106 can take place for a duration of no greater than about 4 minutes, no greater than about 3.8 minutes, no greater than about 3.5 minutes, no greater than about 3.2 minutes, no greater than about 3 minutes, no greater than about 2.8 minutes, no greater than about 2.5 minutes, no greater than about 2.2 minutes, no greater than about 2 minutes, or no greater than about 1.8 minutes. In one or more illustrative examples, the process of annealing of single stranded DNA molecules to primers of the primer sets 106 can take place for durations from about 0.5 minutes to about 4 minutes, from about 0.5 minutes to about 3.5 minutes, from about 0.5 minutes to about 3 minutes, from about 0.5 minutes to about2.5 minutes, from about 0.5 minutes about 2 minutes, from about 0.5 minutes to about1.5 minutes, from about 0.5 minutes to about 1 minute, from about 1 minute to about 4 minutes, from about 1 minute to about 3 minutes, from about 1 minute to about 2.5 minutes, from about 1 minute to about 2 minutes, from about 1.5 minutes to about 4 minutes, from about 1 .5 minutes to about 3.5 minutes, from about 1 .5 minutes to about 3 minutes, from about 1.5 minutes to about 2.5 minutes, or from about 2 minutes to about 4 minutes.

[0094] After annealing of the primers included in the primer sets 106 to target sequences of the single stranded DNA molecules derived from the extracted nucleic acids 118, the primers can be extended using a DNA polymerase and the dNTPs included in the PCR components. The extension of the primers can include adding nucleotides in the 5’ to 3’ direction starting at the 3’ end of each primer and continuing until it reaches the end of the template DNA strand or until the extension operation ends. The extension operation can take place at temperatures that are higher than the temperatures of the annealing process and lower than the temperatures of the denaturing process. To illustrate, the extension of primers bound to single stranded DNA molecules derived from the extracted nucleic acids 118 and having at least one target sequence can take place at temperatures of at least about 65 °C, at least about 66 °C, at least about 67 °C, at least about 68 °C, at least about 69 °C, at least about 70 °C, at least about 71 °C, at least about 72 °C, at least about 73 °C, or at least about 74 °C. In one or more additional examples, extension of primers bound to single stranded DNA molecules derived from the extracted nucleic acids 118 and having at least one target sequence can take place at temperatures no greater than about 82 °C, no greater than about 81 °C, no greater than about 80 °C, no greater than about6356.002W0179 °C, no greater than about 78 °C, no greater than about 77 °C, no greater than about 76 °C, or no greater than about 75 °C. In one or more illustrative examples, extension of primers bound to single stranded DNA molecules derived from the extracted nucleic acids 118 and having at least one target sequence can take place at temperatures from about 65 °C to about 82 °C, from about 65 °C to about 80 °C, from about 65 °C to about 75 °C, from about 68 °C, to about 80 °C, from about 68 °C to about 78 °C, from about 68 °C to about 75 °C, from about 68 °C to about 72 °C, from about 70 °C to about 80 °C, from about 72 °C to about 78 °C, or from about 72 °C to about 76 °C.

[0095] In at least some examples, the process of extending primers bound to single stranded DNA molecules derived from the extracted nucleic acids 118 and having at least one target sequence can take place for a duration of at least about 0.3 minutes, at least about 0.4 minutes, at least about 0.5 minutes, at least about 0.6 minutes, at least about 0.7 minutes, at least about 0.8 minute, at least about 0.9 minutes, at least about 1 minute, at least about 1.1 minutes, or at least about 1 .2 minutes. Additionally, the process of extending primers bound to single stranded DNA molecules derived from the extracted nucleic acids 118 and having at least one target sequence can take place for a duration of no greater than about 3 minutes, no greater than about 2.8 minutes, no greater than about 2.5 minutes, no greater than about 2.2 minutes, no greater than about 2 minutes, no greater than about 1.8 minutes, or no greater than about 1.5 minutes. In one or more illustrative examples, the process of extending primers bound to single stranded DNA molecules derived from the extracted nucleic acids 118 and having at least one target sequence can take place for durations from about 0.3 minutes to about 3 minutes, from about 0.3 minutes to about 2.5 minutes, from about 0.3 minutes to about 2 minutes, from about 0.3 minutes to about 1.5 minutes, from about 0.3 minutes about 1 minute, from about 0.5 minutes to about 2 minutes, from about 0.5 minutes to about 1 .5 minute, from about 0.5 minutes to about 1 minute, from about 0.8 minutes to about 2 minutes, from about 0.8 minutes to about 1.5 minutes, from about 0.8 minutes to about 1.2 minutes, from about 1 minute to about 2 minutes, or from about 1 minute to about 1 .5 minutes.

[0096] In one or more examples, the nucleic acid amplification process 120 can have a number of cycles. Individual cycles of the nucleic acid amplification process 120 can include a denaturation step, an annealing step, and an extension step. In situations where the nucleic acid amplification process 120 includes a plurality of cycles, extension steps of one cycle of the nucleic acid amplification process 120 are followed6356.002W01 by denaturation steps of the subsequent cycles, except for the final extension step. In one or more illustrative examples, the nucleic acid amplification process 120 can include at least 2 cycles, at least 3 cycles, at least 4 cycles, at least 5 cycles, at least 6 cycles, at least 7 cycles, at least 8 cycles, at least 9 cycles, at least 10 cycles, at least 11 cycles, at least 12 cycles, at least 13 cycles, at least 14 cycles, or at least 15 cycles. In one or more additional illustrative examples, the nucleic acid amplification process 120 can include no greater than 30 cycles, no greater than 28 cycles, no greater than 26 cycles, no greater than 24 cycles, no greater than 22 cycles, no greater than 20 cycles, no greater than 18 cycles, or no greater than 16 cycles. In still other examples, the number of cycles of the nucleic acid amplification process 120 can include from 2 cycles to 30 cycles, from 2 cycles to 25 cycles, from 2 cycles to 20 cycles, from 2 cycles to 15 cycles, from 2 cycles to 10 cycles, from 2 cycles to 8 cycles, from 2 cycles to 5 cycles, from 5 cycles to 30 cycles, from 5 cycles to 25 cycles, from 10 cycles to 20 cycles, from 10 cycles to 15 cycles, from 8 cycles to 30 cycles, from 8 cycles to 25 cycles, from 8 cycles, to 20 cycles, from 8 cycles to 15 cycles, from 10 cycles to 30 cycles, from 10 cycles to 25 cycles, from 10 cycles to 20 cycles, from 12 cycles to 30 cycles, from 12 cycles to 25 cycles, or from 12 cycles to 20 cycles.

[0097] In one or more examples, the amplification product 122 can include a number of copies of individual nucleic acids included in the extracted nucleic acids 118 that include a target sequence. In various examples, the amplification product 122 can include at least 2 copies, at least 4 copies, at least 6 copies, at least 10 copies, at least 15 copies, at least 20 copies, at least 30 copies, at least 40 copies, at least 50 copies, at least 100 copies, at least 250 copies, at least 500 copies, or at least 1000 copies individual nucleic acids included in the extracted nucleic acids 118 that include a target sequence. In addition, the amplification product 122 can include no greater than 100,000 copies, no greater than 80,000 copies, no greater than 60,000 copies, no greater than 40,000 copies, no greater than 20,000 copies, no greater than 10,000 copies, no greater than 5000 copies, no greater than 4000 copies, no greater than 3000 copies, or no greater than 2000 copies of individual nucleic acids included in the extracted nucleic acids 118 that include a target sequence. Further, the amplification product 120 can include from about 2 copies to about 500,000 copies, from about 2 copies to about 100,000 copies, from about 2 copies to about 50,000 copies, from about 2 copies to about 10,000 copies, from about 2 copies to about 5000 copies, from about 2 copies to about 1000 copies, from about 2 copies to about 100 copies, from6356.002W01 about 2 copies to about 50 copies, from about 2 copies to about 20 copies, from about 10 copies to about 1000 copies, from about 10 copies to about 500 copies, from about 10 copies to about 100 copies, from about 10 copies to about 50 copies, from about 10 copies to about 30 copies, from about 50 copies to about 1000 copies, from about 50 copies to about 500 copies, from about 50 copies to about 100 copies, from about 1000 copies to about 100,000 copies, from about 1000 copies to about 50,000 copies, from about 1000 copies to about 10,000 copies, or from about 10,000 copies to about 100,000 copies.

[0098] Based on a first primer from the primer pairs included in the primer sets 108 including a phosphate group at the 5’ end, a first portion of the amplicons included in the amplification produce 122 can comprise a 5’ phosphorylated strand. In addition, based on a second primer from the primer pairs included in the primer sets 108 including a biotin molecule at the 5’ end, a second portion of the amplicons included in the amplification produce 122 can comprise a 5’ biotinylated strand. In various examples, after the completion of a final extension operation of the nucleic acid amplification process 120, the amplification produce 122 can include double stranded DNA molecules comprised of a 5’ phosphorylated strand or a 5’ biotinylated strand.

[0099] The process 100 can also include, at 126, performing nucleic acid digestion. The nucleic acid digestion 126 can include contacting nucleic acids included in the amplification product 122 with an exonuclease. The exonuclease can remove nucleotides from individual nucleic acid molecules included in the amplification product 122 to deconstruct a portion of the individual nucleic acid molecules of the amplification product 122. In one or more examples, the exonuclease can remove nucleotides from nucleic acids included in the amplification product 122 that have a 5’ phosphorylated strand. In addition, the exonuclease can be inactive with respect to nucleic acids included in the amplification product 122 that have a 5’biotinylated strand. In these scenarios, the number of nucleic acids included in the amplification product 122 that have a 5’ biotinylated strand that are digested by the exonuclease are minimized. In one or more illustrative examples, the nucleic acid digestion process 126 can be performed using a lambda exonuclease. The nucleic acid digestion process 126 can produce a nucleic acid digestion product 128 that includes 5’ biotinylated single stranded DNA molecules derived from the amplification product 122.6356.002W01

[0100] In one or more examples, the digestion mixture can be comprised of components that have been lyophilized. In various examples, the lyophilized components used in the nucleic acid digestion process 126 can include one or more buffer solutions, an HPBCD solution, a mannitol solution, a nuclease, and additional water. A commercial example of components that can be used in the nucleic acid digestion process 126 is a Lambda Exonuclease available from ThermoFisher Scientific.

[0101] In one or more examples, a volume of lyophilized components used in the nucleic acid digestion process 126 can be at least about 15 microliters pL, at least about 18 pL, at, at least about 20 pL at least about 22 pL, at least about 24 pL, at least about 26 pL, at least about 28 pL, at least about 30 pL, at least about 32 pL, at least about 34 pL, at least about 36 pL, or at least about 38 pL. In one or more additional examples, a volume of lyophilized components used in the nucleic acid digestion process 126 can be no greater than about 60, no greater than about 58 pL, no greater than about 56 pL, no greater than about 54 pL, no greater than about 52 pL, no greater than about 50 pL, no greater than about 48 pL, no greater than about 46 pL, no greater than about 44 pL, no greater than about 42 pL, or no greater than about 40 pL, In one or more further examples, a volume of lyophilized components used in the nucleic acid digestion process 126 can be from about 15 pL to about 60 pL, from about 15 pL to about 55 pL, from about 15 pL to about 50 pL, from about 15 pL to about 45 pL, from about 15 pL to about 40 pL, from about 15 pL to about 35 pL, from about 15 pL to about 30 pL, from about 20 pL to about 60 pL, from about 20 pL to about 55 pL, from about 20 pL to about 50 pL, from about 20 to about 45 pL, from about 20 pL to about 40 pL, from about 20 pL to about 35 pL, from about 20 pL to about 30 pL, from about 25 pL to about 60 pL, from about 25 pL to about 55 pL, from about 25 pL to about 50 pL, from about 25 pL to about 45 pL, from about 25 pL to about 40 pL, from about 25 pL to about 35 pL, from about 30 pL to about 60 pL, from about 30 pL to about 55 pL, from about 30 pL to about 50 pL, from about 30 pL to about 45 pL, from about 30 pL to about 40 pL, from about 35 pL to about 60 pL, from about 35 pL to about 55 pL, from about 35 pL to about 50 pL, from about 35 pL to about 45 pL, from about 35 pL to about 40 pL, from about 40 pL to about 60 pL, from about 40 pL to about 55 pL, from about 40 pL to about 50 pL, from about 45 pL to about 60 pL, from about 45 pL to about 55 pL, or from about 50 pL to about 60 pL.6356.002W01

[0102] In still other examples, the lyophilized components used in the nucleic acid digestion process 126 can be included in a bead having a volume from about 20 pL to about 50 pL, from about 20 pL to about 40 pL, from about 20 pL to about 30 pL, from about 30 pL to about 50 pL, from about 30 pL to about 40 pL, from about 35 pL to about 50 pL or from about 35 pL to about 45 pL. In one or more examples, the bead can include a component comprised of a HPBCD solution having from about 20% by weight to about 40% by weight HPBCD, from about 25% by weight to about 35% by weight HPBCD, or from about 20% by weight to about 30% by weight HPBCD. Additionally, the bead can include a component comprised of a mannitol solution having from about 20% by weight to about 40% by weight mannitol, from about 25% by weight to about 35% by weight mannitol, or from about 20% by weight to about 30% by weight mannitol.

[0103] In various examples, the bead can include from about 20% by volume to about 50% by volume of the HPBCD solution, from about 20% by volume to about 45% by volume of the HPBCD solution, from about 20% by volume to about 40% by volume of the HPBCD solution, from about 20% by volume to about 35% by volume of the HPBCD solution, from about 20% by volume to about 50% by volume of the HPBCD solution, from about 25% by volume to about 45% by volume of the HPBCD solution, from about 25% by volume to about 40% by volume of the HPBCD solution, from about 25% by volume to about 35% by volume of the HPBCD solution, from about 30% by volume to about 50% by volume of the HPBCD solution, from about 30% by volume to about 45% by volume of the HPBCD solution, from about 30% by volume to about 40% by volume of the HPBCD solution, from about 35% by volume to about 50% by volume of the HPBCD solution of the HPBCD solution, from about 35% by volume to about 45% by volume of the HPBCD solution, or from about 40% by volume to about 50% by volume of the HPBCD solution.

[0104] In addition, the bead can include from about 20% by volume to about 50% by volume of the mannitol solution, from about 20% by volume to about 45% by volume of the mannitol solution, from about 20% by volume to about 40% by volume of the mannitol solution, from about 20% by volume to about 35% by volume of the mannitol solution, from about 20% by volume to about 50% by volume of the mannitol solution, from about 25% by volume to about 45% by volume of the mannitol solution, from about 25% by volume to about 40% by volume of the mannitol solution, from about 25% by volume to about 35% by volume of the mannitol solution, from about6356.002W0130% by volume to about 50% by volume of the mannitol solution, from about 30% by volume to about 45% by volume of the mannitol solution, from about 30% by volume to about 40% by volume of the mannitol solution, from about 35% by volume to about 50% by volume of the mannitol solution of the mannitol solution, from about 35% by volume to about 45% by volume of the mannitol solution, or from about 40% by volume to about 50% by volume of the mannitol solution.

[0105] Further, the bead can include from about 20% by volume to about 50% by volume of the lambda exonuclease buffer solution, from about 20% by volume to about 45% by volume of the lambda exonuclease buffer solution, from about 20% by volume to about 40% by volume of the lambda exonuclease buffer solution, from about 20% by volume to about 35% by volume of the lambda exonuclease buffer solution, from about 20% by volume to about 30% by volume of the lambda exonuclease buffer solution, from about 20% by volume to about 25% by volume of the lambda exonuclease buffer solution, from about 25% by volume to about 50% by volume of the lambda exonuclease buffer solution, from about 25% by volume to about 45% by volume of the lambda exonuclease buffer solution, from about 25% by volume to about 40% by volume of the lambda exonuclease buffer solution, from about 25% by volume to about 35% by volume of the lambda exonuclease buffer solution, from about 25% by volume to about 30% by volume of the lambda exonuclease buffer solution, from about 30% by volume to about 50% by volume of the lambda exonuclease buffer solution, from about 30% by volume to about 45% by volume of the lambda exonuclease buffer solution, from about 30% by volume to about 40% by volume of the lambda exonuclease buffer solution, from about 35% by volume to about 50% by volume of the lambda exonuclease buffer solution, from about 35% by volume to about 45% by volume of the lambda exonuclease buffer solution, from about 35% by volume to about 40% by volume of the lambda exonuclease buffer solution, from about 40% by volume to about 50% by volume of the lambda exonuclease buffer solution, from about 40% by volume to about 45% by volume of the lambda exonuclease buffer solution, or from about 45% by volume to about 50% by volume of the lambda exonuclease buffer solution.

[0106] In still other examples, the bead can include from about 0.005% by volume to about 0.030% by volume of the lambda exonuclease, from about 0.005% by volume to about 0.025% by volume of the lambda exonuclease, from about 0.005% by volume to about 0.020% by volume of the lambda exonuclease, from about 0.005%6356.002W01 by volume to about 0.015% by volume of the lambda exonuclease, from about 0.005% by volume to about 0.010% by volume of the lambda exonuclease, from about 0.010% by volume to about 0.030% by volume of the lambda exonuclease, from about 0.010% by volume to about 0.025% by volume of the lambda exonuclease, from about 0.010% by volume to about 0.020% by volume of the lambda exonuclease, from about 0.010% by volume to about 0.015% by volume of the lambda exonuclease, from about 0.015% by volume to about 0.030% by volume of the lambda exonuclease, from about 0.015% by volume to about 0.025% by volume of the lambda exonuclease, from about 0.015% by volume to about 0.020% by volume of the lambda exonuclease, from about 0.020% by volume to about 0.030% by volume of the lambda exonuclease, or from about 0.020% by volume to about 0.025% by volume of the lambda exonuclease.

[0107] In one or more examples, at least one of the mannitol solution, the HPBCD solution, or the lambda exonuclease buffer solution, can include an amount of water. In at least some examples, the bead can include an amount of water that is in addition to the amount of water included in at least one of the mannitol solution, the HPBCD solution, or the lambda exonuclease buffer solution. In these situations, the amount of additional water can be minimal, such as no greater than about 1 % by volume, no greater than about 0.1 % by volume, no greater than about 0.05% by volume, no greater than about 0.01 % by volume, no greater than about 0.005% by volume, or no greater than about 0.001 % by volume.

[0108] The nucleic acid digestion process 126 can include heating a mixture that includes the nucleic acid digestion components and the amplification product 122. In various examples, the nucleic acid digestion process 126 can include a first heating step to cause denaturation of the double stranded nucleic acids included in the amplification product 122 followed by one or more additional heating steps at lower temperatures in relation to the digestion of single stranded 5’ phosphorylated nucleic acids by the exonuclease. In one or more examples, the first heating operation of the nucleic acid digestion process 126 can include heating the nucleic acid digestion components to temperatures of at least about 80 °C, at least about 82 °C, at least about 85 °C, at least about 88 °C, at least about 90 °C, at least about 92 °C, at least about 95 °C, at least about 98 °C, at least about 100 °C, or at least about 102 °C. In one or more additional examples, the first heating operation of the nucleic acid digestion process 126 can include heating the nucleic acid digestion components to temperatures no greater than about 115 °C, no greater than about 112 °C, no greater6356.002W01 than about 110 °C, no greater than about 109 °C, no greater than about 108 °C, no greater than about 107 °C, no greater than about 106 °C, no greater than about 105 °C, no greater than about 104 °C, or no greater than about 103 °C. In one or more illustrative examples, the first heating operation of the nucleic acid digestion process 126 can include heating the nucleic acid digestion components to temperatures from about 80 °C to about 115 °C, from about 85 °C to about 110 °C, from about 88 °C to about 109 °C, from about 90 °C, to about 108 °C, from about 92 °C to about 107 °C, from about 95 °C to about 106 °C, or from about 98 °C to about 105 °C.

[0109] In various examples, the nucleic acid digestion components can be heated at temperatures to cause double stranded DNA of the amplification product 122 to denature for a duration of at least about 0.5 minutes, at least about 0.8 minutes, at least about 1 minute, at least about 1 .2 minutes, at least about 1 .5 minutes, at least about 1.8 minutes, at least about 2 minutes, at least about 2.2 minutes, at least about2.5 minutes, at least about 2.8 minutes, at least about 3 minutes, at least about 3.2 minutes, at least about 3.5 minutes, at least about 3.8 minutes, or at least about 4 minutes. Additionally, the nucleic acid digestion components can be heated at temperatures to cause double stranded DNA of the amplification product 122 to denature for a duration of no greater than about 8 minutes, no greater than about 7.8 minutes, no greater than about 7.5 minutes, no greater than about 7.2 minutes, no greater than about 7 minutes, no greater than about 6.8 minutes, no greater than about6.5 minutes, no greater than about 6.2 minutes, no greater than about 6 minutes, no greater than about 5.8 minutes, no greater than about 5.5 minutes, no greater than about 5.2 minutes, no greater than about 5 minutes, no greater than about 4.8 minutes, no greater than about 4.5 minutes, or no greater than about 4.2 minutes. In one or more illustrative examples, the nucleic acid digestion components can be heated at temperatures to cause double stranded DNA of the amplification product 122 to denature for durations from about 0.5 minutes to about 8 minutes, from about 1 minute to about 7 minutes, from about 2 minutes to about 6 minutes, from about 0.5 minutes to about 5 minutes, from about 0.5 minutes about 2 minutes, from about 1 minute to about 5 minutes, from about 1 minute to about 4 minutes, from about 2 minutes to about 8 minutes, from about 2 minutes to about 4 minutes, from about 3 minutes to about 8 minutes, from about 3 minutes to about 5 minutes, from about 4 minutes to about 8 minutes, from about 4 minutes to about 6 minutes, or from about 5 minutes to about 8 minutes.6356.002W01

[0110] After denaturing double stranded DNA molecules included in the amplification product 122 to produce a number of phosphorylated single stranded DNA molecules and a number of biotinylated single stranded DNA molecules, digestion of the phosphorylated single stranded DNA molecules using lambda exonuclease can take place in two steps. The two steps can include a first step that takes place at lower temperatures and a shorter duration than the second step. Temperatures maintained during at least one of the first step or the second step can be sufficiently high for the lambda exonuclease to be activated.

[0111] The first step of the process to digest phosphorylated single stranded DNA molecules derived from the amplification product 122 can take place at temperatures of at least about 30 °C, at least about 32 °C, at least about 35 °C, at least about 38 °C, at least about 40 °C, at least about 42 °C, at least about 45 °C, at least about 48 °C, at least about 50 °C, at least about 52 °C, or at least about 55 °C. In one or more additional examples, the first step of the digestion of phosphorylated single stranded DNA molecules derived from the amplification product 122 can take place at temperatures no greater than about 65 °C, no greater than about 64 °C, no greater than about 63 °C, no greater than about 62 °C, no greater than about 61 °C, no greater than about 60 °C, no greater than about 59 °C, no greater than about 58 °C, no greater than about 57 °C, or no greater than about 56 °C. In one or more illustrative examples, the first step of the digestion of phosphorylated single stranded DNA molecules derived from the amplification product 122 can take place at temperatures from about 30 °C to about 65 °C, from about 35 °C to about 65 °C, from about 35 °C to about 60 °C, from about 35 °C, to about 55 °C, from about 35 °C to about 50 °C, from about 35 °C to about 45 °C, from about 40 °C to about 65 °C, from about 40 °C to about 60 °C, from about 40 °C to about 55 °C, from about 40 °C to about 50 °C, from about 45 °C to about 65 °C, from about 45 °C to about 60 °C, from about 45 °C to about 55 °C, from about 50 °C to about 65 °C, or from about 50 °C to about 60 °C.

[0112] In one or more examples, the first step of the digestion of phosphorylated single stranded DNA molecules derived from the amplification product 122 can for a duration of at least about 0.5 minutes, at least about 0.8 minutes, at least about 1 minute, at least about 1.2 minutes, at least about 1.5 minutes, at least about 1.8 minutes, at least about 2 minutes, at least about 2.2 minutes, at least about 2.5 minutes, at least about 2.8 minutes, at least about 3 minutes, at least about 3.26356.002W01 minutes, at least about 3.5 minutes, at least about 3.8 minutes, or at least about 4 minutes. Additionally, the first step of the digestion of phosphorylated single stranded DNA molecules derived from the amplification product 122 can be for a duration of no greater than about 10 minutes, no greater than about 9.5 minutes, no greater than about 9 minutes, no greater than about 8.5 minutes, no greater than about 8 minutes, no greater than about 7.5 minutes, no greater than about 7 minutes, no greater than about 6.5 minutes, no greater than about 6 minutes, no greater than about 5.5 minutes, no greater than about 5 minutes, or no greater than about 4.5 minutes. In one or more illustrative examples, the first step of the digestion of phosphorylated single stranded DNA molecules derived from the amplification product 122 can be for durations from about 0.5 minutes to about 4 minutes, from about 0.5 minutes to about 3.5 minutes, from about 0.5 minutes to about 3 minutes, from about 0.5 minutes to about 2.5 minutes, from about 0.5 minutes about 2 minutes, from about 0.5 minutes to about 1 .5 minutes, from about 0.5 minutes to about 1 minute, from about 1 minute to about 4 minutes, from about 1 minute to about 3 minutes, from about 1 minute to about 2.5 minutes, from about 1 minute to about 2 minutes, from about 1.5 minutes to about 4 minutes, from about 1 .5 minutes to about 3.5 minutes, from about 1 .5 minutes to about 3 minutes, from about 1.5 minutes to about 2.5 minutes, or from about 2 minutes to about 4 minutes.

[0113] The second step of the process to digest phosphorylated single strandedDNA molecules derived from the amplification product 122 can take place at temperatures of at least about 55 °C, at least about 58 °C, at least about 60 °C, at least about 62 °C, at least about 65 °C, at least about 68 °C, at least about 70 °C, at least about 72 °C, or at least about 75 °C. In one or more additional examples, the second step of the digestion of phosphorylated single stranded DNA molecules derived from the amplification product 122 can take place at temperatures no greater than about 90 °C, no greater than about 88 °C, no greater than about 86 °C, no greater than about 84 °C, no greater than about 82 °C, no greater than about 80 °C, no greater than about 78 °C, or no greater than about 76 °C, no greater than about 57 °C, or no greater than about 56 °C. In one or more illustrative examples, the second step of the digestion of phosphorylated single stranded DNA molecules derived from the amplification product 122 can take place at temperatures from about 55 °C to about 90 °C, from about 60 °C to about 90 °C, from about 60 °C to about 85 °C, from about 60 °C, to about 80 °C, from about 60 °C to about 75 °C, from about 60 °C to about 706356.002W01°C, from about 65 °C to about 90 °C, from about 65 °C to about 85 °C, from about 65 °C to about 80 °C, from about 65 °C to about 75 °C, from about 70 °C to about 90 °C, from about 70 °C to about 85 °C, from about 70 °C to about 80 °C, from about 75 °C to about 90 °C, from about 75 °C to about 85 °C, or from about 80 °C to about 90 °C.

[0114] In one or more examples, the second step of the digestion of phosphorylated single stranded DNA molecules derived from the amplification product 122 can be for a duration of at least about 4 minutes, at least about 4.2 minutes, at least about 4.5 minute, at least about 4.8 minutes, at least about 5 minutes, at least about 5.2 minutes, at least about 5.5 minutes, at least about 5.8 minutes, at least about 6 minutes, at least about 6.2 minutes, at least about 6.5 minutes, at least about 6.8 minutes, at least about 7 minutes, at least about 7.2 minutes, at least about 7.5 minutes, at least about 7.8 minutes, at least about 8 minutes, at least about 8.2 minutes, at least about 8.5 minutes, at least about 8.8 minutes, or at least about 9 minutes. Additionally, the second step of the digestion of phosphorylated single stranded DNA molecules derived from the amplification product 122 can be for a duration of no greater than about 15 minutes, no greater than about 14.5 minutes, no greater than about 14 minutes, no greater than about 13.5 minutes, no greater than about 13 minutes, no greater than about 12.5 minutes, no greater than about 12 minutes, no greater than about 11.5 minutes, no greater than about 11 minutes, no greater than about 10.5 minutes, no greater than about 10 minutes, or no greater than about 9.5 minutes. In one or more illustrative examples, the second step of the digestion of phosphorylated single stranded DNA molecules derived from the amplification product 122 can be for durations from about 4 minutes to about 15 minutes, from about 5 minutes to about 15 minutes, from about 5 minutes to about 12 minutes, from about 5 minutes to about 10 minutes, from about 5 minutes about 8 minutes, from about 6 minutes to about 15 minutes, from about 6 minutes to about 12 minutes, from about 6 minutes to about 10 minutes, from about 7 minutes to about 15 minutes, from about 7 minutes to about 12 minutes, from about 7 minutes to about 10 minutes, from about 8 minutes to about 15 minutes, from about 8 minutes to about 12 minutes, from about 8 minutes to about 10 minutes, from about 9 minutes to about 15 minutes, from about 9 minutes to about 12 minutes, from about 10 minutes to about 15 minutes, or from about 10 minutes to about 12 minutes.

[0115] The nucleic acid digestion product 128 produced by the nucleic acid digestion process 126 can be contacted with a sensing device 130. The sensing6356.002W01 device 130 can include an array of sensors 132. The sensors 132 can correspond to a region of the sensing device 130 that includes components that detect the presence of one or more target analytes. In one or more examples, the sensors 132 can include a number of layers. In various examples, the sensors 132 can include The sensors 132 can be coupled to electrical components that produce signals in the presence of one or more target analytes and one or more electrical components that process the signals.

[0116] A number of sensing technologies can be implemented by the sensing device 130. For example, the sensing device 130 can implement one or more polymerase chain reaction technologies that use fluorophores to produce signals in the presence of one or more target analytes. To illustrate, the sensing device 130 can implement at least one of Taqman qPCR, droplet digital PCR (ddPCR), or qPCR. In addition, the sensing device 130 can implement magnetic resonance technologies that use magnetic particles to produce signals in the presence of one or more target analytes. Further, the sensing device 130 can implement electrochemical technologies that use ferrocene to produce signals in the presence of one or more target analytes. In one or more additional examples, the sensing device 130 can implement Fluorescent In Situ Hybridization (FISH) technologies that use fluorophores to produce signals in the presence of one or more target analytes. In still other examples, the sensing device 130 can implement polymerase chain reaction technologies with beads using fluorophore-labeled microbeads to produce signals in the presence of one or more target analytes. In some other examples, sensing device 130 can implement geometry based microparticle identification technology. In various examples, the sensing device 130 can implement a number of magnetoresistance technologies that use changes in resistance in the presence of a magnetic field to produce signals in the presence of one or more target analytes. In one or more illustrative examples, the sensing device 130 can implement giant magnetoresistance (GMR) technologies to produce signals in the presence of one or more target analytes. In one or more additional illustrative examples, the sensing device 130 can implement tunnel magnetoresistance (TMR) technologies to produce signals in the presence of one or more target analytes. In one or more further illustrative examples, the sensing device 130 can implement anisotropic magnetoresistance (AMR) to produce signals in the present of one or more target analytes. In still other illustrative examples, the sensing6356.002W01 device 130 can implement magneto-optical Kerr effect (MOKE) technologies to produce signals in the presence of one or more target analytes.

[0117] In one or more examples, the array of sensors 132 can include a biosurface disposed on one or more substrates. In one or more illustrative examples, the biosurface can include a polymer composition and probes coupled to the polymer composition.

[0118] In at least some examples, the polymer composition can comprise at least two hydrophilic polymers and a crosslinking reagent. In some embodiments, at least one of the at least two hydrophilic polymers contain polar or charged functional groups, rendering them soluble in aqueous solution, such as in water. Numerous hydrophilic polymers that are available to the skilled artisan may be employed to generate polymer compositions and biosurfaces, such as, for example: acrylics, including acrylic acids, acrylamides, poly(2-hydroxyethyl methacrylate) (PHEMA), and maleic anhydride polymers and copolymers; amine-functional polymers include allylamine, ethyleneimine, oxazoline, and other polymers containing amine groups in their main- or side- chains; starches; chitins; alginates; dextrans; and celluloses.

[0119] In one or more examples, a polymer composition can comprise at least two hydrophilic polymers selected from the group consisting of a polyethylene glycol (PEG) polymer, a polysaccharide, and a poly(2-hydroxy ethyl methacrylate) (PHEMA) polymer; and a crosslinking reagent crosslinking the at least two hydrophilic polymers. In at least some examples, such polymer compositions do not comprise a block copolymer of a PEG polymer and a PHEMA polymer.

[0120] Additionally, a polymer composition can comprise at least two hydrophilic polymers selected from the group consisting of a polyethylene glycol (PEG) polymer, a cellulose, a starch, a chitin, and alginate, and a dextran, and a poly(2- hydroxyethyl methacrylate) (PHEMA) polymer; and a crosslinking reagent crosslinking the at least two hydrophilic polymers. In various examples, such polymer compositions do not comprise a block co-polymer of a PEG polymer and a PHEMA polymer.

[0121] Further, at least one of the at least two hydrophilic polymers can comprise a polyethylene glycol (PEG) polymer. For example, at least one of the at least two hydrophilic polymers can comprise a derivatized polyethylene glycol (PEG) polymer. In some embodiments, at least one of the at least two hydrophilic polymers comprises a polyethylene glycol (PEG) polymer that is derivatized with one of more functional groups selected from the group consisting of an aldehyde, an alkyne, an6356.002W01 amine, an azide, a biotin, a carboxylic acid, a hydroxyl, a maleimide, an epoxy, an N- hydroxysuccinimide (NHS) ester, an orthopyridyl disulfide (OPSS), a sulfonate, a toluenesulfonate (tosyl), a methanesulfonate (mesyl), a 2,2,2-trifluoroethanesulfonate (tresyl), and a thiol. In some embodiments, at least one of the at least two hydrophilic polymers comprises a polyethylene glycol (PEG) polymer that is derivatized with one or more N- hydroxysuccinimide (NHS) esters. As used herein and throughout, a “derivatized polyethylene glycol (PEG) polymer” is, itself, a “polyethylene glycol (PEG) polymer” or a “PEG polymer”, used interchangeably throughout.

[0122] In various examples, the PEG polymer can be branched, such as multiarm PEG polymers. Example compounds of this type include, without limitation, 4-arm PEG, 8-arm PEG, 4-arm PEG-OH, 8-arm PEG-OH, 4-arm PEG-acrylate, 8-arm PEG- acrylate, 4-arm PEG- acrylamide, 8-arm PEG-acryl amide, 4-arm PEG-amine, 8-arm PEG-amine, 4-arm PEG-thiol, 8- arm PEG thiol, 4-arm PEG maleimide, 8-arm PEG maleimide, 4-arm PEG-succinimidyl caboxymethyl ester (NHS), 8-arm PEG- succinimidyl caboxymethyl ester (NHS), 4-arm PEG- succinimidyl glutarate ester, 8- arm PEG-succinimidyl glutarate ester, 4-arm PEG-succinimidyl succinate ester, 8-arm PEG-succinimidyl succinate ester, 4-arm PEG-glutaramide succinimidyl ester, 8-arm PEG-glutaramide succinimidyl ester, 4-arm PEG-succinimide succinimidyl ester, 8- arm PEG-succinimide succinimidyl ester, 4-arm PEG-epoxide, 8-arm PEG-epoxide, 4-arm PEG 4- nitrophenyl carbonate (NPC), 8-arm PEG 4-nitrophenyl carbonate (NPC), 4-arm PEG-acetic acid, 8-arm PEG-acetic acid, 4-arm PEG-glutaric acid, 8- arm PEG-glutaric acid, 4-arm PEG-succinic acid, 8-arm PEG-succinic acid, 4-arm PEG-glutaramide acid, 8-arm PEG-glutaramide acid, 4-arm PEG-succinimide acid, 8- arm PEG-succinimide acid, 4-arm PEG-azide, 8-arm PEG-azide, 4-arm PEG-alkyne, or 8-arm PEG-alkyne. Examples of multi-arm PEG polymers can be commercially available from, for example, Creative PEGWorks (Chapel Hill, NC).

[0123] The terminal PEG moieties can comprise a leaving group, such as halo, mesylate, tosylate, amine, carboxyl, epoxy, aziridine, thiol, and the like. The terminal PEG group or groups can be converted to a variety of functional group handles including, without limitation, amine, carboxyl, thiol, epoxy, hydroxy, alkyne, azide, alkyne, and the like. These functional group handles can serve as covalent bonding attachment points, for example, for securing biomolecules to the biosurface. In at least some examples, the terminal group can be a precursor for cycloaddition chemistry, such as [4+2], [4+1 ] or [3+2] cycloadditions, and the like. Example reactions for6356.002W01 securing biomolecules to attachment points of the polymer compositions can include, azide-alkyne cycloaddition (Huisgen 1 ,3 dipolar cycloaddition), Diels- Alder reaction (diene and alkene, both normal mode and inverse electron demand), thiol-ene reaction, and the like. The PEG can terminate in a functional handle that enable so- called click chemistry. Example functional groups that can facilitate click chemistry include, without limitation, azides, alkynes, thiol, alkenes, isonitrile, and tetrazine.

[0124] In one or more examples, at least one of the at least two hydrophilic polymers comprises a PEG polymer. The PEG polymer can have a number average molecular weight of: about 100 Da to about 50,000 Da; about 100 Da to about 45,000 Da; about 100 Da to about 40,000 Da; about 100 Da to about 35,000 Da, about 100 Da to about 30,000 Da; about 100 Da to about 25,000 Da; about 100 Da to about 20,000 Da; about 100 Da to about 15,000 Da; about 100 Da to about 10,000 Da; about 100 Da to about 8,000 Da, about 100 Da to about 6,000 Da; about 100 Da to about 4,000 Da; about 100 Da to about 2,000 Da; about 100 Da to about 1 ,000 Da; about 100 Da to about 900 Da; about 100 Da to about 800 Da; about 100 Da to about 700 Da; about 150 Da to about 700 Da; about 200 Da to about 700 Da; or about 200 Da to about 600 Da.

[0125] In one or more additional examples, PEG polymers can terminate in other organic functional groups including, hydroxyl groups (-OH). Terminal functional group on the PEG polymer can be converted into other functional groups before or after crosslinking. In various examples, the terminal groups of the PEG polymer portion of the polymer compositions disclosed herein can serve as the attachment point for various biomolecules to provide a biosurface. For example, the NHS of an NHS-PEG- NHS group can be utilized for attachment of a biomolecule. In one or more illustrative examples, one or more biomolecules can be linked to a polymer composition through terminal PEG chemical moieties. In one or more additional illustrative examples, at least one of the at least two hydrophilic polymers comprises a poly(2- hydroxy ethyl methacrylate) (PHEMA) polymer.

[0126] The PHEMA polymer can have a number average molecular weight range of: about 2,000 Daltons (Da) to about 100,000 Da; about 4,000 Da to about 95,000 Da; about 6,000 Da to about 90,000 Da; about 8,000 Da to about 85,000 Da, about 10,000 Da to about 80,000 Da; about 12,000 Da to about 75,000 Da; about 14,000 Da to about 70,000 Da; about 16,000 Da to about 65,000 Da; about 16,000 Da to about 65,000 Da; about 16,000 Da to about 60,000 Da, about 16,000 Daltons to6356.002W01 about 55,000 Da; about 16,000 Da to about 50,000 Da; about 16,000 Da to about 45,000 Da; about 16,000 Da to about 40,000 Da; about 16,000 Da to about 35,000 Da; about 16,000 Daltons to about 30,000 Da; about 16,000 Da to about 25,000 Da; about 18,000 Da to about 22,000 Da; or about 18,000 Daltons to about 20,000 Da.

[0127] In one or more further examples, a polymer composition can comprise a PEG polymer, wherein the PEG polymer has a number average molecular weight of: about 100 Da to about 50,000 Da; about 100 Da to about 45,000 Da; about 100 Da to about 40,000 Da; about 100 Da to about 35,000 Da, about 100 Da to about 30,000 Da; about 100 Da to about 25,000 Da; about 100 Da to about 20,000 Da; about 100 Da to about 15,000 Da; about 100 Da to about 10,000 Da; about 100 Da to about 8,000 Da, about 100 Da to about 6,000 Da; about 100 Da to about 4,000 Da; about 100 Da to about 2,000 Da; about 100 Da to about 1 ,000 Da; about 100 Da to about 900 Da; about 100 Da to about 800 Da; about 100 Da to about 700 Da; about 150 Da to about 700 Da; about 200 Da to about 700 Da; or about 200 Da to about 600 Da; a poly(2-hydroxyethyl methacrylate) (PHEMA) polymer; and a crosslinking reagent crosslinking the PEG polymer and the PHEMA polymer. In one or more examples, such polymer compositions do not comprise a block co polymer of a PEG polymer and a PHEMA polymer.

[0128] In still other examples, a polymer composition can comprise: a polyethylene glycol (PEG) polymer, wherein the PEG polymer can be derivatized with one of more functional groups selected from the group consisting of an aldehyde, an alkyne, an amine, an azide, a biotin, a carboxylic acid, a hydroxyl, a maleimide, an epoxy, an N-hydroxysuccinimide (NHS) ester, an orthopyridyl disulfide (OPSS), a sulfonate, a toluenesulfonate (tosyl), a methanesulfonate (mesyl), a 2,2,2- trifluoroethanesulfonate (tresyl), and a thiol; and a crosslinking reagent crosslinking the PEG polymer and the PHEMA polymer. In various examples, such polymer compositions do not comprise a block co-polymer of a PEG polymer and a PHEMA polymer. The polyethylene glycol (PEG) polymer can be derivatized with one or more N-hydroxysuccinimide (NHS) esters.

[0129] A polymer composition can comprise: a polyethylene glycol (PEG) polymer, wherein the PEG polymer is derivatized with one of more functional groups selected from the group consisting of an aldehyde, an alkyne, an amine, an azide, a biotin, a carboxylic acid, a hydroxyl, a maleimide, an epoxy, an N-hydroxysuccinimide (NHS) ester, an orthopyridyl disulfide (OPSS), a sulfonate, a toluenesulfonate (tosyl),6356.002W01 a methanesulfonate (mesyl), a 2,2,2-trifluoroethanesulfonate (tresyl), and a thiol, and wherein the derivatized PEG polymer can have a number average molecular weight of: about 100 Daltons (Da) to about 50,000 Da; about 100 Da to about 45,000 Da; about 100 Da to about 40,000 Da; about 100 Da to about 35,000 Da, about 100 Da to about 30,000 Da; about 100 Da to about 25,000 Da; about 100 Da to about 20,000 Da; about 100 Da to about 15,000 Da; about 100 Da to about 10,000 Da; about 100 Da to about 8,000 Da, about 100 Da to about 6,000 Da; about 100 Da to about 4,000 Da; about 100 Da to about 2,000 Da; about 100 Da to about 1 ,000 Da; about 100 Da to about 900 Da; about 100 Da to about 800 Da; about 100 Da to about 700 Da; about 150 Da to about 700 Da; about 200 Da to about 700 Da; or about 200 Da to about 600 Da; a poly(2-hydroxyethyl methacrylate) (PHEMA) polymer, wherein the PHEMA polymer can have a number average molecular weight range of: about 2,000 Daltons (Da) to about 1 ,000,000 Da; about 4,000 Da to about 950,000 Da; about 6,000 Da to about 900,000 Da; about 8,000 Da to about 850,000 Da, about 10,000 Da to about 800,000 Da; about 12,000 Da to about 750,000 Da; about 14,000 Da to about 700,000 Da; about 16,000 Da to about 650,000 Da; about 16,000 Da to about 600,000 Da; about 16,000 Da to about 550,000 Da, about 16,000 Daltons to about 500,000 Da; about 16,000 Da to about 450,000 Da; about 16,000 Da to about 400,000 Da; about 16,000 Da to about 350,000 Da; about 16,000 Da to about 300,000 Da; about 16,000 Daltons to about 250,000 Da; about 16,000 Da to about 200,000 Da; 16,000 Da to about 150,000 Da; about 16,000 Da to about 100,000 Da, about 16,000 Daltons to about 500,000 Da; about 16,000 Da to about 45,000 Da; about 16,000 Da to about 40,000 Da; about 16,000 Da to about 35,000 Da; about 16,000 Da to about 30,000 Da; about 16,000 Daltons to about 25,000 Da; about 16,000 Da to about 22,000 Da about 18,000 Da to about 22,000 Da; or about 18,000 Daltons to about 20,000 Da; and a crosslinking reagent crosslinking the PEG polymer and the PHEMA polymer. In various examples, such polymer compositions do not comprise a block co-polymer of a PEG polymer and a PHEMA polymer.

[0130] In one or more examples, a polymer composition can comprise: a polyethylene glycol (PEG) polymer, wherein the PEG polymer can be derivatized with ab N-hydroxysuccinimide (NHS) ester, and wherein the derivatized PEG polymer can have a number average molecular weight of: about 100 Daltons (Da) to about 50,000 Da; about 100 Da to about 45,000 Da; about 100 Da to about 40,000 Da; about 100 Da to about 35,000 Da, about 100 Da to about 30,000 Da; about 100 Da to about6356.002W0125,000 Da; about 100 Da to about 20,000 Da; about 100 Da to about 15,000 Da; about 100 Da to about 10,000 Da; about 100 Da to about 8,000 Da, about 100 Da to about 6,000 Da; about 100 Da to about 4,000 Da; about 100 Da to about 2,000 Da; about 100 Da to about 1 ,000 Da; about 100 Da to about 900 Da; about 100 Da to about 800 Da; about 100 Da to about 700 Da; about 150 Da to about 700 Da; about 200 Da to about 700 Da; or about 200 Da to about 600 Da; a poly(2-hydroxyethyl methacrylate) (PHEMA) polymer, wherein the PHEMA polymer can have a number average molecular weight range of: about 2,000 Daltons (Da) to about 100,000 Da; about 4,000 Da to about 95,000 Da; about 6,000 Da to about 90,000 Da; about 8,000 Da to about 85,000 Da, about 10,000 Da to about 80,000 Da; about 12,000 Da to about 75,000 Da; about 14,000 Da to about 70,000 Da; about 16,000 Da to about 65,000 Da; about 16,000 Da to about 65,000 Da; about 16,000 Da to about 60,000 Da, about 16,000 Daltons to about 55,000 Da; about 16,000 Da to about 50,000 Da; about 16,000 Da to about 45,000 Da; about 16,000 Da to about 40,000 Da; about 16,000 Da to about 35,000 Da; about 16,000 Daltons to about 30,000 Da; about 16,000 Da to about 25,000 Da; about 18,000 Da to about 22,000 Da; or about 18,000 Daltons to about 20,000 Da; and a crosslinking reagent crosslinking the PEG polymer and the PHEMA polymer. In various examples, such polymer compositions do not comprise a block copolymer of a PEG polymer and a PHEMA polymer.

[0131] In one or more additional examples, a polymer composition can comprise a polyethylene glycol (PEG) polymer, wherein the PEG polymer can be derivatized with an N-hydroxysuccinimide (NHS) ester and wherein the derivatized PEG polymer can have a molecular weight of about 600 Da; a poly(2-hydroxy ethyl methacrylate) (PHEMA) polymer, wherein the PHEMA polymer can have a molecular weight of about 20,000 Da; and a crosslinking reagent crosslinking the PEG polymer and the PHEMA polymer. In various examples, such polymer compositions do not comprise a block co-polymer of a PEG polymer and a PHEMA polymer.

[0132] In one or more illustrative examples, the polymer composition can be formed from a solution including an amount of the at least two hydrophilic polymers and an amount of a crosslinking reagent. For example, the polymeric composition can be formed from a solution having a concentration of a first hydrophilic polymer of at least about 1 m illigram / milliliter (mg / mL), at least about 2 mg / mL, at least about 3 mg / mL, at least about 4 mg / mL, at least about 5 mg / mL, at least about 6 mg / mL, or at least about 7 mg / mL. Additionally, the polymeric composition can be formed from a6356.002W01 solution having a concentration of the first hydrophilic polymer of no greater than about 12 mg / mL, no greater than about 11 mg / mL, no greater than about 10 mg / mL, no greater than about 9 mg / mL, or no greater than about 8 mg / mL. In one or more further illustrative examples, the polymeric composition can be formed from a solution having a concentration of the first hydrophilic polymer from about 1 mg / mL to about 12 mg / mL, from about 2 mg / mL to about 10 mg / mL, from about 3 mg / mL to about 8 mg / mL, from about 4 mg / mL to about 6 mg / mL, from about 2 mg / mL to about 5 mg / mL, from about3 mg / mL to about 6 mg / mL, from about 4 mg / mL to about 8 mg / mL, from about 5 mg / mL to about 9 mg / mL, from about 6 mg / mL to about 10 mg / mL, or from about 2 mg / mL to about 8 mg / mL. In various examples, the first hydrophilic polymer can comprise a PEG polymer.

[0133] In one or more additional illustrative examples, the polymeric composition can be formed from a solution having a concentration of a second hydrophilic polymer of at least about 2 mg / mL, at least about 3 mg / mL, at least about4 mg / mL, at least about 5 mg / mL, at least about 6 mg / mL, at least about 7 mg / mL, at least about 8 mg / mL, or at least about 9 mg / mL. In still other examples, the polymeric composition can be formed from a solution having a concentration of the second hydrophilic polymer of no greater than about 18 mg / mL, no greater than about 17 mg / mL, no greater than about 16 mg / mL, no greater than about 15 mg / mL, no greater than about 14 mg / mL, no greater than about 13 mg / mL, no greater than about 12 mg / mL, no greater than about 11 mg / mL, or no greater than about 10 mg / mL In various examples, the polymeric composition can be formed from a solution having a concentration of the second hydrophilic polymer from about 2 mg / mL to about 18 mg / mL, from about 3 mg / mL to about 16 mg / mL, from about 4 mg / mL to about 14 mg / mL, from about 6 mg / mL to about 12 mg / mL, from about 2 mg / mL to about 10 mg / mL, from about 2 mg / mL to about 8 mg / mL, from about 4 mg / mL to about 12 mg / mL, from about 4 mg / mL to about 10 mg / mL, from about 4 mg / mL to about 8 mg / mL, from about 6 mg / mL to about 12 mg / mL, from about 6 mg / mL to about 10 mg / mL, or from about 7 mg / mL to about 10 mg / mL. In at least some illustrative examples, the second hydrophilic polymer can include a PHEMa polymer.

[0134] In one or more further examples, the polymeric composition can be formed from a solution having a concentration of a crosslinking agent of at least about 0.03 mg / mL, at least about 0.05 mg / mL, at least about 0.08 mg / mL, at least about 0.1 mg / mL, at least about 0.12 mg / mL, at least about 0.15 mg / mL, at least about 0.186356.002W01 mg / mL, at least about 0.20 mg / mL, at least about 0.22 mg / mL, at least about 0.25 mg / mL, at least about 0.28 mg / mL, at least about 0.30 mg / mL, at least about 0.32 mg / mL, or at least about 0.35 mg / mL. In addition, the polymeric composition can be formed from a solution having a concentration of a crosslinking agent of no greater than about 0.60 mg / mL, no greater than about 0.58 mg / mL, no greater than about 0.55 mg / mL, no greater than about 0.52 mg / mL, no greater than about 0.50 mg / mL, no greater than about 0.48 mg / mL, no greater than about 0.45 mg / mL, no greater than about 0.42 mg / mL, no greater than about 0.40 mg / mL, or no greater than about 0.38 mg / mL. Further, the polymeric composition can be formed from a solution having a concentration of a crosslinking agent from about 0.03 mg / mL to about 0.60 mg / mL, from about 0.05 mg / mL to about 0.50 mg / mL, from about 0.10 mg / mL to about 0.40 mg / mL, from about 0.15 mg / mL to about 0.30 mg / mL, from about 0.10 mg / mL to about 0.50 mg / mL, from about 0.10 mg / mL to about 0.40 mg / mL, from about 0.10 mg / mL to about 0.30 mg / mL, from about 0.20 mg / mL to about 0.60 mg / mL, from about 0.20 mg / mL to about 0.50 mg / mL, from about 0.20 mg / mL to about 0.40 mg / mL, from about 0.30 mg / mL to about 0.60 mg / mL, or from about 0.30 mg / mL to about 0.50 mg / mL. In one or more illustrative examples, the crosslinking agent can comprise bis[2-( 4- azidosalicylamido )ethyl ]disulfide or dithiobis(phenylazide ).

[0135] The composition of the polymeric composition can be designed and implemented such that the amount of a nucleic acid bound to the surface of the sensors 132 is at least a minimum amount of nucleic acid. In one or more additional examples, the composition of the polymeric composition can be designed and implemented such that the amount of nucleic acid bound to the surface of the sensors is maximized. In at least some examples, the polymer composition can be formed from a solution comprising a PEG polymer, a PHEMA polymer, and a crosslinking agent comprised of bis[2-(4-azidosalicylamido)ethyl ]disulfide or dithiobis(phenylazide). In various examples, the polymer composition can be formed from a solution comprising from about 2 mg / mL to about 10 mg / mL of a PEG polymer, from about 0.3 mg / mL to about 15 mg / mL of a PHEMA polymer, and from about 0.05 mg / mL to about 0.5 mg / mL of the crosslinking agent. Additionally, the polymer composition can be formed from a solution comprising from about 2 mg / mL to about 8 mg / mL of a PEG polymer, from about 3 mg / mL to about 10 mg / mL of a PHEMA polymer, and from about 0.1 to about 0.5 mg / mL of the crosslinking agent. In still other examples, the polymer composition can be formed from a solution comprising from about 3 mg / mL to about 6 mg / mL of a6356.002W01PEG polymer, from about 4 mg / mL to about 8 mg / mL of a PHEMA polymer, and from about 0.2 mg / mL to about 0.5 mg / mL of the crosslinking agent.

[0136] In various examples, an amount by weight of a first hydrophilic polymer present in the polymer composition can be at least about 1.1 times, at least about 1 .3 times, at least about 1.5 times, at least about 1.8 times, at least about 2.0 times, at least about 2.2 times, at least about 2.5 times, at least about 2.8 times, at least about 3.0 times, at least about 3.2 times, at least about 3.5 times, at least about 3.8 times, at least about 4.0 times, at least about 4.2 times, at least about 4.5 times, at least about 4.8 times, or at least about 5.0 times an amount by weight of a second hydrophilic polymer in the polymer composition. In one or more illustrative examples, an amount by weight of a first hydrophilic polymer present in the polymer composition can be from about 1.1 times to about 8 times, from about 1.5 times to about 6 times, from about 2 times to about 4 times, from about 1 .2 times to about 4 times, or from about 1.5 times to about 3 times an amount by weight of the second hydrophilic polymer in the polymer composition.

[0137] In addition, an amount by weight of a first hydrophilic polymer can be at least about 4 times, at least about 6 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 125 times, at least about 150 times, at least about 175 times, at least about 200 times, at least about 225 times, or at least about 250 times an amount by weight of crosslinking reagent present in the polymer composition. Further, an amount by weight of the first hydrophilic polymer in the polymer composition can be from about 4 times to about 400 times, from about 6 times to about 200 times, from about 10 times to about 100 times, from about 10 times to about 40 times, from about 6 times to about 50 times, from about 10 times to about 100 times, from about 50 times to 150 times, from about 100 times to about 200 times, from about 150 times to about 250 times, from about 200 times to about 300 times, from about 250 times to about 350 times, or from about 300 times to about 400 times an amount by weight of crosslinking reagent present in the polymer composition.

[0138] Further, an amount by weight of a second hydrophilic polymer can be at least about 2 times, at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 30 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 125 times, or at least about 150 times an amount by weight of crosslinking reagent present in the polymer6356.002W01 composition. Additionally, an amount by weight of the second hydrophilic polymer in the polymer composition can be from about 2 times to about 250 times, from about 4 times to about 100 times, from about 10 times to about 80 times, from about 4 times to about 40 times, from about 10 times to about 25 times, from about 50 times to 100 times, from about 100 times to about 150 times, from about 150 times to about 200 times, or from about 200 times to about 250 times an amount by weight of crosslinking reagent present in the polymer composition.

[0139] In one or more illustrative examples, the first hydrophilic polymer can comprise a PHEMA polymer, the second hydrophilic polymer can comprise a PEG polymer, and the crosslinking reagent can comprise bis[2-(4-azidosalicylamido)ethyl ]disulfide or dithiobis(phenylazide ).

[0140] In at least some examples, the polymer composition comprises a crosslinking reagent represented by Formula (I):PA-L-PA (I) wherein each PA is a photo-activated group or a metal-activated, and L is a linking group.

[0141] In one or more examples, each PA can independently comprise an azide (-N3), or a diazo (-N2) group. In one or more additional examples, each PA is the same and in other instances each PA is different. In various examples, PA can be photoactivated or metal-activated to form a nitrene intermediate capable of C- H and / or O- H insertion. See, for example, “Photogenerated reactive intermediates and their properties,” Chapter 2 in Laboratory Techniques in Biochemistry and Molecular Biology , Elsevier Press, 12:8-24 (1983). In still other examples, PA can be metal activated to form a carbene or carbenoid intermediate capable of C-H and / or O-H insertion. See, for example, Doyle et al. “Catalytic Carbene Insertion into C-H Bonds,” Chem. Rev. 2:704-724 (2010).

[0142] In one or more examples, each PA can be an azide (-N3) moiety and photoactivation generates nitrene intermediates capable of C-H and / or O-H insertion thereby mediating crosslinking of PEG and PHEMA polymers. In one or more additional examples, each PA can be a diazo (-N2) and metal catalyzed decomposition reaction that forms a carbene or carbenoid intermediate capable of C-H and / or O-H insertion thereby mediating crosslinking of PEG and PHEMA polymers. Both azide and diazo preparations are well known in the art, and in the case of azide are readily6356.002W01 prepared by SN2 displacement reaction of azide anion, N3 with an appropriate organic moiety possessing a leaving group.

[0143] In various examples, L in Formula (I) can be any organic fragment that will support the presence of many PA moieties on one L. For example, the L can be a hydrocarbon, including a hydrocarbon that is linear, branched, cyclic, or a combination thereof; aromatic, non-aromatic, or a combination thereof; monocyclic, polycyclic, carbocyclic, heterocyclic, or a combination thereof; benzene or a derivative thereof; or combinations thereof. L can be a simple C2-C20 hydrocarbon chain that is straight chained or branched. In addition, L can be a C2-C6 straight chain alkyl group. Any of the foregoing hydrocarbons can include fluorinated variants with any degree of fluorine substitution. In one or more examples, L can include aromatic hydrocarbons including, without limitation, benzene, naphthalene, biphenyl, binaphthyl, or combinations of aromatic structures with C2-C20 hydrocarbon chains. Thus, in at least some scenarios, L can be alkyl, aryl, or aralkyl in structure. Alkyl linking groups can have one or more carbons in their chains substituted with oxygen (0), or an amine (NR), where R is H or Ci-Ce alkyl. The linking groups can also comprise one or more unsaturations and thus include one or more alkenyl and / or alkynyl moieties.

[0144] In at least some examples, L can comprise at least one Y and at least one X. In one or more examples, each at least one Y can be independently selected from the group consisting of: an optionally substituted divalent alkylene; an optionally substituted arylene; and optionally substituted divalent heteroaromatic ring moiety; having from 1 to 20 atoms.

[0145] In one or more examples, each at least on Y can be, independently: an alkylene, -(CR2)p-, wherein p is an integer from 1 to 10, 1 to 6, or 1 to 4, and wherein R2can be independently selected from the group consisting of H and lower alkyl, C1- Cs alkyl, and C1-C3 alkyl.

[0146] In one or more additional examples, each at least one Y can be, independently, an arylene.

[0147] In one or more further examples, each at least one Y can be, independently, a divalent heteroaromatic ring having from 4 to 20 carbon atoms and includes at least one heteroatom selected from the group consisting of O, N, and S.

[0148] In still other examples, each at least one X can be, independently, selected from the group consisting of alkylene, -NR1-, -O-, -S-, -S-S-, -CO-NR1-, - NR1- CO-, -CO-O-, -O-CO-, -CO-, and a bond, wherein R1can be independently selected6356.002W01 from the group consisting of H and lower alkyl. In one or more examples, each of the at least one Y and each of the at least one X can be, independently, optionally substituted.

[0149] In various examples, one or more of the at least one X can be the same.

[0150] Additionally, one or more of the at least one Y can be the same.

[0151] Further, one or more of the at least one X are different.

[0152] In addition, one or more of the at least one Y are different.

[0153] The term "alkyl," as used herein, alone or in combination, refers to a straight-chain or branched-chain alkyl radical containing from 2 to 20 carbon atoms. In various examples, the alkyl can comprise from 2 to 10 carbon atoms. In further examples, the alkyl group can comprise from 2 to 6 carbon atoms. Alkyl groups can be optionally substituted as defined herein below. Examples of alkyl group (given as radicals) can include, without limitation methyl, ethyl, n- propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, octyl, nonyl and the like. The term "alkenyl," as used herein, alone or in combination, can refer to a straight-chain or branched-chain hydrocarbon radical having one or more double bonds and containing from 2 to 20 carbon atoms. In one or more examples, the alkenyl group can comprise from 2 to 6 carbon atoms. The term "alkenylene" can refer to a carbon-carbon double bond system attached at two or more positions such as ethenylene [( — CH=CH — ), ( — C::C — )]. Examples of suitable alkenyl radicals can include propenyl, 2- methylpropenyl, 1 ,4-butadienyl and the like.

[0154] The term "alkynyl," as used herein, alone or in combination, can refer to a straight-chain or branched chain hydrocarbon radical having one or more triple bonds and including from 4 to 20 carbon atoms. In various examples, said alkynyl comprises from 4 to 6 carbon atoms. Examples of alkynyl groups can include butyn- 1 -yl, butyn-2-yl, pentyn-1-yl, 3-methylbutyn-1-yl, hexyn-2-yl, and the like.

[0155] The term "aryl," as used herein, alone or in combination, can mean a carbocyclic aromatic system containing one, two or three rings wherein such rings can be attached together in a pendent manner or can be fused. In at least some examples, “Aryl” groups include groups having one or more 5- or 6-member aromatic rings. Aryl groups contain no heteroatoms in the aryl rings. Aryl groups can be optionally substituted with one or more non-hydrogen substituents.6356.002W01

[0156] The term "aryl" can include aromatic radicals such as benzyl, phenyl, naphthyl, anthracenyl, phenanthryl, indanyl, indenyl, annulenyl, azulenyl, tetrahydronaphthyl, and biphenyl.

[0157] The term “arylene,” can refer to a divalent aromatic radical which consists of the elements carbon and hydrogen. The divalent aromatic radical may include only one benzene ring, or a plurality of benzene rings as in diphenyl, naphthyl, oranthracyl.

[0158] The term "aralkyl," as used herein, alone or in combination, can refer to an aryl group attached to the parent molecular moiety through an alkyl group.

[0159] The term “heteroaryl” and “heteroaromatic rings,” as used herein, can refer to and include groups having one or more aromatic rings in which at least one ring includes a heteroatom (a non-carbon ring atom). Heteroaryl groups can include those having one or two heteroaromatic rings carrying 1 , 2 or 3 heteroatoms. Heteroaryl groups can contain 5-20, 5-12 or 5-10 ring atoms. Heteroaryl groups can include those having one aromatic ring contains a heteroatom and one aromatic ring containing carbon ring atoms. Heteroaryl groups can include those having one or more 5- or 6- member aromatic heteroaromatic rings and one or more 6-member carbon aromatic rings. Heteroaromatic rings can include one or more N, O, or S atoms in the ring. Heteroaromatic rings can include those with one, two or three N, those with one or two O, and those with one or two S, or combinations of one or two or three N, O or S. Specific heteroaryl groups include furyl, pyridinyl, pyrazinyl, pyrimidinyl, quinolinyl, and purinyl groups.

[0160] The term “lower alkyl” can refer to, for example, C1-C9 alkyl, Ci-Cs alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, or C1-C2 alkyl.

[0161] The term "optionally substituted" can mean the anteceding group can be substituted or unsubstituted. When substituted, the substituents of an "optionally substituted" group can include, without limitation, one or more substituents independently selected from the following groups or a particular designated set of groups, alone or in combination: lower alkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl, lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lower haloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl, phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, lower acyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester, lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, lower alkylamino, arylamino, amido, nitro, thiol, lower6356.002W01 alkylthio, lower haloalkylthio, lower perhaloalkylthio, arylthio, sulfonate, sulfonic acid, tri substituted silyl, N3, SH, SCFb, C(O)CH3, CO2CH3, CO2H, pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. Two substituents may be joined together to form a fused five-, six-, or seven-membered carbocyclic or heterocyclic ring consisting of zero to three heteroatoms, for example forming methylenedioxy or ethyl enedioxy. An optionally substituted group may be unsubstituted (e.g., — CH2CH3), fully substituted (e.g., — CF2CF3), monosubstituted (e.g., — CH2CH2F) or substituted at a level anywhere in-between fully substituted and monosub stituted (e.g., — CH2CF3). Where substituents are recited without qualification as to substitution, both substituted and unsubstituted forms are encompassed. Where a substituent is qualified as "substituted," the substituted form is specifically intended. Additionally, different sets of optional substituents to a particular moiety may be defined as needed; in these cases, the optional substitution will be as defined, often immediately following the phrase, "optionally substituted with." The term "lower," as used herein, alone or in combination, means containing from 1 to and including 6 carbon atoms.

[0162] In one or more examples, the polymer composition can comprise a crosslinking reagent represented by Formula (I):PA-L-PA (I) wherein L is a linking group and each PA independently comprises an azide (-N3), a diazo (-N2) group, an aryl azide, an acyl azide, an azidoformate, a sulfonyl azide, a phosphoryl azide, a diazoalkane, a diazoketone, a diazoacetate, a diazirine, an aliphatic azo, an aryl ketone, benzophenone, acetophenone, anthraquinone, or an anthrone.

[0163] In at least some examples, each PA independently comprises an azide (-N3), or a diazo (-N2) group.

[0164] In one or more additional examples, the polymer composition comprises a crosslinking reagent represented by Formula (I):PA-L-PA (I) wherein each PA independently comprises an azide (-N3), a diazo (-N2) group, an aryl azide, an acyl azide, an azidoformate, a sulfonyl azide, a phosphoryl azide, a diazoalkane, a diazoketone, a diazoacetate, a diazirine, an aliphatic azo, an aryl ketone, benzophenone, acetophenone, anthraquinone, and anthrone; L comprises at least one Y and one or more X, wherein: a) each at least one Y is independently selected from the group consisting of: an optionally substituted divalent alkylene; an6356.002W01 optionally substituted arylene; and optionally substituted divalent heteroaromatic ring moiety; having from 1 to 20 atoms; an alkylene, -(CR2)p-, wherein p is an integer from 1 to 10, 1 to 6, or 1 to 4, and wherein R2is independently selected from the group consisting of H and lower alkyl, C1-C5 alkyl, and C1-C3 alkyl; and / or a divalent heteroaromatic ring having from 4 to 20 carbon atoms and contains at least one heteroatom selected from the group consisting of O, N, and S; and b) each X is independently selected from the group consisting of alkylene, -NR1-, -O-, -S-, -S-S-, - CO-NR1-, - NR1-CO-, -CO-O-, -O-CO-, -CO-, and a bond, wherein R1is independently selected from the group consisting of H and lower alkyl.

[0165] In various examples, the polymer composition comprises a crosslinking reagent represented by Formula (II):PA-Y1-X1-X2-X3-X4-X5-X6-X7-X8-X9-Y2-PA (II) wherein each PA is a photo-activated group or a metal-activated group, and Y1-X1-X2- X3-X4-X5-X6-X7-X8-X9-Y2is a linking group, wherein; each PA, independently, comprises an azide (-N3), a diazo (-N2) group, an aryl azide, an acyl azide, an azidoformate, a sulfonyl azide, a phosphoryl azide, a diazoalkane, a diazoketone, a diazoacetate, a diazirine, an aliphatic azo, an aryl ketone, benzophenone, acetophenone, anthraquinone, and anthrone. In some embodiments, each PA independently comprises an azide (-N3), or a diazo (-N2) group.

[0166] In one or more examples, Y1and Y2can each, independently be: an alkylene, -(CR2)p-, wherein p is an integer from 1 to 10, 1 to 6, or 1 to 4, and wherein R2is independently selected from the group consisting of H and lower alkyl, C1-C5 alkyl, and C1-C3 alkyl.

[0167] In one or more additional examples, Y1and Y2can each, independently be, an arylene.

[0168] In one or more further examples, Y1and Y2can each, independently, be a divalent heteroaromatic ring having from 4 to 20 carbon atoms and includes at least one heteroatom selected from the group consisting of O, N, and S.

[0169] In at least some examples, X1, X2, X3, X4, X5, X6, X7, X8, and X9can each, independently, be selected from the group consisting of alkylene, -NR1-, -O-, -S-, -S- S-, -CO-NR1-, - NR1-CO-, - CO-O-, -O-CO-, -CO-, and a bond, wherein R1is independently selected from the group consisting of H and lower alkyl.

[0170] In still other examples, each of Y1and Y2, X1, X2, X3, X4, X5, X6, X7, X8, and X9can, independently, be optionally substituted.6356.002W01

[0171] In some embodiments, one or more of X1, X2, X3, X4, X5, X6, X7, X8, and X9are the same. In certain embodiments, Y1and Y2are the same.

[0172] In certain embodiments, one or more X1, X2, X3, X4, X5, X6, X7, X8, and X9isdifferent. In certain embodiments, Y1and Y2are different.

[0173] In some embodiments, the polymer composition comprises a crosslinking reagent represented by Formula (II):PA-Y1-X1-X2-X3-X4-X5-X6-X7-X8-X9-Y2-PA (II) wherein each PA is a photo-activated group or a metal-activated group, and Y1-X1-X2- X3-X4-X5-X6-X7-X8-X9-Y2is a linking group, wherein; each PA, independently, comprises an azide (-N3), a diazo (-N2) group, an aryl azide, an acyl azide, an azidoformate, a sulfonyl azide, a phosphoryl azide, a diazoalkane, a diazoketone, a diazoacetate, a diazirine, an aliphatic azo, an aryl ketone, benzophenone, acetophenone, anthraquinone, and anthrone; (a) Y1and Y2are each, independently, selected from the group consisting of: an optionally substituted divalent alkylene; an optionally substituted arylene; and optionally substituted divalent heteroaromatic ring moiety; having from 1 to 20 atoms; an alkylene, -(CR2)p-, wherein p is an integer from 1 to 10, 1 to 6, or 1 to 4, and wherein R2is independently selected from the group consisting of H and lower alkyl, C1-C5 alkyl, and C1-C3 alkyl; and / or a divalent heteroaromatic ring having from 4 to 20 carbon atoms and contains at least one heteroatom selected from the group consisting of O, N, and S; and c) each of1X2, X3, X4, X5, X6, X7, X8, and X9is independently selected from the group consisting of alkylene, -NR1-, -O-, -S-, -S-S-, -CO-NR1-, - NR1-CO-, -CO-O-, -O-CO-, -CO-, and a bond, wherein R1is independently selected from the group consisting of H and lower alkyl.

[0174] In some embodiments, provided are polymer compositions comprising: a polyethylene glycol (PEG) polymer; a poly(2-hydroxyethyl methacrylate) (PHEMA) polymer; and a crosslinking reagent represented by either Formula (I) or Formula (II).

[0175] In some embodiments, the crosslinking reagent comprises bis[2-(4- azidosalicylamido)ethyl]disulfide or dithiobis(phenylazide).

[0176] In some embodiments, a polymer composition comprises: a polyethylene glycol (PEG) polymer; a poly(2-hydroxyethyl methacrylate) (PHEMA) polymer; and a crosslinking reagent represented by Formula (I):6356.002W01PA-L-PA (I) wherein: each PA independently can comprise an azide (-N3), a diazo (-N2) group, an aryl azide, an acyl azide, an azidoformate, a sulfonyl azide, a phosphoryl azide, a diazoalkane, a diazoketone, a diazoacetate, a diazirine, an aliphatic azo, an aryl ketone, benzophenone, acetophenone, anthraquinone, and anthrone; L can comprise at least one Y and one or more X, wherein: a) each at least one Y is independently selected from the group consisting of: an optionally substituted divalent alkylene; an optionally substituted arylene; and optionally substituted divalent heteroaromatic ring moiety; having from 1 to 20 atoms; an alkylene, -(CR2)p-, wherein p is an integer from 1 to 10, 1 to 6, or 1 to 4, and wherein R2is independently selected from the group consisting of H and lower alkyl, C1-C5 alkyl, and C1-C3 alkyl; and / or a divalent heteroaromatic ring having from 4 to 20 carbon atoms and contains at least one heteroatom selected from the group consisting of O, N, and S; and b) each X is independently selected from the group consisting of alkylene, -NR1-, -O-, -S-, -S-S-, - CO-NR1-, - NR1-CO-, -CO-O-, -O-CO-, -CO-, and a bond, wherein R1is independently selected from the group consisting of H and lower alkyl.

[0177] In one or more examples, the crosslinking reagent can comprise bis[2- (4- azidosalicylamido)ethyl]disulfide or dithiobis(phenylazide).

[0178] In still other examples, a polymer composition can comprise: a polyethylene glycol (PEG) polymer; a poly(2-hydroxyethyl methacrylate) (PHEMA) polymer; and a crosslinking reagent represented by Formula (II):PA-Y1-X1-X2-X3-X4-X5-X6-X7-X8-X9-Y2-PA (II) wherein each PA is a photo-activated group or a metal-activated group, and Y1-X1-X2- X3-X4-X5-X6-X7-X8-X9-Y2is a linking group, wherein: each PA independently comprises an azide (-N3), a diazo (-N2) group, an aryl azide, an acyl azide, an azidoformate, a sulfonyl azide, a phosphoryl azide, a diazoalkane, a diazoketone, a diazoacetate, a diazirine, an aliphatic azo, an aryl ketone, benzophenone, acetophenone, anthraquinone, and anthrone; each of Y1, and Y2is independently selected from the group consisting of: an optionally substituted divalent alkylene; an optionally substituted arylene; and optionally substituted divalent heteroaromatic ring moiety; having from 1 to 20 atoms; an alkylene, -(CR2)p-, wherein p is an integer from 1 to 10, 1 to 6, or 1 to 4, and wherein R2is independently selected from the group consisting of H and lower alkyl, C1-C5 alkyl, and C1-C3 alkyl; and / or a divalent heteroaromatic ring having from 4 to 20 carbon atoms and contains at least one heteroatom selected from6356.002W01 the group consisting of 0, N, and S; and each of X1, X2, X3, X4, X5, X6, X7, X8, and X9is independently selected from the group consisting of alkylene, -NR1-, -O-, -S-, -S-S- , -CO-NR1-, - NR1-CO-, -CO-O-, -O-CO-, -CO-, and a bond, wherein R' is independently selected from the group consisting of H and lower alkyl. In some embodiments, the crosslinking reagent comprises bis[2-(4- azidosalicylamido)ethyl]disulfide or dithiobis(pheny 1 -azide) .

[0179] In some embodiments, the PEG polymer is derivatized with an N- hydroxysuccinimide (NHS) ester and wherein the derivatized PEG polymer has a molecular weight of about 600 Da. In some embodiments, there are provided polymer compositions disposed as a layer on a sensor as a biosurface prepared by a method comprising crosslinking the at least two hydrophilic polymers of the polymer composition with the crosslinking reagent via an attachment atom.

[0180] In some such embodiments, the crosslinking agent is represented by each A in Formula (I) or Formula (II).

[0181] In some such embodiments, each A in Formula (I) and Formula (II) represents an attachment atom derived from the decomposition reaction of an azide (-N3), a diazo (-N2) group, an aryl azide, an acyl azide, an azidoformate, a sulfonyl azide, a phosphoryl azide, a diazoalkane, a diazoketone, a diazoacetate, a diazirine, an aliphatic azo, an aryl ketone, benzophenone, acetophenone, anthraquinone, or an anthrone.

[0182] In addition to compounds of Formula (I) and Formula (II), other compounds that can serve as crosslinkers include, without limitation, the crosslinkers disclosed in U.S. Patent Nos. 10,315,987, 10,253,193, 9,994,721 , 9,487,663, and 8,889,760, all of which are incorporated herein by reference in their entirety.

[0183] In one or more examples, biosurfaces are provided that comprise a polymer composition disposed as a layer on a sensor, wherein the biosurface is prepared by a method comprising crosslinking at least two hydrophilic polymers of the polymer composition with the crosslinking reagent. In various examples, the crosslinking can comprise a decomposition reaction of an attachment atom on the crosslinking reagent. The decomposition reaction can occur by a method comprising photocatalysis. In addition, the decomposition reaction can occur by a method comprising metal catalysis. In one or more of the preceding examples, the crosslinking can be photocatalyzed. In still other examples, the photocatalysis can comprise6356.002W01 irradiating the polymer composition with for example, ultraviolet light (i.e., light has a wavelength in the ultraviolet wavelength range).

[0184] In one or more further examples, a biosurface is provided comprising a polymer composition disposed as a layer on a sensor, wherein said biosurface is prepared by a method comprising crosslinking at least two hydrophilic polymers with a crosslinking reagent represented by Formula (I) or Formula (II), wherein each A is an attachment atom derived from the decomposition reaction of an azide (-N3), a diazo (-N2) group, an aryl azide, an acyl azide, an azidoformate, a sulfonyl azide, a phosphoryl azide, a diazoalkane, a diazoketone, a diazoacetate, a diazirine, an aliphatic azo, an aryl ketone, benzophenone, acetophenone, anthraquinone, or an anthrone. In at least some examples, the crosslinking can comprise a decomposition reaction of an attachment atom on the crosslinking reagent. In one or more additional examples, the decomposition reaction can occur by a method comprising photocatalysis. In one or more further examples, the decomposition reaction can occur by a method comprising metal catalysis. In one or more of the preceding examples, the crosslinking can be photocatalyzed. In still other examples, the photocatalysis can comprise irradiating the polymer composition with, for example, ultraviolet light (i.e., light has a wavelength in the ultraviolet wavelength range).

[0185] A polymer composition and / or a biosurface further can comprise a covalently attached biomolecule. For example, the covalently attached molecule can comprise a covalently attached biomolecule comprising a protein, a transcription factor, a nucleic acid, a deoxyribonucleic acid, a ribonucleic acid, a polynucleotide, a double-stranded DNA (dsDNA), a single stranded DNA (ssDNA), a hybrid doublestranded polynucleotide comprising a ssDNA and a ssRNA, an oligonucleotide, a carbohydrate, a hormone, a glycoprotein, an immunoglobulins, an antibody, or antigen-binding antibody fragments.

[0186] In one or more illustrative examples, the covalently attached molecule is a protein.

[0187] In one or more additional illustrative examples, the covalently attached biomolecule can comprise an antibody, an immunoglobulin, or an antigen-binding antibody fragment.

[0188] In one or more further examples, the covalently attached molecule can comprise a double-stranded DNA (dsDNA).6356.002W01

[0189] In one or more of the preceding examples, the PEG polymer can further comprise one or two terminal N-hydroxysuccinimde (NHS) groups.

[0190] In one or more of the preceding examples, at least one of the at least two hydrophilic polymers can be provided in a solvent. For example, a PEG polymer and / or a PHEMA polymer can provided in a solvent. To illustrate, the solvent can comprise isopropyl alcohol, water, or mixtures thereof.

[0191] In one or more examples, methods of forming a biosurface on a sensor can comprise coating the sensor with at least two hydrophilic polymers and a crosslinking reagent as disclosed herein. In one or more examples, methods for forming a biosurface on a sensor can comprise coating the sensor with at least one hydrophilic polymer selected from the group consisting of a polyethylene glycol (PEG) polymer, a cellulose, a starch, a chitin, and alginate, and a dextran, and at least one poly(2-hydroxyethyl methacrylate) (PHEMA) polymer. In at least some examples, one hydrophilic polymer can comprise a PEG polymer and another of the at least two hydrophilic polymers can comprise a PHEMA polymer.

[0192] In one or more additional examples, methods of forming a biosurface on a sensor can comprise coating the sensor with at least two hydrophilic polymers and a crosslinking reagent represented by Formula (I) or Formula (II).

[0193] In one or more further examples, hydrophilic polymers and the crosslinking reagent can be mixed together in a solvent, and the coating step of each can occur at the same time or substantially simultaneously. In one or more illustrative examples, the hydrophilic polymers and the crosslinking reagent can each be provided in a separate solvent, and the coating step of each can be performed sequentially. In one or more of the preceding examples, the solvent can be selected from the group consisting of isopropyl alcohol, water, and mixtures thereof. In various examples, other solvents such as methanol, acetone can be employed. In still other examples, one or more of the hydrophilic polymers and the crosslinking reagent can be provided as a solid, for example, a powder, and can be dissolved in a solvent.

[0194] In various examples, one hydrophilic polymer can be coated first on a sensor, then the crosslinking reagent, then the other hydrophilic polymer. In one or more examples, a PEG polymer can be coated first on a sensor, then a crosslinking reagent, then a PHEMA polymer. In one or more additional examples, a PHEMA polymer can be coated first on a sensor, then a crosslinking reagent, then a PEG polymer. In one or more further examples, a PEG polymer can be coated first on a6356.002W01 sensor, then a crosslinking reagent, then a PHEMA polymer. In at least some examples, coating of sensors can be performed by any combination or permutation of the above.

[0195] In one or more examples, the coating step comprises microprinting. As a non-limiting example, a sensor or other substrate may be coated with a PEG polymer, PHEMA polymer, and / or crosslinking reagent via non-contact capillary dispensing or contact printing. The thickness of the component may be controlled by the number of drops and / or drop volume from the dispensing capillary.

[0196] In one or more additional examples, a coating step can comprise dip coating. As a non-limiting example, a sensor or other substrate can be dipped into a container of any or all of a PEG polymer, a PHEMA polymer, and / or a crosslinking reagent. The desired sensor area or surface can be submerged in a solution of these components and then removed. Thickness can be controlled by the speed of the dip and removal process.

[0197] In one or more further examples, the coating step can comprise spin coating. As a non-limiting example, any or all of a PEG polymer, a PHEMA polymer, and / or a crosslinking reagent can be deposited onto a sensor or other substrate by attachment of the sensor or substrate to a spin coater, with the aid of vacuum suction or other fixture. The sensor or substrate can be spun while the polymer composition components are deposited. The thickness of the deposition can be controlled by speed and time of spinning and the volume of the polymer composition components.

[0198] In still other examples, the coating step can comprise aerosol coating. As a non-limiting example, aerosol coating equipment can be loaded with any or all of a PEG polymer, a PHEMA polymer, and / or a crosslinking reagent and used to coat the sensor or other substrate.

[0199] In at least some examples, when individual polymer composition components are added sequentially, any combination of the aforementioned coating techniques can be employed for each of the polymers and / or the crosslinking reagent.

[0200] In various examples, after coating, the polymer composition can be crosslinked. In one or more examples, the crosslinking can be photocatalyzed. In one or more additional examples, the crosslinking can be metal-catalyzed. In one or more further examples, photocatalyzed crosslinking can employ UV radiation. Following crosslinking the surface can be washed with solvent such as isopropyl alcohol, water or mixtures thereof.6356.002W01

[0201] In one or more of the preceding implementations, methods can further comprise attaching a biomolecule to form the biosurface.

[0202] In one or more of the preceding implementations, a thickness of the formed biosurface may vary from about 0.5 nm to about 5 micrometers. In one or more examples, the biosurface can have a thickness of at least 0.5 nanometers (nm), at least 1 nm, at least 2 nm, at least 3 nm, at least 4 nm, at least 5 nm, at least 6 nm, at least 7 nm, at least 8 nm, at least 9 nm, at least 10 nm, at least 11 nm, at least 12 nm, at least 13 nm, at least 14 nm, or at least 15 nm. In one or more additional examples, the biosurface can have a thickness no greater than 100 nm, no greater than 90 nm, no greater than 80 nm, no greater than 70 nm, no greater than 60 nm, no greater than 50 nm, no greater than 40 nm, no greater than 30 nm, or no greater than 20 nm. In one or more further examples, the biosurface can have a thickness from about 0.5 nm to about 100 nm, from about 2 nm to about 80 nm, from about 5 nm to about 60 nm, from about 8 nm to about 40 nm, from about 10 nm to about 20 nm, from about 1 nm to about 10 nm, from about 5 nm to about 15 nm, from about 15 nm to about 25 nm, from about 20 nm to about 30 nm, from about 25 nm to about 35 nm, from about 30 nm to about 40 nm, from about 35 nm to about 45 nm, or from about 40 nm to about 50 nm.

[0203] In various examples, probes can be coupled to the polymeric composition having nucleotide sequences that corresponds to a portion of a target sequence. For example, the probes can include nucleotide sequences that are complementary to at least a portion of a target sequence of an analyte of interest. To illustrate, the probes can include nucleotide sequences that are complementary to at least a portion of the 5’ biotinylated nucleic acid strands produced by the nucleic acid digestion process 126.

[0204] In one or more examples, individual sensors 132 can include probes that having nucleotide sequences that correspond to different target nucleic acid sequences. For example, a first portion of the sensors 132 of the sensing device 130 can include first probes having first nucleotide sequences that are complementary to one or more first target nucleotide sequences, a second portion of the sensors 132 can include second probes having second nucleotide sequences that are complementary to one or more second target nucleotide sequences, and a third portion of the sensors 132 can include third probes having third nucleotide sequences that are complementary to one or more third target nucleotide sequences. In one or6356.002W01 more illustrative examples, individual sensors 132 can include probes having nucleotide sequences that are complementary with individual target nucleotide sequences. To illustrate, a first sensor can include first probes with first nucleotide sequences complementary to a first target nucleotide sequence, a second sensor can include second probes with second nucleotide sequences complementary to a second target nucleotide sequence, a third sensor can include third probes with third nucleotide sequences complementary to a third target nucleotide sequence. Additional individual sensors 132 can include probes that correspond to additional individual target nucleotide sequences.

[0205] Examples of probe sequences can be found below in Table 2:Table 2. Sequences of Probes to Capture Target Nucleic Acids6356.002W01

[0206] Although, the sensing device 130 has been described with respect to three sensors and their corresponding probes, the sensing device 130 can include a greater number of individual sensors 132 or fewer individual sensors 132. Additionally, the sensing device 130 can include multiple sensors that detect a single target nucleotide sequence. In one or more illustrative examples, the sensing device 130 can include at least one sensor, at least 2 sensors, at least 2 sensors, at least 4 sensors,6356.002W01 at least 5 sensors, at least 6 sensors, at least 7 sensors, at least 8 sensors, at least 9 sensors, at least 10 sensors, at least 11 sensors, at least 12 sensors, at least 13 sensors, at least 14 sensors, at least 15 sensors, or at least 16 sensors. In one or more additional illustrative examples, the sensing device 130 can include no greater than 50 sensors, no greater than 48 sensors, no greater than 46 sensors, no greater than 44 sensors, no greater than 42 sensors, no greater than 40 sensors, no greater than 38 sensors, no greater than 36 sensors, no greater than 34 sensors, no greater than 32 sensors, no greater than 30 sensors, no greater than 28 sensors, no greater than 26 sensors, no greater than 24 sensors, no greater than 22 sensors, no greater than 20 sensors, or no greater than 18 sensors. In one or more illustrative examples, the sensing device 130 can have from 1 sensor to 50 sensors, from 1 sensor to 40 sensors, from 1 sensor to 30 sensors, from 1 sensor to 20 sensors, from 1 sensor to10 sensors, from 5 sensors to 50 sensors, from 5 sensors to 45 sensors, from 5 sensors to 40 sensors, from 5 sensors to 35 sensors, from 5 sensors to 30 sensors, from 5 sensors to 25 sensors, from 5 sensors to 20 sensors, from 5 sensors to 15 sensors, from 10 sensors to 50 sensors, from 10 sensors to 45 sensors, from 10 sensors to 40 sensors, from 10 sensors to 35 sensors, from 10 sensors to 30 sensors, from 10 sensors to 25 sensors, from 10 sensors to 20 sensors, from 15 sensors to 50 sensors, from 15 sensors to 45 sensors, from 15 sensors to 40 sensors, from 15 sensors to 35 sensors, from 15 sensors to 30 sensors, from 15 sensors to 25 sensors, from 20 sensors to 50 sensors, from 20 sensors to 45 sensors, from 20 sensors to 40 sensors, from 20 sensors to 35 sensors, from 20 sensors to 30 sensors, from 25 sensors to 50 sensors, from 25 sensors to 45 sensors, from 25 sensors to 40 sensors, from 25 sensors to 35 sensors, from 30 sensors to 50 sensors, from 30 sensors to 45 sensors, from 30 sensors to 40 sensors, from 35 sensors to 50 sensors, from 35 sensors to 45 sensors, or from 40 sensors to 50 sensors.

[0207] The sensing device 130 can be configured according to a shape. In one or more examples, the sensing device 130 can have a linear shape. In one or more additional examples, the sensing device 130 can have a circular shape. In one or more additional examples, the sensing device 130 can have a serpentine shape. In various examples, the sensors 132 can be arranged an array of rows and columns along a length of the sensing device 130. In one or more illustrative examples, the sensors 132 can be arranged in a single row along a length of the sensing device 130 In one or more additional illustrative examples, the sensors 132 can be arranged in two rows6356.002W01 along a length of the sensing device 130. In one or more further illustrative examples, the sensors 132 can be arranged in three rows along a length of the sensing device 130. In still other illustrative examples, the sensors 132 can be arranged in four rows along a length of the sensing device 130. In various illustrative examples, the sensors 132 can be arranged in five rows along a length of the sensing device 130.

[0208] The sensing device 130 can have a length of at least 5 millimeters (mm), at least 10 mm, at least 15 mm, at least 20 mm, at least 25 mm, or at least 30 mm. Additionally, the sensing device 130 can have a length no greater than 100 mm, no greater than 90 mm, no greater than 80 mm, no greater than 70 mm, no greater than 60 mm, no greater than 50 mm, or no greater than 40 mm. Further, the sensing device 130 can have a length from 5 mm to 100 mm, from 5 mm to 90 mm, from 5 mm to 80 mm, from 5 mm to 70 mm, from 5 mm to 60 mm, from 5 mm to 50 mm, from 5 mm to 40 mm, from 5 mm to 30 mm, from 5 mm to 20 mm, from 5 mm to 10 mm, from 10 mm to 100 mm, from 10 mm to 90 mm, from 10 mm to 80 mm, from 10 mm to 70 mm, from 10 mm to 60 mm, from 10 mm to 50 mm, from 10 mm to 40 mm, from 10 mm to 30 mm, from 10 mm to 20 mm, from 20 mm to 100 mm, from 20 mm to 90 mm from 20 mm to 80 mm, from 20 mm to 70 mm, from 20 mm to 60 mm, from 20 mm to 50 mm, from 20 mm to 40 mm, from 20 mm to 30 mm, from 30 mm to 100 mm , from 30 mm to 90 mm, from 30 mm to 80 mm, from 30 mm to 70 mm, from 30 mm to 60 mm, from 30 mm to 50 mm, from 30 mm to 40 mm, from 50 mm to 100 mm, from 50 mm to 90 mm, from 50 mm to 80 mm, from 50 mm to 70 mm, of from 50 mm to 60 mm.

[0209] The sensing device 130 can also have a width of at least 1 mm, at least 2 mm, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7 mm, at least 8 mm, at least 9 mm, at least 10 mm, at least 11 mm, at least 12 mm, at least 13 mm, at least 14 mm, at least 15 mm, at least 16 mm, at least 17 mm, at least 18 mm, at least 19 mm, or at least 20 mm. Additionally, the sensing device 130 can have a width no greater than 50 mm, no greater than 45 mm, no greater than 40 mm, no greater than 35 mm, no greater than 30 mm, or no greater than 25 mm. Further, the sensing device 130 can have a width from 1 mm to 50 mm, from 1 mm to 40 mm, from 1 mm to 30 mm, from 1 mm to 20 mm, from 1 mm to 10 mm, from 5 mm to 50 mm, from 5 mm to 40 mm, from 5 mm to 30 mm, from 5 mm to 20 mm, from 5 mm to 10 mm, from 10 mm to 50 mm, from 10 mm to 40 mm, from 10 mm to 30 mm, from 10 mm to 20 mm, from 15 mm to 50 mm, from 15 mm to 45 mm, from 15 mm to 40 mm, from 15 mm to 35 mm, from 15 mm to 30 mm, from 15 mm to 25 mm, from 20 mm to6356.002W0150 mm from 20 mm to 45 mm, from 20 mm to 40 mm, from 20 mm to 35 mm, from 20 mm to 30 mm, from 30 mm to 50 mm, from 30 mm to 45 mm, from 30 mm to 40 mm, from 35 mm to 50 mm, from 35 mm to 45 mm, or from 40 mm to 50 mm.

[0210] In one or more examples, the probes can include at least 12 base pairs, at least 13 base pairs, at least 14 base pairs, at least 15 base pairs, at least 16 base pairs, at least 17 base pairs, at least 18 base pairs, at least 19 base pairs, or at least20 base pairs. In one or more additional examples, the probes can include no greater than 40 base pairs, no greater than 38 base pairs, no greater than 36 base pairs, no greater than 34 base pairs, no greater than 32 base pairs, no greater than 30 base pairs, no greater than 28 base pairs, no greater than 26 base pairs, no greater than24 base pairs, or no greater than 22 base pairs. In one or more illustrative examples, the probes can have from 12 base pairs to 40 base pairs, from 12 base pairs to 35 base pairs, from 12 base pairs to 30 base pairs, from 12 base pairs to 25 base pairs, from 12 base pairs to 20 base pairs, from 15 base pairs to 40 base pairs, from 15 base pairs to 35 base pairs, from 15 base pairs to 30 base pairs, from 15 base pairs to 25 base pairs, from 20 base pairs to 40 base pairs, from 20 base pairs to 35 base pairs, or from 20 base pairs to 30 base pairs.

[0211] The probes can have a 5’ end that includes a probe linker group. The probe linker group can cause the payload portion of the probes having sequences that are complementary to target sequences to be positioned a distance away from the surface of the sensor array of the sensing device 130 such that the probes have a greater probability of binding to nucleic acids included in the digestion product. In one or more illustrative examples, probe linker groups can be attached to the 5’ end of the probes included in Table 2 and have SEQ IDs 38 - 60.

[0212] The probe linker group can include a number of thymine nucleotides. For example, the 5’ end of the probes can include at least 3 thymine nucleotides, at least 4 thymine nucleotides, at least 5 thymine nucleotides, at least 6 thymine nucleotides, at least 7 thymine nucleotides, at least 8 thymine nucleotides, at least 9 thymine nucleotides, or at least 10 thymine nucleotides. Additionally, the 5’ end of the probes can include no greater than 18 thymine nucleotides, no greater than 17 thymine nucleotides, no greater than 16 thymine nucleotides, no greater than 15 thymine nucleotides, no greater than 14 thymine nucleotides, no greater than 13 thymine nucleotides, no greater than 12 thymine nucleotides, or no greater than 11 thymine nucleotides. In one or more illustrative examples, the 5’ end of the probes can have6356.002W01 from 3 thymine nucleotides to 18 thymine nucleotides, from 3 thymine nucleotides to 16 thymine nucleotides, from 3 thymine nucleotides to 14 thymine nucleotides, from 3 thymine nucleotides to 12 thymine nucleotides, from 3 thymine nucleotides to 10 thymine nucleotides, from 5 thymine nucleotides to 18 thymine nucleotides, from 5 thymine nucleotides to 16 thymine nucleotides, from 5 thymine nucleotides to 14 thymine nucleotides, from 5 thymine nucleotides to 12 thymine nucleotides, from 5 thymine nucleotides to 10 thymine nucleotides, from 8 thymine nucleotides to 18 thymine nucleotides, from 8 thymine nucleotides to 16 thymine nucleotides, from 8 thymine nucleotides to 14 thymine nucleotides, from 8 thymine nucleotides to 12 thymine nucleotides, from 10 thymine nucleotides to 18 thymine nucleotides, from 10 thymine nucleotides to 16 thymine nucleotides, or from 10 thymine nucleotides to 14 thymine nucleotides.

[0213] Additionally, a 5’ end of the probes can be capped by a thymine nucleotide having an amino group substituted at position 6. The amino group at position 6 of the thymine nucleotide can bind to the polymeric material coated on the array of sensors 132.

[0214] In one or more further examples, The probe linker group can include a number of adenine nucleotides. For example, the 5’ end of the probes can include at least 3 adenine nucleotides, at least 4 adenine nucleotides, at least 5 adenine nucleotides, at least 6 adenine nucleotides, at least 7 adenine nucleotides, at least 8 adenine nucleotides, at least 9 adenine nucleotides, or at least 10 adenine nucleotides. Additionally, the 5’ end of the probes can include no greater than 18 adenine nucleotides, no greater than 17 adenine nucleotides, no greater than 16 adenine nucleotides, no greater than 15 adenine nucleotides, no greater than 14 adenine nucleotides, no greater than 13 adenine nucleotides, no greater than 12 adenine nucleotides, or no greater than 11 adenine nucleotides. In one or more illustrative examples, the 5’ end of the probes can have from 3 adenine nucleotides to 18 adenine nucleotides, from 3 adenine nucleotides to 16 adenine nucleotides, from 3 adenine nucleotides to 14 adenine nucleotides, from 3 adenine nucleotides to 12 adenine nucleotides, from 3 adenine nucleotides to 10 adenine nucleotides, from 5 adenine nucleotides to 18 adenine nucleotides, from 5 adenine nucleotides to 16 adenine nucleotides, from 5 adenine nucleotides to 14 adenine nucleotides, from 5 adenine nucleotides to 12 adenine nucleotides, from 5 adenine nucleotides to 10 adenine nucleotides, from 8 adenine nucleotides to 18 adenine nucleotides, from 86356.002W01 adenine nucleotides to 16 adenine nucleotides, from 8 adenine nucleotides to 14 adenine nucleotides, from 8 adenine nucleotides to 12 adenine nucleotides, from 10 adenine nucleotides to 18 adenine nucleotides, from 10 adenine nucleotides to 16 adenine nucleotides, or from 10 adenine nucleotides to 14 adenine nucleotides.

[0215] Additionally, a 5’ end of the probes can be capped by an adenine nucleotide having an amino group substituted at position 6. The amino group at position 6 of the adenine nucleotide can bind to the polymeric material coated on the array of sensors 132.

[0216] The sensors 132 can be formed on a biosurface of the sensing device 130 using one or more liquid phase deposition processes. In one or more examples, individual sensors 132 can be formed on a surface of the sensing device 130 by implementing one or more droplet deposition processes. One or more droplets comprised of a solution including one or more probes can be deposited on a location of a surface of the sensing device 130 to form a sensor 132. Droplets deposited on a surface of the sensing device 130 can have diameters from about 10 pm to about 500 pm, from about 10 pm to about 450 pm, from about 10 pm to about 400 pm, from about 10 pm to about 350 pm, from about 10 pm to about 300 pm, from about 10 pm to about 250 pm, from about 10 pm to about 200 pm, from about 10 pm to about 150 pm, from about 10 pm to about 100 pm, from about 10 pm to about 50 pm, from about 25 pm to about 500 pm, from about 25 pm to about 450 pm, from about 25 pm to about 400 pm, from about 25 pm to about 350 pm, from about 25 pm to about 300 pm, from about 25 pm to about 250 pm, from about 25 pm to about 200 pm, from about 25 pm to about 150 pm, from about 25 pm to about 100 pm, from about 25 pm to about 50 pm, from about 50 pm to about 500 pm, from about 50 pm to about 450 pm, from about 50 pm to about 400 pm, from about 50 pm to about 350 pm, from about 50 pm to about 300 pm, from about 50 pm to about 250 pm, from about 50 pm to about 200 pm, from about 50 pm to about 150 pm, from about 50 pm to about 100 pm, from about 75 pm to about 500 pm, from about 75 pm to about 450 pm, from about 75 pm to about 400 pm, from about 75 pm to about 350 pm, from about 75 pm to about 300 pm, from about 75 pm to about 250 pm, from about 75 pm to about 200 pm, from about 75 pm to about 150 pm, from about 75 pm to about 100 pm, from about 100 pm to about 500 pm, from about 100 pm to about 450 pm, from about 100 pm to about 400 pm, from about 100 pm to about 350 pm, from about 100 pm to about 300 pm, from about 100 pm to about 250 pm, from about 100 pm to about 200 pm, from about6356.002W01100 m to about 150 pm, from about 200 pm to about 500 pm, from about 200 pm to about 450 pm, from about 200 pm to about 400 pm, from about 200 pm to about 350 pm, from about 200 pm to about 300 pm, from about 300 pm to about 500 pm, from about 300 pm to about 450 pm, from about 300 pm to about 400 pm, or from about 400 pm to about 500 pm.

[0217] A solution used to form the sensors 132 on a biosurface of the sensing device 130 can have a probe concentration of at least about 0.1 micromolar (pM), at least about 0.2 pM, at least about 0.3 pM, at least about 0.4 pM, at least about 0.5 pM, at least about 0.6 pM, at least about 0.7 pM, at least about 0.8 pM, at least about 0.9 pM, at least about 1 pM, at least about 2 pM, at least about 3 pM, at least about 4 pM, at least about 5 pM, at least about 6 pM, at least about 7 pM, at least about 8 pM, at least about 9 pM, or at least about 10 pM. Additionally, a solution used to form the sensors 132 can have a probe concentration of no greater than about 30 pM, no greater than about 28 pM, no greater than about 26 pM, no greater than about 24 pM, no greater than about 22 pM, no greater than about 20 pM, no greater than about 18 pM, no greater than about 16 pM, no greater than about 14 pM, or no greater than about 12 pM. Further, a solution used to form the sensors 132 can have a probe concentration from about 0.1 pM to about 30 pM, from about 0.1 pM to about 25 pM, from about 0.1 pM to about 20 pM, from about 0.1 pM to about 15 pM, from about 0.1 pM to about 10 pM, from about 0.1 pM to about 5 pM, from about 0.1 pM to about 1 pM, from about 0.5 pM to about 30 pM, from about 0.5 pM to about 25 pM, from about 0.5 pM to about 20 pM, from about 0.5 pM to about 15 pM, from about 0.5 pM to about 10 pM, from about 0.5 pM to about 5 pM, from about 0.5 pM to about 1 pM, from about 1 pM to about 30 pM, from about 1 pM to about 25 pM, from about 1 pM to about 20 pM, from about 1 pM to about 15 pM, from about 1 pM to about 10 pM, from about 1 pM to about 5 pM, from about 2 pM to about 30 pM, from about 2 pM to about 25 pM, from about 2 pM to about 2 pM, from about 2 pM to about 2 pM, from about 2 pM to about 10 pM, from about 2 pM to about 5 pM, from about 5 pM to about 30 pM, from about 5 pM to about 25 pM, from about 5 pM to about 20 pM, from about 5 pM to about 15 pM, from about 5 pM to about 10 pM, from about 8 pM to about 30 pM, from about 8 pM to about 25 pM, from about 8 pM to about 20 pM, from about 8 pM to about 15 pM, from about 8 pM to about 12 pM, from about 10 pM to about 30 pM, from about 10 pM to about 28 pM, from about 10 pM to about 26 pM, from about 10 pM to about 24 pM, from about 10 pM to about 22 pM, from about 10 pM to about 20 pM,6356.002W01 from about 10 pM to about 18 pM, from about 10 pM to about 15 pM, from about 15 pM to about 30 pM, from about 15 pM to about 28 pM, from about 15 pM to about 26 pM, from about 15 pM to about 24 pM, from about 15 pM to about 22 pM, from about 15 pM to about 20 pM, from about 20 pM to about 28 pM, from about 20 pM to about 26 pM, from about 20 pM to about 24 pM, from about 20 pM to about 30 pM, from about 20 pM to about 28 pM, from about 20 pM to about 26 pM, from about 20 pM to about 24 pM, or from about 25 pM to about 30 pM.

[0218] In addition, a solution used to form the sensors 132 can include one or more additional components. For example, a solution used to form the sensors 132 can include trisodium phosphate (NasPCM). A concentration of trisodium phosphate present in a solution used to form the sensors 132 can be at least about 5 millimolar (mM), at least about 8 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, or at least about 45 mM. Additionally, a concentration of trisodium phosphate present in a solution used to form the sensors 132 can be no greater than about 80 mM, no greater than about 75 mM, no greater than about 70 mM, no greater than about 65 mM, no greater than about 60 mM, no greater than about 55 mM, or no greater than about 50 mM. In one or more illustrative examples, a concentration of trisodium phosphate present in a solution used to form the sensors 132 can be from about 5 mM to about 80 mM, from about 5 mM to about 70 mM, from about 5 mM to about 60 mM, from about 5 mM to about 50 mM, from about 5 mM to about 40 mM, from about 5 mM to about 30 mM, from about 5 mM to about 20 mM, from about 5 mM to about 10 mM, from about 10 mM to about 80 mM, from about 10 mM to about 70 mM, from about 10 mM to about 60 mM, from about 10 mM to about 50 mM, from about 10 mM to about 40 mM, from about 10 mM to about 30 mM, from about 10 mM to about 20 mM, from about 20 mM to about 80 mM, from about 20 mM to about 70 mM, from about 20 mM to about 60 mM, from about 20 mM to about 50 mM, from about 20 mM to about 40 mM, from about 20 mM to about 30 mM, from about 30 mM to about 80 mM, from about 30 mM to about 70 mM, from about 30 mM to about 60 mM, from about 30 mM to about 50 mM, from about 30 mM to about 40 mM, from about 40 mM to about 80 mM, from about 40 mM to about 70 mM, from about 40 mM to about 60 mM, from about 40 mM to about 50 mM, from about 50 mM to about 80 mM, from about 50 mM to about 70 mM, from about 50 mM to about 60 mM, from6356.002W01 about 60 mM to about 80 mM, from about 60 mM to about 70 mM, or from about 70 mM to about 80 mM.

[0219] In one or more further examples, a solution used to form the sensors 132 can include one or more salts. For example, a solution used to form the sensors 132 can include sodium chloride (NaCI). A concentration of one or more salts present in a solution used to form the sensors 132 can be at least about 0.05 molar (M), at least about 0.06 M, at least about 0.07 M, at least about 0.08 M, at least about 0.09 M, at least about 0.1 M, at least about 0.2 M, at least about 0.3 M, at least about 0.4 M, or at least about 0.5 M. Additionally, a concentration of one or more salts present in a solution used to form the sensors 132 can be no greater than about 1.2 M, no greater than about 1.1 M, no greater than about 1 M, no greater than about 0.9 M, no greater than about 0.8 M, no greater than about 0.7 M, or no greater than about 0.6 M. In one or more illustrative examples, a concentration of one or more salts present in a solution used to form the sensors 132 can be from about 0.05 M to about 1 .2 M, from about 0.05 M to about 1 M, from about 0.05 M to about 0.8 M, from about 0.05 M to about 0.6 M, from about 0.05 M to about 0.4 M, from about 0.05 M to about 0.3 M, from about 0.05 M to about 0.2 M, from about 0.05 M to about 0.1 M, from about 0.1 M to about 1.2 M, from about 0.1 M to about 1 M, from about 0.1 M to about 0.8 M, from about 0.1 M to about 0.6 M, from about 0.1 M to about 0.4 M, from about 0.1 M to about 0.3 M, from about 0.2 M to about 1 .2 M, from about 0.2 M to about 1 M, from about 0.2 M to about 0.8 M, from about 0.2 M to about 0.6 M, from about 0.2 M to about 0.4 M, from about 0.4 M to about 1.2 M, from about 0.4 M to about 1 M, from about 0.4 M to about 0.8 M, from about 0.4 M to about 0.6 M, from about 0.6 M to about 1 .2 M, from about 0.6 M to about 1 M, from about 0.8 M to about 1 .2 M.

[0220] In various examples, the sensing device 130 can produce signals in response to nucleic acids derived from a sample being bound to probes coupled to a surface of the sensors 132. A magnitude of the signal can be based on a number of nucleic acids bound to the sensors 132. In one or more illustrative examples, a sample solution can be delivered to the sensing device 130 that includes nucleic acids that have been subjected to the nucleic acid digestion process 126. As the sample is delivered to the sensing device 130, nucleic acids included in the sample solution can become bound to probes on the surface of one or more sensors 132. In at least some examples, the nucleic acids bound to the probes can be modified to include one or6356.002W01 more reporting moieties. The one or more reporting moieties can include fluorophores, magnetic particles, chemical compounds, such as ferrocene, and the like.

[0221] In one or more additional illustrative examples, nucleic acids bound to the surface of the sensors 132 can be labeled using magnetic beads. The magnetic beads can be comprised of metallic materials that have magnetic properties in the presence of a magnetic field. For example, the magnetic beads can be comprised of iron, cobalt, nickel, alloys of iron, alloys of cobalt, alloys of nickel, platinum, alloys of platinum, or one or more combinations thereof. In one or more examples, the magnetic beads can have superparamagnetic properties. In various examples, the magnetic beads can be comprised of iron oxide particles. In one or more further illustrative examples, the magnetic beads can be comprised of Fe2Os or FesCM particles. Additionally, the magnetic beads can be comprised of at least one of iron-platinum particles or cobalt-platinum particles.

[0222] In at least some examples, the magnetic beads can have a spherical shape and have diameters from about 2 nanometers (nm) to about 1000 nm, from about 10 nm to about 800 nm, from about 25 nm to about 600 nm, from about 50 nm to about 400 nm, from about 100 nm to about 200 nm, from about 200 nm to about 300 nm, from about 300 nm to about 400 nm, from about 400 nm to about 500 nm, from about 500 nm to about 600 nm, from about 600 nm to about 700 nm, from about 700 nm to about 800 nm, from about 800 nm to about 900 nm, from about 900 nm to about 1000 nm, from about 10 nm to about 50 nm, from about 10 nm to about 45 nm, from about 10 nm to about 40 nm, from about 10 nm to about 35 nm, from about 10 nm to about 30 nm, from about 10 nm to about 25 nm, from about 10 nm to about 20 nm, from about 15 nm to about 50 nm, from about 15 nm to about 45 nm, from about 15 nm to about 40 nm, from about 15 nm to about 35 nm, from about 15 nm to about 30 nm, from about 15 nm to about 25 nm, from about 20 nm to about 50 nm, from about 20 nm to about 45 nm, from about 20 nm to about 40 nm, from about 20 nm to about 35 nm, from about 20 nm to about 30 nm, from about 25 nm to about 50 nm, from about 25 nm to about 45 nm, from about 25 nm to about 40 nm, from about 25 nm to about 35 nm, from about 30 nm to about 50 nm, from about 30 nm to about 45 nm, from about 30 nm to about 40 nm, from about 35 nm to about 50 nm, from about 35 nm to about 45 nm, or from about 40 nm to about 50 nm.

[0223] The magnetic beads can be amine functionalized and can be labeled with streptavidin. The streptavidin can be bound to the amine functionalized magnetic6356.002W01 beads by thiolating the streptavidin particles and then activating the amine functionalized magnetic beads. The streptavidin particles can be thiolated using a solution including 2-iminothiolane, which can also be known as Traut’s reagent. Additionally, the magnetic beads can be activated using a solution comprising succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 -carboxylate (SMCC). The amine functional groups of the magnetic beads can then be bound to the thiol groups of the modified streptavidin particles. A solution comprising a hybridization buffer and the streptavidin labeled magnetic beads can be delivered to the sensing device 130. In various examples, a number of washes and rinses can be performed at 126 to remove unbound nucleic acids and weakly bound nucleic acids to improve signal quality. Additionally, temperatures within the sensing device 130 can be increased to differentiate between specific and non-specific binding of nucleic acids to probes of the sensors 132.

[0224] At 134, the process 100 can include signal analysis operations. Magnetic beads that are bound to the surface of the sensing device 130 can cause a change in magnetoresistance that can be quantified. For example, a change in resistance detected with respect to individual sensors 132 can be analyzed with respect to a reference resistance. As the difference between the reference resistance and a change in resistance detected in relation to individual sensors 132 increases, the amount of a target analyte detected can also increase. In one or more illustrative examples, the signal analysis performed at 134 can be performed in accordance with implementations described in U.S. patent application number 15 / 734,395, entitled “System and method for processing analyte signals in GMR-based detection of biomarkers”, which is incorporated by reference herein.

[0225] In one or more examples, the sensing device 130 can include one or more magnetic field generators that produces a magnetic field that can activate magnetic particles located near the sensors 132. The sensing device 130 can also include a control unit that controls the opening and closing of valves in the system to control the flow of fluids through the sensing device 130. The control unit can also control the activation of the one or more magnetic field generators. For example, a control unit can be configured to control, among other things, a giant magnetic resistance (GMR) sensor chip and / or a memory chip associated with one or more devices used to perform implementations of the process 100 and a pneumatic system or other fluid supply control system included in one or more devices used to perform6356.002W01 implementations of the process 100 as well as the controls from one or more user interfaces, driving one or more magnetic field generators, and receiving and / or sending signals from / to a sensor chip and / or memory associated with one or more devices used to perform implementations of the process 100, for example.

[0226] One or more devices used to perform implementations of the process 100 can transmit and receive data using wireless or wired technology. Power can be supplied to the devices via a power source in the form of an internal battery or in the form of a connector that receives power via an external source that is connected thereto (e.g., via a cord and a plug). A power source can supply power to parts of the devices, when activated. For example, a power source may supply power to a control unit and a printed circuit board (PCB) assembly, a magnetic field generator, a display device and / or user interface, and a pneumatic system or other fluid control system (including, for example, any motors, valves, and / or pumps associated therewith).

[0227] A pneumatic system or other fluid control system included in the devices can be used to process and prepare a sample by means of moving and directing fluids inside various chambers of the devices (e.g., via a pneumatic connection, through its channels and connecting to direct elastomeric valves). The pneumatic system may be a system and / or device for moving fluid, which could use, for example, plungers and / or pistons in contact with fluids. The magnetic field generator may be an external magnetic coil or other field generating device that is mounted in the devices or integrated in some fashion with one or more of the chips inserted into the analyte detection devices, such as a sample cartridge. The magnetic field generator can be used to stimulate magnetic nanoparticles.

[0228] At 136, the process 100 can include providing an indicator of a biological condition. The indicator of the biological condition can be determined based on a strength of a signal produced by one or more of the sensors 132. In one or more examples, the indicator of the biological condition can indicate the presence of one or more fungi within a sample. In one or more additional examples, the indicator of the biological condition can indicate the presence of one or more bacteria within a sample. In one or more further examples, the indicator of the biological condition can indicate the presence of one or more viruses within a sample. In one or more illustrative examples, individual sensors 132 can correspond to individual fungi, bacteria, and / or viruses. In response to detection of at least a threshold signal at a given sensor 132, the presence of the fungi, bacteria, or virus corresponding to the sensor 132 can be6356.002W01 identified for a sample. Based on determining that one or more analytes are present in a sample, a determination can be made that a biological condition is present in the subject that provided the sample. In situations where one or more fungi, one or more bacteria, one or more viruses, or one or more combinations thereof are present in relation to a sample, a subject providing the sample can be diagnosed with one or more fungal infections, one or more bacterial infections, one or more viral infections, one or more infectious diseases, one or more autoimmune diseases, one or more neurological diseases, one or more types of cancer, one or more types of cardiovascular disease, one or more combinations thereof, and so forth. In one or more illustrative examples, based on the indication of a biological condition being identified by the process 100 with respect to a subject, one or more treatments can be administered to a subject.

[0229] In one or more illustrative examples, process 100 can include providing an apparatus that includes (i) an array of giant magnetoresistance (GMR) sensors 130 and (i) a plurality of chambers for performing amplification of nucleic acids. The process 100 can also include causing a plurality of probes to be attached to the array of GMR sensors 130. The plurality of probes can correspond to a plurality of fungal species. In addition, the process 100 can include causing a first solution to be supplied to a first chamber of the plurality of chambers where the first solution includes a first set of primers that correspond to a first group of fungi included in the plurality of fungal species. Further, the process 100 can include causing a second solution to be supplied to a second chamber of the plurality of chambers. The second solution can include a second set of primers that correspond to a second group of fungi included in the plurality of fungal species, different from the first group of fungi

[0230] In various examples, the process 100 can also include causing a first sample portion to be supplied to the first chamber and a second sample portion to be supplied to the second chamber. The first sample portion can include first nucleic acids and the second sample portion including second nucleic acids. In one or more examples, the first sample portion and the second sample portion can be subsamples obtained from a single sample obtained from a subject. In one or more additional examples, the first sample portion and the second sample portion can correspond to multiple samples obtained from a subject. Additionally, the process 100 can include performing a first amplification process in the first chamber to produce a first amplification product that includes first double stranded amplicons and performing a6356.002W01 second amplification process in the second chamber to produce a second amplification product that includes second double stranded amplicons. In at least some examples, the second amplification process can be performed at least one of concurrently or simultaneously with respect to the first amplification process.

[0231] In still other examples, the process 100 can include causing the first amplification product and the second amplification product to be supplied to a chamber that includes the array of GMR sensors 130. The process 100 can also include determining a change in electrical signals produced by at least one GMR sensor 132 of the array of GMR sensors 130 based on a first number nucleic acids derived from the first double stranded amplicons coupled to one or more first probes attached to the at least one GMR sensor 132 or a second number derived from the second double stranded amplicons coupled to one or more second probes attached to the at least one GMR sensor 132, the one or more first probes corresponding to the first group of fungi and the one or more second probes corresponding to the second group of fungi.

[0232] In one or more illustrative examples, individual GMR sensors 132 of the array of GMR sensors 130 can have probes with nucleic acid sequences corresponding to an individual fungal species included in the first group of fungi or the second group of fungi. In one or more additional illustrative examples, individual first fungal species included in the first group of fungi have no greater than about 80% no greater than about 70%, no greater than about 60%, no greater than about 50%, no greater than about 40%, no greater than about 30%, no greater than about 20%, or no greater than about 10% homology with respect to one another. Individual second fungal species included in the second group of fungi have no greater than about 80%, no greater than about 70%, no greater than about 60%, no greater than about 50%, no greater than about 40%, no greater than about 30%, no greater than about 20%, or no greater than about 10% homology with respect to one another.

[0233] In one or more further illustrative examples, the first group of primers and the second group of primers can be included in a plurality of primer sets that include a third group of primers and a fourth group of primers. The first group of primers can correspond to first genomic regions of a first group of fungi including Aspergillus flavus, Candida albicans, Candida auris, and Candida glabrata, Scedosporium spp, the second group of primers can correspond to second genomic regions of a second group of fungi including Aspergillus fumigatus, Aspergillus niger, Candida krusei, and Cryptococcus, the third group of primers can correspond to third genomic regions of a6356.002W01 third group of fungi including Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, and Fusarium solani, and the fourth group of primers can correspond to fourth genomic regions of a fourth group of fungi including Candida tropicalis, Coccidioides, Histoplasma capulatun, Pneumocystis jirovecii, Mucorales, and Blastomyces.

[0234] Figure 2 is a diagrammatic representation of an example process 200 of delivering a number of solutions across a sensor array 202 comprised of a number of sensors 204 to cause nucleic acids that correspond to target sequences to bind to a surface of the sensor array, according to one or more example implementations. In one or more illustrative examples, the sensor array 202 and the sensors 204 can correspond to the sensing device 130 and the sensors 132 described in relation to Figure 1 .

[0235] The process 200 can include, at 206, sample delivery. Sample delivery can include providing a sample solution 208 including nucleic acids to the sensor array 202. In one or more examples, the sample solution 208 can include nucleic acids that correspond to biotinylated strands produced by a nucleic acid amplification process and a nucleic acid digestion process, such as the nucleic acid amplification process 120 and the nucleic acid digestion process 126 described in relation to Figure 1. The sample solution 208 can include one or more salts and one or more additional components. For example, the sample solution 208 can include at least one of NaCI, KCI, and MgCh The sample solution 208 can also include one or more surfactants. To illustrate, the sample solution 208 can include one or more non-ionic surfactants. In one or more illustrative examples, the sample solution 208 can include Tween 20. In still other examples, the sample solution 208 can include gelatin. In one or more further examples, the sample solution 208 can include a glycine-KOH compound. In various examples, delivering the sample solution 208 to the sensor array 202 can result in nucleic acids including target sequences being bound to probes located on the sensors 204.

[0236] In at least some illustrative examples, the sample solution 208 can comprise NaCI in concentrations from 200 mM to 300 mM, from 200 mM to 280 mM, from 200 mM to 260 mM, from 200 mM to 240 mM, from 220 mM to 300 mM, from 220 mM to 280 mM, from 220 mM to 260 mM, from 240 mM to 300 mM, from 240 mM to 280 mM, or from 260 mM to 300 mM. Additionally, the sample solution 208 can comprise KCI in concentrations from 10 mM to 100 mM, from 10 mM to 90 mM, from6356.002W0110 mM to 80 mM, from 10 mM to 70 mM, from 10 mM to 60 mM, from 10 mM to 50 mM, from 10 mM to 40 mM, from 10 mM to 30 mM, from 20 mM to 100 mM, from 20 mM to 90 mM, from 20 mM to 80 mM, from 20 mM to 70 mM, from 20 mM to 60 mM, from 20 mM to 50 mM, from 20 mM to 40 mM, from 30 mM to 100 mM, from 30 mM to 90 mM, from 30 mM to 80 mM, from 30 mM to 70 mM, from 30 mM to 60 mM, from 30 mM to 50 mM, from 40 mM to 100 mM, from 40 mM to 90 mM, from 40 mM to 80 mM, from 40 mM to 70 mM, from 40 mM to 60 mM, from 50 mM to 100 mM, from 50 mM to 90 mM, from 50 mM to 80 mM, from 50 mM to 70 mM, from 60 mM to 100 mM, from 60 mM to 90 mM, from 60 mM to 80 mM, from 70 mM to 100 mM, from 70 mM to 90 mM, or from 80 mM to 100 mM. Further, the sample solution 208 can comprise MgCl2 in concentrations from 0.5 mM to 5 mM, from 0.5 mM to 4.5 mM, from 0.5 mM to 4 mM, from 0.5 mM to 3.5 mM, from 0.5 mM to 3 mM, from 0.5 mM to 2.5 mM, from 0.5 mM to 2 mM, from 0.5 mM to 1.5 mM, from 0.5 mM to 1 mM, from 0.8 mM to 5 mM, from 0.8 mM to 4.5 mM, from 0.8 mM to 4 mM, from 0.8 mM to 3.5 mM, from 0.8 mM to 3 mM, from 0.8 mM to 2.5 mM, from 0.8 mM to 2 mM, from 0.8 mM to 1 .5 mM, from 1 mM to 5 mM, from 1 mM to 4.5 mM, from 1 mM to 4 mM, from 1 mM to 3.5 mM, from 1 mM to 3 mM, from 1 mM to 2.5 mM, from 1 mM to 2 mM, from 1 mM to 1 .5 mM, from 1 .5 mM to 5 mM, from 1 .5 mM to 4.5 mM, from 1 .5 mM to 4 mM, from 1 .5 mM to 3.5 mM, from 1 .5 mM to 3 mM, from 1 .5 mM to 2.5 mM, from 1 .5 mM to 2 mM, from 2 mM to 5 mM, from 2 mM to 4.5 mM, from 2 mM to 4 mM, from 2 mM to 3.5 mM, from 2 mM to 3 mM, from 2 mM to 2.5 mM, from 2.5 mM to 5 mM, from 2.5 mM to 4.5 mM, from 2.5 mM to 4 mM, from 2.5 mM to 3.5 mM, from 2.5 mM to 3 mM, from 3 mM to 5 mM, from 3.5 mM to 4.5 mM, from 3.5 mM to 4 mM, from 4 mM to 5 mM, or from 4 mM to 4.5 mM.

[0237] In still other examples, the sample solution 208 can include from about 0.010% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.010% by weight to about 0.035% by weight of one or more nonionic surfactants, from about 0.010% by weight to about 0.030% by weight of one or more nonionic surfactants, from about 0.010% by weight to about 0.025% by weight of one or more nonionic surfactants, from about 0.010% by weight to about 0.020% by weight of one or more nonionic surfactants, from about 0.010% by weight to about 0.015% by weight of one or more nonionic surfactants, from about 0.015% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.015% by weight to about 0.035% by weight of one or more nonionic surfactants, from about 0.015%6356.002W01 by weight to about 0.030% by weight of one or more nonionic surfactants, from about 0.015% by weight to about 0.025% by weight of one or more nonionic surfactants, from about 0.015% by weight to about 0.020% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.035% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.030% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.025% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.035% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.030% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.040% by weight of one or more nonionic surfactants, or from about 0.035% by weight to about 0.040% by weight of one or more nonionic surfactants. In one or more additional examples, the sample solution 208 can include from about 0.0005% by weight to about 0.0015% by weight gelatin, from about 0.0005% by weight to about 0.0012% by weight gelatin, from about 0.0005% by weight to about 0.0010% by weight gelatin, from about 0.0005% by weight to about 0.0008% by weight gelatin, from about 0.0008% by weight to about 0.0015% by weight gelatin, from about 0.0008% by weight to about 0.0012% by weight gelatin, from about 0.0008% by weight to about 0.0010% by weight gelatin, from about 0.0010% by weight to about 0.0015% by weight gelatin, or from about 0.010% by weight to about 0.0012% by weight gelatin. In one or more further examples, the sample solution 208 can include glycine-KOH in concentrations from about 50 mM to about 75 mM, from about 50 mM to about 70 mM, from about 50 mM to about 65 mM, from about 50 mM to about 60 mM, from about 55 mM to about 75 mM, from about 55 mM to about 70 mM, from about 55 mM to about 65 mM, from about 55 mM to about 60 mM, from about 60 mM to about 75 mM, from about 60 mM to about 70 mM, from about 60 mM to about 65 mM, from about 65 mM to about 75 mM, from about 65 mM to about 70 mM, or from about 70 mM to about 75 mM.

[0238] In at least some examples, the sample solution 208 can be delivered to the sensor array 202 at flow rate from about 2 microliters (pL) I minute to about 10 pL / minute, from about 2 pL / minute to about 8 pL / minute, from about 2 pL / minute to about 6 pL / minute, from about 2 pL / minute to about 4 pL / minute, from about 4 pL / minute to about 10 pL / minute, from about 4 pL / minute to about 8 pL / minute, from6356.002W01 about 4 pL / minute to about 6 pL / minute, from about 6 pL / minute to about 10 pL / minute, from about 6 pL / minute to about 8 pL / minute, or from about 8 pL / minute to about 10 pL / minute. In addition, the sample solution 208 can be delivered to the sensor array 202 at temperatures from about 40 °C to about 80 °C, from about 40 °C to about 75 °C, from about 40 °C to about 70 °C, from about 40 °C to about 65 °C, from about 40 °C to about 60 °C, from about 40 °C to about 55 °C, from about 40 °C to about 50 °C, from about 45 °C to about 80 °C, from about 45 °C to about 75 °C, from about 45 °C to about 70 °C, from about 45 °C to about 65 °C, from about 45 °C to about 60 °C, from about 45 °C to about 55 °C, from about 45 °C to about 50 °C, from about 50 °C to about 80 °C, from about 50 °C to about 75 °C, from about 50 °C to about 70 °C, from about 50 °C to about 65 °C, from about 50 °C to about 60 °C, from about 50 °C to about 55 °C, from about 55 °C to about 80 °C, from about 55 °C to about 75 °C, from about 55 °C to about 70 °C, from about 55 °C to about 65 °C, from about 55 °C to about 60 °C, from about 60 °C to about 80 °C, from about 60 °C to about 75 °C, from about 60 °C to about 70 °C, from about 60 °C to about 65 °C, from about 65 °C to about 80 °C, from about 65 °C to about 75 °C, from about 65 °C to about 70 °C, from about 70 °C to about 80 °C, from about 70 °C to about 75 °C, or from about 75 °C to about 80 °C.

[0239] The process 200 can include, at 212, a sample wash. The sample wash can include delivering a wash solution 214 to the sensor array 202. In one or more examples, the wash solution 214 can include one or more salts. For example, the wash solution 214 can include NaCI. Additionally, the wash solution 214 can include an amount of bovine serum albumin (BSA). The wash solution 214 can also include one or more surfactants. To illustrate, the wash solution 214 can include one or more nonionic surfactants. In one or more illustrative examples, the wash solution 214 can include Tween 20. In still other examples, the wash solution 214 can include NaNs. In various examples, delivering the wash solution 214 to the sensor array 202 can result in removing unbound nucleic acids included in the wash solution 214 from the sensor array 202.

[0240] In at least some illustrative examples, the wash solution 214 can comprise NaCI in concentrations from 400 mM to 600 mM, from 400 mM to 575 mM, from 400 mM to 550 mM, from 400 mM to 525 mM, from 400 mM to 500 mM, from 400 mM to 475 mM, from 400 mM to 450 mM, from 400 mM to 425 mM, from 425 mM to 600 mM, from 425 mM to 575 mM, from 425 mM to 550 mM, from 425 mM to 5256356.002W01 mM, from 425 mM to 500 mM, from 425 mM to 475 mM, from 425 mM to 450 mM, from 450 mM to 600 mM, from 450 mM to 575 mM, from 450 mM to 550 mM, from 450 mM to 525 mM, from 450 mM to 500 mM, from 450 mM to 475 mM, from 500 mM to 600 mM, from 500 mM to 575 mM, from 500 mM to 550 mM, from 500 mM to 525 mM, from 525 mM to 600 mM, from 525 mM to 575 mM, from 525 mM to 550 mM, from 550 mM to 600 mM, from 550 mM to 575 mM, or from 575 mM to 600. Additionally, the wash solution 214 can comprise from about 0.020% by weight to about 0.075% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.045% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.035% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.030% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.050% from about 0.025% by weight to about 0.045% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.035% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.045% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.065% by weight of one or more nonionic surfactants, from about6356.002W010.035% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.045% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.050% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.050% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.050% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.050% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.055% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.055% by weight to about 0.065% by weight of one or more nonionic surfactants, or from about 0.060% by weight to about 0.070% by weight of one or more nonionic surfactants. In one or more additional examples, the wash solution 214 can include from about 0.05% by weight to about 0.15% by weight NaNs, from about 0.05% by weight to about 0.12% by weight NaNs, from about 0.05% by weight to about 0.10% by weight NaNs, from about 0.05% by weight to about 0.08% by weight NaNs, from about 0.08% by weight to about 0.15% by weight NaNs, from about 0.08% by weight to about 0.12% by weight NaNs, from about 0.08% by weight to about 0.10% by weight NaNs, from about 0.10% by weight to about 0.15% by weight NaNs, or from about 0.10% by weight to about 0.12% by weight NaNs. In one or more further examples, the wash solution 214 can include from about 0.5% by weight to about 1 .5% by weight BSA, from about 0.5% by weight to about 1.2% by weight BSA, from about 0.5% by weight to about 1 % by weight BSA, from about 0.5% by weight to about 0.8% by weight6356.002W01BSA, from about 0.8% by weight to about 1.5% by weight BSA, from about 0.8% by weight to about 1 .2% by weight BSA, from about 0.8% by weight to about 1 % by weight BSA, from about 1 % by weight to about 1.5% by weight BSA, or from about 1 % by weight to about 1 .2% by weight BSA.

[0241] In at least some examples, the wash solution 214 can be delivered to the sensor array 202 at flow rate from about 15 pL / minute to about 40 pL / minute, from about 15 pL / minute to about 35 pL / minute, from about 15 pL / minute to about 30 pL / minute, from about 15 pL / minute to about 25 pL / minute, from about 15 pL / minute to about 20 pL / minute, from about 20 pL / minute to about 40 pL / minute, from about 20 pL / minute to about 35 pL / minute, from about 20 pL / minute to about 30 pL / minute, from about 20 pL / minute to about 25 pL / minute, from about 25 pL / minute to about 40 pL / minute, from about 25 pL / minute to about 35 pL / minute, from about 25 pL / minute to about 30 pL / minute, from about 30 pL / minute to about 40 pL / minute, from about 30 pL / minute to about 35 pL / minute, or from about 35 pL / minute to about 40 pL / minute. In addition, the wash solution 214 can be delivered to the sensor array 202 at temperatures from about 40 °C to about 80 °C, from about 40 °C to about 75 °C, from about 40 °C to about 70 °C, from about 40 °C to about 65 °C, from about 40 °C to about 60 °C, from about 40 °C to about 55 °C, from about 40 °C to about 50 °C, from about 45 °C to about 80 °C, from about 45 °C to about 75 °C, from about 45 °C to about 70 °C, from about 45 °C to about 65 °C, from about 45 °C to about 60 °C, from about 45 °C to about 55 °C, from about 45 °C to about 50 °C, from about 50 °C to about 80 °C, from about 50 °C to about 75 °C, from about 50 °C to about 70 °C, from about 50 °C to about 65 °C, from about 50 °C to about 60 °C, from about 50 °C to about 55 °C, from about 55 °C to about 80 °C, from about 55 °C to about 75 °C, from about 55 °C to about 70 °C, from about 55 °C to about 65 °C, from about 55 °C to about 60 °C, from about 60 °C to about 80 °C, from about 60 °C to about 75 °C, from about 60 °C to about 70 °C, from about 60 °C to about 65 °C, from about 65 °C to about 80 °C, from about 65 °C to about 75 °C, from about 65 °C to about 70 °C, from about 70 °C to about 80 °C, from about 70 °C to about 75 °C, or from about 75 °C to about 80 °C.

[0242] The process 200 can include, at 218, a bead delivery process. The bead delivery process can include delivering a bead delivery solution 220 to the sensor array 202. In one or more examples, the bead delivery solution 220 can include a mannitol containing solution, an HPBCD-containing solution, and a solution comprising6356.002W01 magnetic beads Delivering the bead delivery solution 220 to the sensor array 202 can result in, at 222, the magnetic beads binding to biotin labeled amplicons bound to the sensors 204. In one or more illustrative examples, at least a portion of the components of the bead delivery solution 220 can be lyophilized before being delivered to the sensor array 202. In various examples, the lyophilized components can be provided as beads that are reconstituted and used to form the bead delivery solution 220.

[0243] In one or more examples, the lyophilized components used to produce the bead delivery solution 220 can be included in a bead having a volume from about 2 pL to about 20 pL, from about 4 pL to about 18 pL, from about 6 pL to about 16 pL, from about 8 pL to about 14 pL, from about 5 pL to about 15 pL, from about 10 pL to about 20 pL or from about 2 pL to about 10 pL. In one or more examples, the lyophilized bead can include a component comprised of a HPBCD solution having from about 20% by weight to about 40% by weight HPBCD, from about 25% by weight to about 35% by weight HPBCD, or from about 20% by weight to about 30% by weight HPBCD. Additionally, the lyophilized bead can include a component comprised of a mannitol solution having from about 20% by weight to about 40% by weight mannitol, from about 25% by weight to about 35% by weight mannitol, or from about 20% by weight to about 30% by weight mannitol.

[0244] In various examples, the lyophilized bead can include from about 20% by volume to about 40% by volume of the HPBCD solution, from about 20% by volume to about 35% by volume of the HPBCD solution, from about 20% by volume to about 30% by volume of the HPBCD solution, from about 20% by volume to about 25% by volume of the HPBCD solution, from about 25% by volume to about 40% by volume of the HPBCD solution, from about 25% by volume to about 35% by volume of the HPBCD solution, from about 25% by volume to about 30% by volume of the HPBCD solution, from about 30% by volume to about 40% by volume of the HPBCD solution, or from about 35% by volume to about 40% by volume of the HPBCD solution.

[0245] In addition, the lyophilized bead can include from about 20% by volume to about 40% by volume of the mannitol solution, from about 20% by volume to about 35% by volume of the mannitol solution, from about 20% by volume to about 30% by volume of the mannitol solution, from about 20% by volume to about 25% by volume of the mannitol solution, from about 25% by volume to about 40% by volume of the mannitol solution, from about 25% by volume to about 35% by volume of the mannitol solution, from about 25% by volume to about 30% by volume of the mannitol solution,6356.002W01 from about 30% by volume to about 40% by volume of the mannitol solution, or from about 35% by volume to about 40% by volume of the mannitol solution.

[0246] Further, the lyophilized bead can include from about 0.5% by volume to about 2% by volume of the magnetic beads, from about 0.5% by volume to about 1 .8% by volume of the magnetic beads, from about 0.5% by volume to about 1.5% by volume of the magnetic beads, from about 0.5% by volume to about 1 .2% by volume of the magnetic beads, from about 0.5% by volume to about 1 % by volume of the magnetic beads, from about 0.5% by volume to about 0.8% by volume of the magnetic beads, from about 0.8% by volume to about 2% by volume of the magnetic beads, from about 0.8% by volume to about 1 .8% by volume of the Taq polymerase solution magnetic beads, from about 0.8% by volume to about 1 .5% by volume of the magnetic beads, from about 0.8% by volume to about 1.2% by volume of the magnetic beads, from about 1 % by volume to about 2% by volume of the magnetic beads, from about 1 % by volume to about 1.8% by volume of the magnetic beads, from about 1 % by volume to about 1 .5% by volume of the magnetic beads, from about 1 .2% by volume to about 2% by volume of the magnetic beads, from about 1.2% by volume to about 1.8% by volume of the magnetic beads, or from about 1.5% by volume to about 2% by volume of the magnetic beads.

[0247] In one or more examples, at least one of the mannitol solution, the HPBCD solution, or the magnetic beads solution can include an amount of water. In at least some examples, the lyophilized bead can include an amount of water that is in addition to the amount of water included in at least one of the mannitol solution, the HPBCD solution, and the magnetic bead solution. For example, the bead can include from about 2% by volume to about 15% by volume of additional water, from about 2% by volume to about 12% by volume of additional water, from about 2% by volume to about 10% by volume of additional water, from about 2% by volume to about 8% by volume of additional water, from about 2% by volume to about 6% volume of additional water, from about 2% by volume to about 4% by volume of additional water, from about 5% by volume to about 15% by volume of additional water, from about 5% by volume to about 12% by volume of additional water, from about 5% by volume to about 10% by volume of additional water, from about 5% by volume to about 8% by volume of additional water, from about 8% by volume to about 15% by volume of additional water, from about 8% by volume to about 12% by volume of additional water, from about 8% by volume to about 10% by volume of additional water, from about 10% by volume to6356.002W01 about 15% by volume of additional water, from about 10% by volume to about 12% by volume of additional water, or from about 12% by volume to about 15% by volume of additional water.

[0248] In at least some examples, the bead delivery solution 220 can be delivered to the sensor array 202 at flow rate from about 2 pL / m inute to about 25 pL / minute, from about 2 pL / minute to about 20 pL / minute, from about 2 pL / minute to about 15 pL / minute, from about 2 pL / minute to about 10 pL / minute, from about 2 pL / minute to about 5 pL / minute, from about 5 pL / minute to about 25 pL / minute, from about 5 pL / minute to about 20 pL / minute, from about 5 pL / minute to about 25 pL / minute, from about 5 pL / minute to about 20 pL / minute, from about 5 pL / minute to about 15 pL / minute, from about 5 pL / minute to about 8 pL / minute, from about 10 pL / minute to about 25 pL / minute, from about 10 pL / minute to about 20 pL / minute, from about 10 pL / minute to about 15 pL / minute, from about 15 to about 25 pL / minute , from about 15 pL / minute to about 20 pL / minute, or from about 20 pL / minute to about 40 25 / minute. In addition, the sample solution 208 can be delivered to the sensor array 202 at temperatures from about 40 °C to about 80 °C, from about 40 °C to about 75 °C, from about 40 °C to about 70 °C, from about 40 °C to about 65 °C, from about 40 °C to about 60 °C, from about 40 °C to about 55 °C, from about 40 °C to about 50 °C, from about 45 °C to about 80 °C, from about 45 °C to about 75 °C, from about 45 °C to about 70 °C, from about 45 °C to about 65 °C, from about 45 °C to about 60 °C, from about 45 °C to about 55 °C, from about 45 °C to about 50 °C, from about 50 °C to about 80 °C, from about 50 °C to about 75 °C, from about 50 °C to about 70 °C, from about 50 °C to about 65 °C, from about 50 °C to about 60 °C, from about 50 °C to about 55 °C, from about 55 °C to about 80 °C, from about 55 °C to about 75 °C, from about 55 °C to about 70 °C, from about 55 °C to about 65 °C, from about 55 °C to about 60 °C, from about 60 °C to about 80 °C, from about 60 °C to about 75 °C, from about 60 °C to about 70 °C, from about 60 °C to about 65 °C, from about 65 °C to about 80 °C, from about 65 °C to about 75 °C, from about 65 °C to about 70 °C, from about 70 °C to about 80 °C, from about 70 °C to about 75 °C, or from about 75 °C to about 80 °C.

[0249] The process 200 can include, at 224, performing a bead wash using a bead wash solution 226. Delivering the bead wash solution 226 to the sensor array 202 can cause, at 228, weakly bound nucleic acids to be removed from the sensory array 202. In one or more examples, the weakly bound nucleic acids may not have6356.002W01 magnetic beads attached. In one or more additional examples, the weakly bound nucleic acids can have magnetic beads attached. In one or more illustrative examples, the bead wash solution 226 can include one or more salts. For example, the bead wash solution 226 can include NaCI. Additionally, the bead wash solution 226 can include an amount of bovine serum albumin (BVA). The bead wash solution 226 can also include one or more surfactants. To illustrate, the bead wash solution 226 can include one or more non-ionic surfactants. In one or more illustrative examples, the bead wash solution 226 can include Tween 20. In still other examples, the bead wash solution 226 can include NaNs.

[0250] In at least some illustrative examples, the bead wash solution 226 can comprise NaCI in concentrations from 400 mM to 600 mM, from 400 mM to 575 mM, from 400 mM to 550 mM, from 400 mM to 525 mM, from 400 mM to 500 mM, from 400 mM to 475 mM, from 400 mM to 450 mM, from 400 mM to 425 mM, from 425 mM to 600 mM, from 425 mM to 575 mM, from 425 mM to 550 mM, from 425 mM to 525 mM, from 425 mM to 500 mM, from 425 mM to 475 mM, from 425 mM to 450 mM, from 450 mM to 600 mM, from 450 mM to 575 mM, from 450 mM to 550 mM, from 450 mM to 525 mM, from 450 mM to 500 mM, from 450 mM to 475 mM, from 500 mM to 600 mM, from 500 mM to 575 mM, from 500 mM to 550 mM, from 500 mM to 525 mM, from 525 mM to 600 mM, from 525 mM to 575 mM, from 525 mM to 550 mM, from 550 mM to 600 mM, from 550 mM to 575 mM, or from 575 mM to 600. Additionally, the bead wash solution 226 can comprise from about 0.020% by weight to about 0.075% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.045% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.035% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.030% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.055% by weight of one or more nonionic surfactants,6356.002W01 from about 0.025% by weight to about 0.050% from about 0.025% by weight to about 0.045% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.035% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.045% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.045% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.050% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.050% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.050% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.050% by weight to about 0.055% by weight of one or more nonionic surfactants, from about6356.002W010.055% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.055% by weight to about 0.065% by weight of one or more nonionic surfactants, or from about 0.060% by weight to about 0.070% by weight of one or more nonionic surfactants. In one or more additional examples, the bead wash solution 226 can include from about 0.05% by weight to about 0.15% by weight NaNs, from about 0.05% by weight to about 0.12% by weight NaNs, from about 0.05% by weight to about 0.10% by weight NaNs, from about 0.05% by weight to about 0.08% by weight NaNs, from about 0.08% by weight to about 0.15% by weight NaNs, from about 0.08% by weight to about 0.12% by weight NaNs, from about 0.08% by weight to about 0.10% by weight NaNs, from about 0.10% by weight to about 0.15% by weight NaNs, or from about 0.10% by weight to about 0.12% by weight NaNs. In one or more further examples, the bead wash solution 226 can include from about 0.5% by weight to about 1 .5% by weight BSA, from about 0.5% by weight to about 1 .2% by weight BSA, from about 0.5% by weight to about 1 % by weight BSA, from about 0.5% by weight to about 0.8% by weight BSA, from about 0.8% by weight to about 1 .5% by weight BSA, from about 0.8% by weight to about 1.2% by weight BSA, from about 0.8% by weight to about 1 % by weight BSA, from about 1 % by weight to about 1.5% by weight BSA, or from about 1 % by weight to about 1 .2% by weight BSA.

[0251] In at least some examples, the bead wash solution 226 can be delivered to the sensor array 202 at flow rate from about 2 pL / m inute to about 25 pL / minute, from about 2 pL / minute to about 20 pL / minute, from about 2 pL / minute to about 15 pL / minute, from about 2 pL / minute to about 10 pL / minute, from about 2 pL / minute to about 5 pL / minute, from about 5 pL / minute to about 25 pL / minute, from about 5 pL / minute to about 20 pL / minute, from about 5 pL / minute to about 25 pL / minute, from about 5 pL / minute to about 20 pL / minute, from about 5 pL / minute to about 15 pL / minute, from about 5 pL / minute to about 8 pL / minute, from about 10 pL / minute to about 25 pL / minute, from about 10 pL / minute to about 20 pL / minute, from about 10 pL / minute to about 15 pL / minute, from about 15 to about 25 pL / minute , from about 15 pL / minute to about 20 pL / minute, or from about 20 pL / minute to about 40 25 / minute. In addition, the bead wash solution 226 can be delivered to the sensor array 202 at temperatures from about 40 °C to about 80 °C, from about 40 °C to about 75 °C, from about 40 °C to about 70 °C, from about 40 °C to about 65 °C, from about 40 °C to about 60 °C, from about 40 °C to about 55 °C, from about 40 °C to about 50 °C, from about 45 °C to about 80 °C, from about 45 °C to about 75 °C, from about 45 °C to6356.002W01 about 70 °C, from about 45 °C to about 65 °C, from about 45 °C to about 60 °C, from about 45 °C to about 55 °C, from about 45 °C to about 50 °C, from about 50 °C to about 80 °C, from about 50 °C to about 75 °C, from about 50 °C to about 70 °C, from about 50 °C to about 65 °C, from about 50 °C to about 60 °C, from about 50 °C to about 55 °C, from about 55 °C to about 80 °C, from about 55 °C to about 75 °C, from about 55 °C to about 70 °C, from about 55 °C to about 65 °C, from about 55 °C to about 60 °C, from about 60 °C to about 80 °C, from about 60 °C to about 75 °C, from about 60 °C to about 70 °C, from about 60 °C to about 65 °C, from about 65 °C to about 80 °C, from about 65 °C to about 75 °C, from about 65 °C to about 70 °C, from about 70 °C to about 80 °C, from about 70 °C to about 75 °C, or from about 75 °C to about 80 °C.

[0252] The process 200 can include, at 230, performing a final wash using a final wash solution 232. Delivering the final wash solution 232to the sensor array 202 can cause weakly bound nucleic acids to be removed from the sensory array 202. In one or more examples, the weakly bound nucleic acids may not have magnetic beads attached. In one or more additional examples, the weakly bound nucleic acids can have magnetic beads attached. In one or more illustrative examples, the bead wash solution 226 can include one or more salts. For example, the final wash solution 232 can include NaCI. Additionally, the final wash solution 232 can include an amount of bovine serum albumin (BVA). The final wash solution 232 can also include one or more surfactants. To illustrate, the final wash solution 232 can include one or more non-ionic surfactants. In one or more illustrative examples, the final wash solution 232 can include Tween 20. In still other examples, the final wash solution 232 can include NaN3.

[0253] In at least some illustrative examples, the final wash solution 232 can comprise NaCI in concentrations from 400 mM to 600 mM, from 400 mM to 575 mM, from 400 mM to 550 mM, from 400 mM to 525 mM, from 400 mM to 500 mM, from 400 mM to 475 mM, from 400 mM to 450 mM, from 400 mM to 425 mM, from 425 mM to 600 mM, from 425 mM to 575 mM, from 425 mM to 550 mM, from 425 mM to 525 mM, from 425 mM to 500 mM, from 425 mM to 475 mM, from 425 mM to 450 mM, from 450 mM to 600 mM, from 450 mM to 575 mM, from 450 mM to 550 mM, from 450 mM to 525 mM, from 450 mM to 500 mM, from 450 mM to 475 mM, from 500 mM to 600 mM, from 500 mM to 575 mM, from 500 mM to 550 mM, from 500 mM to 525 mM, from 525 mM to 600 mM, from 525 mM to 575 mM, from 525 mM to 550 mM,6356.002W01 from 550 mM to 600 mM, from 550 mM to 575 mM, or from 575 mM to 600. Additionally, the final wash solution 232 can comprise from about 0.020% by weight to about 0.075% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.045% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.035% by weight of one or more nonionic surfactants, from about 0.020% by weight to about 0.030% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.050% from about 0.025% by weight to about 0.045% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.025% by weight to about 0.035% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.045% by weight of one or more nonionic surfactants, from about 0.030% by weight to about 0.040% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.035% by weight to about 0.045% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.070% by weight6356.002W01 of one or more nonionic surfactants, from about 0.040% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.040% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.045% by weight to about 0.050% by weight of one or more nonionic surfactants, from about 0.050% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.050% by weight to about 0.065% by weight of one or more nonionic surfactants, from about 0.050% by weight to about 0.060% by weight of one or more nonionic surfactants, from about 0.050% by weight to about 0.055% by weight of one or more nonionic surfactants, from about 0.055% by weight to about 0.070% by weight of one or more nonionic surfactants, from about 0.055% by weight to about 0.065% by weight of one or more nonionic surfactants, or from about 0.060% by weight to about 0.070% by weight of one or more nonionic surfactants. In one or more additional examples, the final wash solution 232 can include from about 0.05% by weight to about 0.15% by weight NaNs, from about 0.05% by weight to about 0.12% by weight NaNs, from about 0.05% by weight to about 0.10% by weight NaNs, from about 0.05% by weight to about 0.08% by weight NaNs, from about 0.08% by weight to about 0.15% by weight NaNs, from about 0.08% by weight to about 0.12% by weight NaNs, from about 0.08% by weight to about 0.10% by weight NaNs, from about 0.10% by weight to about 0.15% by weight NaNs, or from about 0.10% by weight to about 0.12% by weight NaNs. In one or more further examples, the final wash solution 232 can include from about 0.5% by weight to about 1 .5% by weight BSA, from about 0.5% by weight to about 1 .2% by weight BSA, from about 0.5% by weight to about 1 % by weight BSA, from about 0.5% by weight to about 0.8% by weight BSA, from about 0.8% by weight to about 1 .5% by weight BSA, from about 0.8% by weight to about 1.2% by weight BSA, from about 0.8% by weight to about 1 % by weight BSA, from about 1 % by weight to about 1.5% by weight BSA, or from about 1 % by weight to about 1 .2% by weight BSA.6356.002W01

[0254] In at least some examples, the final wash solution 232 can be delivered to the sensor array 202 at flow rate from about 2 microliters (pL) I minute to about 10 pL / minute, from about 2 pL / minute to about 8 pL / minute, from about 2 pL / minute to about 6 pL / minute, from about 2 pL / minute to about 4 pL / minute, from about 4 pL / minute to about 10 pL / minute, from about 4 pL / minute to about 8 pL / minute, from about 4 pL / minute to about 6 pL / minute, from about 6 pL / minute to about 10 pL / minute, from about 6 pL / minute to about 8 pL / minute, or from about 8 pL / minute to about 10 pL / minute. In addition, the final wash solution 232 can be delivered to the sensor array 202 at temperatures from about 40 °C to about 80 °C, from about 40 °C to about 75 °C, from about 40 °C to about 70 °C, from about 40 °C to about 65 °C, from about 40 °C to about 60 °C, from about 40 °C to about 55 °C, from about 40 °C to about 50 °C, from about 45 °C to about 80 °C, from about 45 °C to about 75 °C, from about 45 °C to about 70 °C, from about 45 °C to about 65 °C, from about 45 °C to about 60 °C, from about 45 °C to about 55 °C, from about 45 °C to about 50 °C, from about 50 °C to about 80 °C, from about 50 °C to about 75 °C, from about 50 °C to about 70 °C, from about 50 °C to about 65 °C, from about 50 °C to about 60 °C, from about 50 °C to about 55 °C, from about 55 °C to about 80 °C, from about 55 °C to about 75 °C, from about 55 °C to about 70 °C, from about 55 °C to about 65 °C, from about 55 °C to about 60 °C, from about 60 °C to about 80 °C, from about 60 °C to about 75 °C, from about 60 °C to about 70 °C, from about 60 °C to about 65 °C, from about 65 °C to about 80 °C, from about 65 °C to about 75 °C, from about 65 °C to about 70 °C, from about 70 °C to about 80 °C, from about 70 °C to about 75 °C, or from about 75 °C to about 80 °C.

[0255] Figure 3 is a diagrammatic representation of a flow diagram of a process 300 to capture nucleic acids having target sequences and analyzing quantitative measures related to the nucleic acids to determine the presence or absence of a biological condition within a subject, according to one or more example implementations.

[0256] At 302, the process 300 can include extracting nucleic acids from a sample. In one or more examples, the sample can include a plasma sample and the nucleic acids can include cell-free DNA molecules. In one or more illustrative examples, one or more peaks of the distribution of lengths of the nucleic acids extracted from the sample can be from about 50 nucleotides to about 500 nucleotides, from about 80 nucleotides to about 450 nucleotides, from about 100 nucleotides to6356.002W01 about 400 nucleotides, from about 120 nucleotides to about 350 nucleotides, from about 150 nucleotides to about 300 nucleotides, from about 180 nucleotides to about 250 nucleotides, from about 50 nucleotides to about 150 nucleotides, from about 100 nucleotides to about 200 nucleotides, from about 150 nucleotides to about 250 nucleotides, from about 200 nucleotides to about 300 nucleotides, from about 100 nucleotides to about 150 nucleotides, from about 150 nucleotides to about 200 nucleotides, from about 200 nucleotides to about 250 nucleotides, or from about 250 nucleotides to about 300 nucleotides. In various additional examples, the sizes of the nucleic acids derived from a sample can be dependent on characteristics of the sample, such as a source of the sample and / or an amount of the sample extracted from the subject.

[0257] In addition, the process 300 can include, at 304, providing the nucleic acids and a plurality of primer sets to one or more containers. The plurality of primer sets can include at least a first primer set and a second primer set selected from two primer groups. The first primer group can be complementary with first genomic regions of a first group of fungi including Aspergillus flavus, Candida albicans, Candida auris, and Candida glabrata, Scedosporium spp, and one or more first control sequence. The second primer group can be complementary with second genomic regions of a second group of fungi including Aspergillus fumigatus, Aspergillus niger, Candida krusei, and Cryptococcus and one or more second control sequences. The third primer group can be complementary with third genomic regions of a third group of fungi including Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, and Fusarium solani and one or more third control sequences. The fourth primer group that are complementary with fourth genomic regions of a fourth group of fungi including Candida tropicalis, Coccidioides, Histoplasma capulatun, Mucorales, Blastomyces, or Pneumocystis jirovecii, and one or more fourth control sequences. In one or more examples, the one or more first control sequences can include one or more Actin ([3- actin, ACTB gene) P3 sequence. In one or more additional examples, the one or more second control sequence can include at least one of one or more RP (Human Ribonuclease P (RNase P) complex ) F3.1 sequences or one or more RP P3 sequences. In one or more further examples, the one or more third control sequences can include one or more RP P1 sequences. In still other examples, the one or more fourth control sequences can include one or more Actin P1 sequences. In various examples, the one or more first control sequences, the one or more second control6356.002W01 sequences, the one or more third control sequences, or the one or more fourth control sequences can include one or more sequences selected from the hg19 human genome.

[0258] The process 300 can include, at 306, performing amplification of a portion of the nucleic acids using the plurality of primer sets to produce an amplification product. The amplification product can include double stranded amplicons of the portion of the nucleic acids complementary to at least two of the first group of fungi, the second group of fungi, the third group of fungi, or the fourth group of fungi corresponding to the first primer set and the second primer set. Individual double stranded amplicons includes a first strand having a first binding group at the 5’ end and a second strand having a second binding group at the 5’ end. In one or more illustrative examples, the first binding group can include a 5’ biotin and the second binding group can include a 5’ phosphate moiety. A group of single stranded nucleic acids can be produced corresponding to the first strands of the double stranded amplicons having the 5’ biotin.

[0259] Further, at 308, the process 300 can include forming a surface of a sensor. The surface of the sensor can include a polymeric coating and a number of probe sequences. Individual probe sequences can include (i) a first section having a nucleotide sequence that is complementary to a genomic region included in the two of the first genomic regions, the second genomic regions, the third genomic regions, or the fourth genomic regions that correspond to the first primer set and the second primer set, and (ii) a second section that includes a linker sequence having at least five nucleotides and a 5’ amino group coupled to the polymeric coating.

[0260] The process 300 can also include, at 310, contacting the surface of the sensor with the group of single stranded nucleic acids to produce a modified surface of the sensor that includes a number of captured single stranded nucleic acids. Individual captured single stranded nucleic acids being bound to an individual probe sequence. In various examples, the surface of the sensor can be contacted with a solution including superparamagnetic streptavidin magnetic particles to produce a number of modified single stranded nucleic acids bound to the modified surface of the sensor. Individual modified single stranded nucleic acids having a streptavidin superparamagnetic particle bound to the 5’ biotin binding group; and

[0261] At 312, the process 300 can include determining an electrical signal that corresponds to a number of the modified single-stranded nucleic acids bound to the6356.002W01 surface of the sensor and, at 314, the process 300 can include determining an indicator of a fungal-related biological condition based on the electrical signal.Capturing Nucleic Acids

[0262] Methods disclosed herein can comprise capturing DNA, such as cfDNA target regions. In one or more examples, the capturing comprises contacting the DNA with primers (e.g., oligonucleotides) specific for the target sequences. Enrichment or capture may be performed on any sample or subsample described herein using any suitable approach known in the art. In various examples, enrichment or capture can be performed after an amplification step.

[0263] In at least some examples, the probes specific for the target regions comprise a capture moiety that facilitates the enrichment or capture of the DNA hybridized to the probes. In one or more examples, the capture moiety is biotin. In some such embodiments, streptavidin attached to a solid support, such as magnetic beads, is used to bind to the biotin. Nonspecifically bound DNA that does not comprise a target region is washed away from the captured DNA. In various examples, DNA is then dissociated from the probes and eluted from the solid support using salt washes or buffers comprising another DNA denaturing agent. In one or more illustrative examples, the probes are also eluted from the solid support by, e.g., disrupting the biotin-streptavidin interaction.

[0264] The capturing step may be performed using conditions suitable for specific nucleic acid hybridization, which generally depend to some extent on features of the probes such as length, base composition, etc. Those skilled in the art will be familiar with appropriate conditions given general knowledge in the art regarding nucleic acid hybridization. In one or more examples, complexes of target-specific probes and DNA are formed.Subjects

[0265] In one or more examples, the DNA (e.g., cfDNA) is obtained from a subject having an infection, transplant rejection, or other disease directly or indirectly affecting the immune system. In various examples, the DNA (e.g., cfDNA) is obtained from a subject suspected of having an infection, transplant rejection, or other disease directly or indirectly affecting the immune system. In some embodiments, the DNA (e.g., cfDNA) is obtained from a subject having an infection. In some embodiments,6356.002W01 the DNA (e.g., cfDNA) is obtained from a subject suspected of having an infection. The present methods can be used to diagnose, prognose, monitor or observe fungal infections, viral infections, bacterial infections, or other diseases.Samples

[0266] A sample can be any biological sample isolated from a subject. A sample can be a bodily sample. Samples can include body tissues, such as known or suspected solid tumors, whole blood, platelets, serum, plasma, stool, red blood cells, white blood cells or leucocytes, endothelial cells, tissue biopsies, cerebrospinal fluid synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid, the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, pleural effusions, cerebrospinal fluid, saliva, mucous, sputum, semen, sweat, urine. Samples are preferably body fluids, particularly blood and fractions thereof, and urine. A sample can be in the form originally isolated from a subject or can have been subjected to further processing to remove or add components, such as cells, or enrich for one component relative to another. Thus, a preferred body fluid for analysis is plasma or serum containing cell-free nucleic acids.

[0267] A sample can be isolated or obtained from a subject and transported to a site of sample analysis. The sample may be preserved and shipped at a desirable temperature, e.g., room temperature, 4°C, -20°C, and / or -80°C. A sample can be isolated or obtained from a subject at the site of the sample analysis. The subject can be a human, a mammal, an animal, a companion animal, a service animal, or a pet. The subject can have a cancer, precancer, infection, transplant rejection, or other disease or disorder related to changes in the immune system. The subject may not have cancer, infection, transplant rejection, or other disease or disorder related to changes in the immune system. The subject may have received one or more treatments for a biological condition. The subject may be in remission. The subject may or may not be diagnosed of having one or more biological conditions related to at least one of a viral infection, a bacterial infection, or a fungal infection.

[0268] In some embodiments, the sample comprises plasma. The volume of plasma obtained can depend on the desired read depth for sequenced regions. Exemplary volumes are 0.4-40 ml, 5-20 ml, 10-20 ml. For examples, the volume can be 0.5 mL, 1 mL, 5 mL 10 mL, 20 mL, 30 mL, or 40 mL. A volume of sampled plasma may be 5 to 20 mL.6356.002W01

[0269] A sample can comprise various amount of nucleic acid that contains genome equivalents. For example, a sample of about 30 ng DNA can contain about 10,000 (104) haploid human genome equivalents and, in the case of cfDNA, about 200 billion (2xlOn) individual polynucleotide molecules. Similarly, a sample of about 100 ng of DNA can contain about 30,000 haploid human genome equivalents and, in the case of cfDNA, about 600 billion individual molecules.

[0270] A sample can comprise nucleic acids from different sources, e.g., from cells and cell-free of the same subject, from cells and cell-free of different subjects. Example amounts of cell-free nucleic acids in a sample before amplification range from about 1 fg to about 1 pg, e.g., 1 pg to 200 ng, 1 ng to 100 ng, 10 ng to 1000 ng. For example, the amount can be up to about 600 ng, up to about 500 ng, up to about 400 ng, up to about 300 ng, up to about 200 ng, up to about 100 ng, up to about 50 ng, or up to about 20 ng of cell-free nucleic acid molecules. The amount can be at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of cell- free nucleic acid molecules. The amount can be up to 1 femtogram (fg), 10 fg, 100 fg, 1 picogram (pg), 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng, or 200 ng of cell-free nucleic acid molecules. The method can comprise obtaining 1 femtogram (fg) to 200 ng cell-free nucleic acid molecules. Cell-free nucleic acids are nucleic acids not contained within or otherwise bound to a cell or in other words nucleic acids remaining in a sample after removing intact cells. Cell- free nucleic acids include DNA, RNA, and hybrids thereof, including genomic DNA, mitochondrial DNA, siRNA, miRNA, circulating RNA (cRNA), tRNA, rRNA, small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), long non-coding RNA (long ncRNA), or fragments of any of these. Cell- free nucleic acids can be double-stranded, single-stranded, or a hybrid thereof. A cell- free nucleic acid can be released into bodily fluid through secretion or cell death processes, e.g., cellular necrosis and apoptosis. Some cell-free nucleic acids are released into bodily fluid from cancer cells e.g., circulating tumor DNA, (ctDNA). Others are released from healthy cells. In some embodiments, cfDNA is cell-free fetal DNA (cffDNA) In some embodiments, cell free nucleic acids are produced by tumor cells. In some embodiments, cell free nucleic acids are produced by a mixture of tumor cells and non-tumor cells.

[0271] Cell-free nucleic acids have an exemplary size distribution of about 100- 500 nucleotides, with molecules of 110 to about 230 nucleotides representing about6356.002W0190% of molecules, with a mode of about 168 nucleotides and a second minor peak in a range between 240 to 440 nucleotides.

[0272] Cell-free nucleic acids can be isolated from bodily fluids through a fractionation step in which cell-free nucleic acids, as found in solution, are separated from intact cells and other non-soluble components of the bodily fluid. Partitioning may include techniques such as centrifugation or filtration. Alternatively, cells in bodily fluids can be lysed and cell-free and cellular nucleic acids processed together. Generally, after addition of buffers and wash steps, nucleic acids can be precipitated with an alcohol. Further clean up steps may be used such as silica-based columns to remove contaminants or salts. Non-specific bulk carrier nucleic acids, such as C 1 DNA, DNA or protein for bisulfite sequencing, hybridization, and / or ligation, may be added throughout the reaction to optimize certain aspects of the procedure such as yield.

[0273] After such processing, samples can include various forms of nucleic acid including double stranded DNA, single stranded DNA, and single stranded RNA. In some embodiments, single stranded DNA and RNA can be converted to double stranded forms so they are included in subsequent processing and analysis steps. Typically, double-stranded molecules are blunt ended by treatment with a polymerase with a 5'-3' polymerase and a 3 '-5' exonuclease (or proof-reading function), in the presence of all four standard nucleotides. Klenow large fragment and T4 polymerase are examples of suitable polymerase.Amplification

[0274] Amplification is typically primed by primers that anneal or bind to primer binding sites in adapters flanking a DNA molecule to be amplified. Amplification methods can involve cycles of denaturation, annealing and extension, resulting from thermocycling or can be isothermal as in transcription-mediated amplification. Other amplification methods include the ligase chain reaction, strand displacement amplification, nucleic acid sequence-based amplification, and self-sustained sequence-based replication.Capture moieties

[0275] As discussed above, nucleic acids in a sample can be subject to a capture step, in which molecules having target regions are captured for subsequent analysis. Target capture can involve use of probes (e.g., oligonucleotides) labeled with6356.002W01 a capture moiety, such as biotin, and a second moiety or binding partner that binds to the capture moiety, such as streptavidin.

[0276] Capture moieties include, without limitation, biotin, avidin, streptavidin, a nucleic acid comprising a particular nucleotide sequence, a hapten recognized by an antibody, and magnetically attractable particles. The extraction moiety can be a member of a binding pair, such as biotin / streptavidin or hapten / antibody. In some embodiments, a capture moiety that is attached to an analyte is captured by its binding pair which is attached to an isolatable moiety, such as a magnetically attractable particle or a large particle that can be sedimented through centrifugation. The capture moiety can be any type of molecule that allows affinity separation of nucleic acids bearing the capture moiety from nucleic acids lacking the capture moiety. Exemplary capture moieties are biotin which allows affinity separation by binding to streptavidin linked or linkable to a solid phase or an oligonucleotide, which allows affinity separation through binding to a complementary oligonucleotide linked or linkable to a solid phase.Customized Therapies and Related Administrations

[0277] In some implementations, the methods disclosed herein relate to identifying and administering therapies to patients having a given disease, disorder or condition. In certain implementations, the customized therapies described herein are typically administered parenterally (e.g., intravenously or subcutaneously). Pharmaceutical compositions containing the immunotherapeutic agent are typically administered intravenously. Certain therapeutic agents are administered orally. However, customized therapies (e.g., immunotherapeutic agents, etc.) may also be administered by any method known in the art, including, for example, buccal, sublingual, rectal, vaginal, intraurethral, topical, intraocular, intranasal, and / or intraarticular, which administration may include tablets, capsules, granules, aqueous suspensions, gels, sprays, suppositories, salves, ointments, or the like.

[0278] Treatments for fungal patients can be provided to patients having a number of conditions, such as patients with compromised immune systems. This can include patients having autoimmune disorders, patients receiving immunosuppressants, and patients that have undergone organ transplants. Example treatments can include echinocandins, such as caspofungin, micafungin, and6356.002W01 anidulafungin. Other example treatments for fungal infections can include lipid formulations of amphotericin B. Additional example treatments of fungal infections can include fluconazole, including oral forms of fluconazole. In still other examples, treatments for fungal infections can include at least one of voriconazole, isavuconazole, itraconazole, or posaconazole. In various additional examples, treatments for fungal infections can include lipid formulation of amphotericin B plus flucytosine.Giant magnetoresistance (GMR) Systems and Apparatuses to Detect Analytes

[0279] A signal preparing unit can comprises a carrier signal generation part, a magnetic field excitation part, a circuit configuration part, a signal pick up part, and a clock synchronization part. A digital / analog (D / A) converter, a carrier signal generator and a carrier signal buffer can form the carrier signal generation part. The D / A converter is configured to receive control signal from the D / A converter control signal output unit of the signal processing control unit, and generate carrier signal generation parameters based on the received control signal. The carrier signal generator is configured to, based on the carrier signal generation parameters from the D / A converter, generate AC carrier signal used in the measurement circuits. A carrier signal buffer is coupled between the carrier signal generator and the measurement circuits, making the carrier signal generator present a very low impedance output relative to the higher impedance of the measurement circuits. Optionally, filters can be disposed at the carrier signal input to the measurement circuits to remove potential harmonics.

[0280] The D / A converter and a magnetic field drive can form the magnetic field excitation part. Based on the control signal received from the D / A converter control signal output unit of the signal processing control unit, the D / A converter generates magnetic field generation parameters. The magnetic field drive is configured to drive the magnetic field generator based on the magnetic field generation parameters, so as to apply AC magnetic field onto the GMR sensors.

[0281] The circuit configuration part includes at least one multiplexer and at least one reference resistor. Based on the multiplexer control signal received from the signal processing control unit, the at least one multiplexer routes one or more GMR sensors or one or more reference resistors in to configure appropriate measurement circuits.6356.002W01

[0282] A measurement signal buffer, a differential amplifier and an A / D converter form the signal pick up part (also called “differential voltage probe” or “voltage probe”). The measurement signal buffer can be coupled between the multiplexer(s) and the differential amplifier and is used to make the measurement circuits present a relatively high impedance at the inputs of the differential amplifier. The differential amplifier operates to capture time series of the voltage observations from the measurement circuits, and send the amplified measurement signals to the A / D converter. The A / D converter is configured to send the analog-to-digital-converted measurement signals to the signal processing unit. Optionally, filters can be used at the differential amplifier and / or at the A / D converter to remove harmonics.

[0283] Preferably, in some embodiments, a clock synchronizer is used to provide synchronization between the carrier signal generation part, the magnetic field excitation part and the signal pick up part. More specifically, the generation of the carrier signal generation parameters and the magnetic field generation parameters by the D / A converter is clocked from the same source as the A / D converter, i.e. by the clock synchronizer.

[0284] A process to generate GMR-based signals can include receiving multiplexer control signal from the signal processing control unit. Then, the multiplexer in the signal preparing unit configures the measurement circuits based on the received multiplexer control signal by routing certain GMR sensors and / or reference resistor(s) in. The signal preparing unit 1540 can then receive the D / A converter control signal from the signal processing control unit. Based on the D / A converter control signal, the D / A converter in the signal preparing unit generates carrier signal generation parameters. Then, the carrier signal is generated based on the generated carrier signal generation parameters, buffered and applied to the measurement circuits. Further, the D / A converter in the signal preparing unit generates magnetic field generation parameters based on the D / A converter control signal. Then, a magnetic field is excited by the magnetic field generator drive based on the magnetic field generation parameters, and applied to the GMR sensors via the magnetic field generator. Further, the measurement signals collected at the configured measurement circuits are buffered, and then amplified by the differential amplifier in the signal preparing unit. The amplified measurement signals are converted into digital signals by the A / D converter in the signal preparing unit. Next, the converted digital signals are sent to the signal processing unit for further processing.6356.002W01

[0285] In at least some examples, the signal processing unit can comprise reference signal generators, a multiplier, an integrator, an integration timing controller, and a close-form solution unit. The reference signal generators are configured to receive control signal from the solution and I / O control unit of the signal processing control unit, generate in-phase and quadrature (rotated 90 degrees) sinusoid reference signals at all frequencies of interest based on the received control signal, and send the generated reference signals to the multiplier. The multiplier is configured to receive the measurement signals from the A / D converter of the signal preparing unit, and multiply the measurement signals by the reference signals from the reference signal generators to produce an in-phase product and a quadrature product at each frequency of interest for each measurement signal. The in-phase products and quadrature products are sent to the integrator. The integrator is configured to accumulate these products under the control of the integration timing controller and send the accumulations to the close-form solution unit. The close-form solution unit is adapted to solve for, from the received accumulations, the phase-accurate GMR sensor resistance and magnetoresistance quantities that are not influenced by the frequency, amplitude or phase of the applied carrier signal, or by the amplitude or phase response of the circuits supplying this.

[0286] In view of the above-described implementations of subject matter this application discloses the following list of examples, wherein one feature of an example in isolation or more than one feature of an example, taken in combination and, optionally, in combination with one or more features of one or more further examples are further examples also falling within the disclosure of this application.

[0287] Example 1 is a method comprising: extracting nucleic acids from a sample, the nucleic acids comprising cell-free deoxyribonucleic acid (cfDNA) molecules; providing the nucleic acids and a plurality of primer sets to one or more containers, the plurality of primer sets including at least a first primer set and a second primer set selected from two of the following groups of primers: first primers that are complementary with first genomic regions of a first group of fungi including Aspergillus flavus, Candida albicans, Candida auris, Candida glabrata, and Scedosporium spp, and one or more first control sequences; second primers that are complementary with second genomic regions of a second group of fungi including Aspergillus fumigatus, Aspergillus niger, Candida krusei, and Cryptococcus and one or more second control sequences; third primers that are complementary with third genomic regions of a third6356.002W01 group of fungi including Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, and Fusarium solani and one or more third control sequences; or fourth primers that are complementary with fourth genomic regions of a fourth group of fungi including Candida tropicalis, Coccidioides, Histoplasma capulatun, Pneumocystis jirovecii, Mucorales, Blastomyces, and one or more fourth control sequences; performing amplification of a portion of the nucleic acids using the plurality of primer sets to produce an amplification product, the amplification product including double stranded amplicons of the portion of the nucleic acids complementary to at least two of the first group of fungi, the second group of fungi, the third group of fungi, or the fourth group of fungi corresponding to the first primer set and the second primer set, and wherein individual double stranded amplicons includes, a first strand having a 1 is missing parent: 1 is missing parent: 1 is missing parent: 1 is missing parent: 1 is missing parent: 5’ biotin and a second strand having a 5’ phosphate group; producing a group of single stranded nucleic acids corresponding to the first strands of the double stranded amplicons having the 5’ biotin; forming a surface of a sensor, wherein the surface includes a polymeric coating and a number of probe sequences, individual probe sequences including (i) a first section having a nucleotide sequence that is complementary to a genomic region included in the two of the first genomic regions, the second genomic regions, the third genomic regions, or the fourth genomic regions that correspond to the first primer set and the second primer set, and (ii) a second section that includes a linker sequence having at least five nucleotides and a 5’ amino group coupled to the polymeric coating; contacting the surface of the sensor with the group of single stranded nucleic acids to produce a modified surface of the sensor that includes a number of captured single stranded nucleic acids, individual captured single stranded nucleic acids being bound to an individual probe sequence; contacting the surface of the sensor with a solution including superparamagnetic streptavidin magnetic particles to produce a number of modified single stranded nucleic acids bound to the modified surface of the sensor, individual modified single stranded nucleic acids having a streptavidin particle bound to the 5’ biotin; and determining an electrical signal that corresponds to a number of the modified single stranded nucleic acids bound to the surface of the sensor.

[0288] In Example 2, the subject matter of Example 1 includes, wherein at least one additional primer of the first primer set is complementary to one or more first6356.002W01 control genomic regions and at least one additional primer of the second primer set is complementary to one or more second control genomic regions.

[0289] In Example 3, the subject matter of Examples 1-2 includes, wherein the plurality of primer sets include a third primer set including one of the first primers, the second primers, the third primers, or the fourth primers that are not included in the first primer set and the second primer set.

[0290] In Example 4, the subject matter of Example 3 includes, wherein the plurality of primer sets include a fourth primer set including one of the first primers, the second primers, the third primers, or the fourth primers that are not included in the first primer set, the second primer set, or the third primer set.

[0291] In Example 5, the subject matter of Example 4 includes, wherein at least one additional primer of the third primer set is complementary to one or more third control genomic regions and where at least one additional fourth primer corresponds to one or more fourth control genomic regions.

[0292] In Example 6, the subject matter of Example 5 includes, wherein the one or more first control genomic regions, the one or more second control genomic regions, the one or more third control genomic regions, or the one or more fourth control genomic regions comprise one or more same nucleotide sequences.

[0293] In Example 7, the subject matter of Examples 1-6 includes, wherein: the first primer set and the second primer set include a plurality of primer pairs, individual primer pairs of the plurality of primer pairs having a forward primer and a reverse primer; and an amount of the forward primer and the reverse primer of the individual primer pairs is from 25 nanomolar (nM) to 200 nM.

[0294] In Example 8, the subject matter of Examples 1-7 includes, providing an exonuclease to produce the group of single stranded nucleic acids corresponding to the first strands of the double stranded amplicons having the 5’ biotin.

[0295] In Example 9, the subject matter of Example 8 includes, wherein the exonuclease is a lambda exonuclease and producing the group of single stranded nucleic acids includes: heating a solution including the exonuclease and the amplification product at first temperatures from about 40 °C to about 50 °C for a first duration of about 3 minutes to about 10 minutes; and heating the solution including the exonuclease and the amplification product at second temperatures from about 70 °C to about 80 °C for a second duration of about 8 minutes to about 15 minutes.6356.002W01

[0296] In Example 10, the subject matter of Examples 1 -9 includes, wherein the linker sequence is comprised of thymine nucleotides or adenine nucleotides.

[0297] In Example 11 , the subject matter of Examples 1 -10 includes, NaCI, and bovine serum albumin (BSA).

[0298] In Example 12, the subject matter of Examples 1-11 wherein the streptavidin particles include iron oxide particles having diameters from 2 nm to 1000 nm.

[0299] In Example 13, the subject matter of Examples 1-12 includes, wherein the streptavidin particles include one or more amine streptavidin functional groups and the method comprises: contacting the streptavidin particles with a crosslinking agent to cause the one or more amine functional groups to bind with one or more thiol groups of modified streptavidin molecules.

[0300] In Example 14, the subject matter of Example 13 includes, - wherein the crosslinking agent includes succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 - carboxylate (Sulfo-SMCC).

[0301] In Example 15, the subject matter of Examples 1-14 includes, performing a first wash after producing the modified surface of the sensor to remove single stranded nucleic acid molecules that are not bound to a probe sequence; and performing a second wash after producing the modified single stranded nucleic acids to remove modified single stranded nucleic acid molecules that are weakly bound to a probe sequence.

[0302] In Example 16, the subject matter of Examples 1-15 includes, performing, before determining the electrical signal, one or more heating cycles with respect to the number of modified single stranded nucleic acids bound to the modified surface of the sensor, wherein individual heating cycles of the one or more heating cycles are performed at temperatures from about 50 °C to about 80 °C and have a duration from about 3 minutes to about 10 minutes.

[0303] In Example 17, the subject matter of Examples 1-16 includes, analyzing the electrical signal to determine an indication of one or more fungi being present with respect to a subject from which the sample was obtained.

[0304] In Example 18, the subject matter of Example 17 includes, wherein the indication of the one or more fungi being present with respect to the subject is determined when between 10 nucleic acids to 50 nucleic acids of at least one of one6356.002W01 or more of the first group of fungi or one or more of the second group of fungi are present in the sample.

[0305] In Example 19, the subject matter of Examples 1-18 includes, wherein the sample includes a plasma sample and the nucleic acids comprise cell-free deoxyribonucleic acid (cfDNA).

[0306] Example 20 is a kit comprising: a plurality of primer sets including at least a first primer set and a second primer set selected from two of the following groups of primers: first primers that are complementary with first genomic regions of a first group of fungi including Aspergillus flavus, Candida albicans, Candida auris, and Candida glabrata, and Scedosporium spp, and one or more first control sequences; second primers that are complementary with second genomic regions of a second group of fungi including Aspergillus fumigatus, Aspergillus niger, Candida krusei, and Cryptococcus and a second control sequence; third primers that are complementary with third genomic regions of a third group of fungi including Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, and Fusarium solani and one or more third control sequences; or fourth primers that are complementary with fourth genomic regions of a fourth group of fungi including Candida tropicalis, Coccidioides, Histoplasma capulatun, Pneumocystis jirovecii, Mucorales, and Blastomyces, and one or more fourth control sequences; and a set of lyophilized components including a polymerase, a number of deoxynucleotide triphosphates, and an exonuclease.

[0307] In Example 21 , the subject matter of Example 20 includes, wherein individual primers included in the first primer set and the second primer set include from 10 nucleotides to 30 nucleotides.

[0308] In Example 22, the subject matter of Examples 20-21 includes, wherein the set of lyophilized components includes a number of streptavidin magnetic particles having diameters from about 20 nanometers (nm) to about 1000 nm.

[0309] In Example 23, the subject matter of Examples 20-22 includes, a third primer set including one of the first primers, the second primers, the third primers, or the fourth primers that are not included in the first primer set and the second primer set; and a fourth primer set including one of the first primers, the second primers, the third primers, or the fourth primers that are not included in the first primer set, the second primer set, or the third primer set.

[0310] Example 24 is a method comprising: forming a surface of a sensor, wherein the surface includes, a polymeric coating and a number of probe sequences,6356.002W01 individual probe sequences including (i) a first section that includes a linker sequence having from 2 to 15 consecutive thymine or adenine molecules and a 5’ amino group coupled to the polymeric coating and (ii) a second section having a nucleotide sequence that is complementary to a genomic region of a fungi selected from Aspergillus flavus, Candida albicans, Candida auris, Candida glabrata, Scedosporium spp, Aspergillus fumigatus, Aspergillus niger, Candida krusei, Cryptococcus, Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, and Fusarium solani, Candida tropicalis, Coccidioides, Histoplasma capulatun, or Pneumocystis jirovecii; Mucorales, Blastomyces, or one or more control sequences; and determining an electrical signal that corresponds to a number of single stranded nucleic acids bound to the surface of the sensor, wherein the single stranded nucleic acid molecules can be derived from a sample and individual single stranded nucleic acid molecules.

[0311] In Example 25, the subject matter of Example 24 includes, extracting nucleic acids from a sample, the nucleic acids comprising cell-free deoxyribonucleic acid (cfDNA) molecules; providing the nucleic acids and a plurality of primer sets to one or more containers.In Example 26, the subject matter of Example 25 includes, wherein the plurality of primer sets include at least a first primer set and a second primer set selected from: first primers that are complementary with first genomic regions of a first group of fungi including Aspergillus flavus, Candida albicans, Candida auris, and Candida glabrata, and Scedosporium spp, and a first control sequence; second primers that are complementary with second genomic regions of a second group of fungi including Aspergillus fumigatus, Aspergillus niger, Candida krusei, and Cryptococcus and a second control sequence; third primers that are complementary with third genomic regions of a third group of fungi including Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, and Fusarium solani and a third control sequence; or fourth primers that are complementary with fourth genomic regions of a fourth group of fungi including Candida tropicalis, Coccidioides, Histoplasma capulatun, Pneumocystis jirovecii, Mucorales, or Blastomyces, and a fourth control sequence.

[0312] In Example 27, the subject matter of Example 26 includes, performing amplification of a portion of the nucleic acids using the plurality of primer sets to produce an amplification product, the amplification product including double stranded amplicons of the portion of the nucleic acids complementary to at least two of the first group of fungi, the second group of fungi, the third group of fungi, or the fourth group6356.002W01 of fungi corresponding to the first primer set and the second primer set, and wherein individual double stranded amplicons includes a first strand having a 5’ biotin and a second strand having a 5’ phosphate group; producing a group of single stranded nucleic acids corresponding to the first strands of the double stranded amplicons having the 5’ biotin.

[0313] Example 28 is , The method of Example 27, comprising: contacting the surface of the sensor with the number of single stranded nucleic acids to produce a modified surface of the sensor that includes, a number of captured single stranded nucleic acids, individual captured single stranded nucleic acids being bound to an individual probe sequence of the number of probe sequences; and contacting the modified surface of the sensor with a solution including superparamagnetic streptavidin magnetic particles to produce a number of modified single stranded nucleic acids bound to the modified surface of the sensor, individual modified single stranded nucleic acids having a streptavidin particle bound to the 5’ biotin.

[0314] In Example 29, the subject matter of Examples 26-28 includes, wherein at least one additional primer of the first primer set is complementary to one or more first control genomic regions and at least one additional primer of the second primer set is complementary to one or more second control genomic regions.

[0315] In Example 30, the subject matter of Examples 26-29 includes, wherein the plurality of primer sets include a third primer set including one of the first primers, the second primers, the third primers, or the fourth primers that are not included in the first primer set and the second primer set.

[0316] In Example 31 , the subject matter of Examples 26-30 includes, wherein the plurality of primer sets include a fourth primer set in...

Claims

6356.002W01CLAIMSWHAT IS CLAIMED IS:1 . A method comprising: providing an apparatus that includes (i) a plurality of giant magnetoresistance (GMR) sensors and (i) a plurality of chambers for performing amplification of nucleic acids; causing a plurality of probes to be attached to the plurality of GMR sensors, wherein the plurality of probes correspond to a plurality of fungal species; causing a first solution to be supplied to a first chamber of the plurality of chambers, the first solution including a first set of primers that correspond to a first group of fungi included in the plurality of fungal species; causing a second solution to be supplied to a second chamber of the plurality of chambers, the second solution including a second set of primers that correspond to a second group of fungi included in the plurality of fungal species, different from the first group of fungi; causing a first sample portion to be supplied to the first chamber and a second sample portion to be supplied to the second chamber, the first sample portion including first nucleic acids and the second sample portion including second nucleic acids; performing a first amplification process in the first chamber to produce a first amplification product that includes first double stranded amplicons; performing a second amplification process in the second chamber to produce a second amplification product that includes second double stranded amplicons, wherein the second amplification process is performed concurrently with respect to the first amplification process; causing the first amplification product and the second amplification product to be supplied to a chamber that includes the plurality of GMR sensors; and determining a change in electrical signals produced by at least one GMR sensor of the plurality of GMR sensors based on a first number nucleic acids derived from the first double stranded amplicons coupled to one or more first probes attached to the at least one GMR sensor or a second number derived from the second double stranded amplicons coupled to one or more second probes attached to the at least one GMR sensor, the one or more first probes corresponding to the first group of fungi and the one or more second probes corresponding to the second group of fungi.6356.002W012. The method of claim 1 , wherein individual GMR sensors of the plurality of GMR sensors have probes with nucleic acid sequences corresponding to an individual fungal species included in the first group of fungi or the second group of fungi.

3. The method of claim 1 , wherein individual first fungal species included in the first group of fungi have no greater than about 80% homology with respect to one another and individual second fungal species included in the second group of fungi have no greater than about 80% homology with respect to one another.

4. The method of claim 1 , wherein the first group of primers and the second group of primers are included in a plurality of primer sets that include a third group of primers and a fourth group of primers.

5. The method of claim 4, wherein the first group of primers corresponds to first genomic regions of a first group of fungi including Aspergillus flavus, Candida albicans, Candida auris, and Candida glabrata, Scedosporium spp, the second group of primers corresponds to second genomic regions of a second group of fungi including Aspergillus fumigatus, Aspergillus niger, Candida krusei, and Cryptococcus, the third group of primers corresponds to third genomic regions of a third group of fungi including Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, and Fusarium solani, and the fourth group of primers corresponds to fourth genomic regions of a fourth group of fungi including Candida tropicalis, Coccidioides, Histoplasma capulatun, Pneumocystis jirovecii, Mucorales, and Blastomyces.

6. The method of claim 1 , wherein individual double stranded amplicons included in the first double stranded amplicons and the second double stranded amplicons include a first strand having a 5’ biotin and a second strand having a 5’ phosphate group; and the method comprises producing a group of single stranded nucleic acids corresponding to the first strands of the first double stranded amplicons and the firsts strands of the second double stranded amplicons having the 5’ biotin.6356.002W017. The method of claim 6, wherein the group of single stranded nucleic acids contacts a surface of at least a portion of the plurality of GMR sensors.

8. The method of claim 6, comprising providing an exonuclease to produce the group of single stranded nucleic acids corresponding to the first strands of the double stranded amplicons having the 5’ biotin.

9. The method of claim 8, wherein the exonuclease is a lambda exonuclease and producing the group of single stranded nucleic acids includes: heating a solution including the exonuclease and the amplification product at first temperatures from about 40 °C to about 50 °C for a first duration of about 3 minutes to about 10 minutes; and heating the solution including the exonuclease and the amplification product at second temperatures from about 70 °C to about 80 °C for a second duration of about 8 minutes to about 15 minutes.

10. The method of claim 6, comprising: contacting the surface of the plurality of GMR sensors with a solution including superparamagnetic streptavidin magnetic particles to produce a number of modified single stranded nucleic acids bound to the modified surface of the sensor, individual modified single stranded nucleic acids having a streptavidin particle bound to the 5’ biotin; and determining an electrical signal that corresponds to a number of the modified single stranded nucleic acids bound to the surface of at least a portion of the plurality of GMR sensors.11 . A method comprising: extracting nucleic acids from a sample, the nucleic acids comprising cell-free deoxyribonucleic acid (cfDNA) molecules; providing the nucleic acids and a plurality of primer sets to one or more containers, the plurality of primer sets including at least a first primer set and a second primer set selected from two of the following groups of primers:6356.002W01 first primers that are complementary with first genomic regions of a first group of fungi including Aspergillus flavus, Candida albicans, Candida auris, and Candida glabrata, Scedosporium spp, and a first control sequence; second primers that are complementary with second genomic regions of a second group of fungi including Aspergillus fumigatus, Aspergillus niger, Candida krusei, and Cryptococcus and a second control sequence; third primers that are complementary with third genomic regions of a third group of fungi including Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, and Fusarium solani and a third control sequence; or fourth primers that are complementary with fourth genomic regions of a fourth group of fungi including Candida tropicalis, Coccidioides, Histoplasma capulatun, Pneumocystis jirovecii, Mucorales, Blastomyces, and a fourth control sequence; performing amplification of a portion of the nucleic acids using the plurality of primer sets to produce an amplification product, the amplification product including double stranded amplicons of the portion of the nucleic acids complementary to at least two of the first group of fungi, the second group of fungi, the third group of fungi, or the fourth group of fungi corresponding to the first primer set and the second primer set, and wherein individual double stranded amplicons includes a first strand having a 5’ biotin and a second strand having a 5’ phosphate group; producing a group of single stranded nucleic acids corresponding to the first strands of the double stranded amplicons having the 5’ biotin; forming a surface of a sensor, wherein the surface includes a polymeric coating and a number of probe sequences, individual probe sequences including (i) a first section having a nucleotide sequence that is complementary to a genomic region included in the two of the first genomic regions, the second genomic regions, the third genomic regions, or the fourth genomic regions that correspond to the first primer set and the second primer set, and (ii) a second section that includes a linker sequence having at least five nucleotides and a 5’ amino group coupled to the polymeric coating; contacting the surface of the sensor with the group of single stranded nucleic acids to produce a modified surface of the sensor that includes a number of captured single stranded nucleic acids, individual captured single stranded nucleic acids being bound to an individual probe sequence; contacting the surface of the sensor with a solution including superparamagnetic streptavidin magnetic particles to produce a number of modified6356.002W01 single stranded nucleic acids bound to the modified surface of the sensor, individual modified single stranded nucleic acids having a streptavidin particle bound to the 5’ biotin; and determining an electrical signal that corresponds to a number of the modified single stranded nucleic acids bound to the surface of the sensor.

12. The method of claim 11 , wherein at least one additional primer of the first primer set is complementary to one or more first control genomic regions and at least one additional primer of the second primer set is complementary to one or more second control genomic regions.

13. The method of claim 11 , wherein the plurality of primer sets include a third primer set including one of the first primers, the second primers, the third primers, or the fourth primers that are not included in the first primer set and the second primer set.

14. The method of claim 13, wherein the plurality of primer sets include a fourth primer set including one of the first primers, the second primers, the third primers, or the fourth primers that are not included in the first primer set, the second primer set, or the third primer set.

15. The method of claim 14, wherein at least one additional primer of the third primer set is complementary to one or more third control genomic regions and where at least one additional fourth primer corresponds to one or more fourth control genomic regions of the human genome.

16. The method of claim 15, wherein the one or more first control genomic regions, the one or more second control genomic regions, the one or more third control genomic regions, or the one or more fourth control genomic regions comprise one or more same nucleotide sequences.

17. The method of claim 11 , wherein:6356.002W01 the first primer set and the second primer set include a plurality of primer pairs, individual primer pairs of the plurality of primer pairs having a forward primer and a reverse primer; and an amount of the forward primer and the reverse primer of the individual primer pairs is from 25 nanomolar (nM) to 200 nM.

18. The method of claim 11 , comprising: providing an exonuclease to produce the group of single stranded nucleic acids corresponding to the first strands of the double stranded amplicons having the 5’ biotin.

19. The method of claim 18, wherein the exonuclease is a lambda exonuclease and producing the group of single stranded nucleic acids includes: heating a solution including the exonuclease and the amplification product at first temperatures from about 40 °C to about 50 °C for a first duration of about 3 minutes to about 10 minutes; and heating the solution including the exonuclease and the amplification product at second temperatures from about 70 °C to about 80 °C for a second duration of about 8 minutes to about 15 minutes.

20. The method of claim 11 , wherein the linker sequence is comprised of thymine nucleotides or adenine nucleotides.

21. The method of claim 11 , wherein the modified surface is formed using a solution including NasPCM, NaCI, and bovine serum albumin (BSA).

22. The method of claim 11 , wherein the streptavidin particles include iron oxide particles having diameters from 2 nm to 1000 nm.

23. The method of claim 11 , wherein the streptavidin particles include one or more amine streptavidin functional groups and the method comprises: contacting the streptavidin particles with a crosslinking agent to cause the one or more amine functional groups to bind with one or more thiol groups of modified streptavidin molecules.6356.002W0124. The method of claim 23, wherein the crosslinking agent includes succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 -carboxylate (Sulfo-SMCC).

25. The method of claim 11 , comprising: performing a first wash after producing the modified surface of the sensor to remove single stranded nucleic acid molecules that are not bound to a probe sequence; and performing a second wash after producing the modified single stranded nucleic acids to remove modified single stranded nucleic acid molecules that are weakly bound to a probe sequence.

26. The method of claim 11 , comprising: performing, before determining the electrical signal, one or more heating cycles with respect to the number of modified single stranded nucleic acids bound to the modified surface of the sensor, wherein individual heating cycles of the one or more heating cycles are performed at temperatures from about 50 °C to about 80 °C and have a duration from about 3 minutes to about 10 minutes.

27. The method of claim 1 , comprising: analyzing the electrical signal to determine an indication of one or more fungi being present with respect to a subject from which the sample was obtained.

28. The method of claim 27, wherein the indication of the one or more fungi being present with respect to the subject is determined when between 10 nucleic acids to 50 nucleic acids of at least one of one or more of the first group of fungi or one or more of the second group of fungi are present in the sample.

29. The method of claim 1 , wherein the sample includes a plasma sample and the nucleic acids comprise cell-free deoxyribonucleic acid (cfDNA).

30. A method comprising: extracting nucleic acids from a sample;6356.002W01 performing a first amplification process in relation to a first number of the nucleic acids to produce first amplicons, the first amplification process being performed using primers that are complementary with first genomic regions of a first group of fungi; performing a second amplification process in relation to a second number of the nucleic acids to produce second amplicons, the second amplification process being performed using primers that are complementary with second genomic regions of a second group of fungi different from the first group of fungi; wherein the first group of fungi and the second group of fungi include two of the following groups of fungi:(1 ) Aspergillus flavus, Candida albicans, Candida auris, Candida glabrata, and Scedosporium spp.,(2) Aspergillus fumigatus, Aspergillus niger, Candida krusei, and Cryptococcus',(3) Aspergillus terreus, Candida parapsilosis, Fusarium verticillioides, and Fusarium solanr', and(4) Candida tropicalis, Coccidioides, Histoplasma capulatun, Pneumocystis jirovecii, Mucorales, and Blastomyces', and determining an electrical signal that corresponds to at least one of a number of single stranded first amplicons or a number of single stranded second amplicons bound to a surface of a sensor.31 . The method of claim 30, wherein: the primers that are complementary with the first genomic regions comprise a first primer set of a plurality of primer sets; and the primers that are complementary with the second genomic regions comprise a second primer set of the plurality of primer sets.

32. The method of claim 31 , wherein: the plurality of primer sets include a third primer set including primers that are complementary with third genomic regions of a third group of fungi; and the plurality of primer sets include a fourth primer set including primers that are complementary with fourth genomic regions of a fourth group of fungi.

33. The method of claim 32, wherein:6356.002W01 at least one additional primer of the first primer set is complementary to one or more first control genomic regions; at least one additional primer of the second primer set is complementary to one or more second control genomic regions; at least one additional primer of the third primer set is complementary to one or more third control genomic regions and at least one additional fourth primer corresponds to one or more fourth control genomic regions of the human genome.

34. The method of claim 33, wherein the one or more first control genomic regions, the one or more second control genomic regions, the one or more third control genomic regions, or the one or more fourth control genomic regions comprise one or more same nucleotide sequences.

35. The method of claim 31 , wherein: the first primer set and the second primer set include a plurality of primer pairs, individual primer pairs of the plurality of primer pairs having a forward primer and a reverse primer; and an amount of the forward primer and the reverse primer of the individual primer pairs is from 25 nanomolar (nM) to 200 nM.

36. The method claim 32, comprising: forming a surface of a sensor, wherein the surface includes a polymeric coating and a number of probe sequences, individual probe sequences including (i) a first section having a nucleotide sequence that is complementary to a genomic region included in the first genomic region, the second genomic region, the third genomic region, or the fourth genomic region that corresponds to the first primer set, the second primer set, the third primer set, or the fourth primer set and (ii) a second section that includes a linker sequence having at least five nucleotides and a 5’ amino group coupled to the polymeric coating; contacting the surface of the sensor with a group of single stranded nucleic acids to produce a modified surface of the sensor that includes a number of captured single stranded nucleic acids, the group of single stranded nucleic acids having a6356.002W015’biotin and individual captured single stranded nucleic acids being bound to an individual probe sequence; contacting the surface of the sensor with a solution including superparamagnetic streptavidin magnetic particles to produce a number of modified single stranded nucleic acids bound to the modified surface of the sensor, individual modified single stranded nucleic acids having a streptavidin particle bound to the 5’ biotin.

37. The method of claim 36, comprising: providing an exonuclease to produce the group of single stranded nucleic acids corresponding to the first strands of the double stranded amplicons having the 5’ biotin.

38. The method of claim 37, wherein the exonuclease is a lambda exonuclease and producing the group of single stranded nucleic acids includes: heating a solution including the exonuclease and the amplification product at first temperatures from about 40 °C to about 50 °C for a first duration of about 3 minutes to about 10 minutes; and heating the solution including the exonuclease and the amplification product at second temperatures from about 70 °C to about 80 °C for a second duration of about 8 minutes to about 15 minutes.

39. The method of claim 36, wherein the linker sequence is comprised of thymine nucleotides or adenine nucleotides.

40. The method of claim 36, wherein the modified surface is formed using a solution including NasPCM, NaCI, and bovine serum albumin (BSA).41 . The method of claim 36, wherein the streptavidin particles include iron oxide particles having diameters from 2 nm to 1000 nm.

42. The method of claim 36, wherein the streptavidin particles include one or more amine streptavidin functional groups and the method comprises:6356.002W01 contacting the streptavidin particles with a crosslinking agent to cause the one or more amine functional groups to bind with one or more thiol groups of modified streptavidin molecules.

43. The method of claim 42, wherein the crosslinking agent includes succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 -carboxylate (Sulfo-SMCC).

44. The method of claim 36, comprising: performing a first wash after producing the modified surface of the sensor to remove single stranded nucleic acid molecules that are not bound to a probe sequence; and performing a second wash after producing the modified single stranded nucleic acids to remove modified single stranded nucleic acid molecules that are weakly bound to a probe sequence.

45. The method of claim 36, comprising: performing, before determining the electrical signal, one or more heating cycles with respect to the number of modified single stranded nucleic acids bound to the modified surface of the sensor, wherein individual heating cycles of the one or more heating cycles are performed at temperatures from about 50 °C to about 80 °C and have a duration from about 3 minutes to about 10 minutes.

46. The method of claim 30, comprising: analyzing the electrical signal to determine an indication of one or more fungi being present with respect to a subject from which the sample was obtained.

47. The method of claim 46, wherein the indication of the one or more fungi being present with respect to the subject is determined when between 10 nucleic acids to 50 nucleic acids of at least one of one or more of the first group of fungi or one or more of the second group of fungi are present in the sample.

48. The method of claim 30, wherein the sample includes a plasma sample and the nucleic acids comprise cell-free deoxyribonucleic acid (cfDNA).

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