Method and apparatus for detecting orthogonal classification of disease biomarkers

Abstract

In one embodiment, a method for screening an individual for head and neck cancer comprises collecting a saliva sample from the individual and analyzing the saliva sample to detect one or more HPVs and at least one of a phenotypic biomarker, a regulatory biomarker, a microbiome-derived biomarker, and a metabolome biomarker that has been dysregulated by HPV infection, wherein the detection of both HPV and at least one of the biomarkers indicates that the individual is at risk for head and neck cancer.

Description

Technical Field

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[0001] (Related Application) This application claims the priority of Provisional Patent Application No. 63 / 463,204, filed on May 1, 2023, and the entire content thereof is incorporated herein by reference.

[0002] (Technical Field) The present invention relates to diagnostic methods and systems for detecting orthogonal classification of disease biomarkers.

Background Art

[0003] The diagnosis and early detection of various diseases can be essential for obtaining good treatment outcomes. Accurate diagnosis of diseases at the point of care enables timely and effective treatment, leading to saving lives. For example, cancer is a multi-step process that can take years (in some cases up to 15 years) to develop. If cancer is detected early, the effectiveness of cancer treatment can be dramatically improved.

[0004] Therefore, there is a need for improved diagnostic methods and systems for accurately, preferably early, detecting various diseases.

Summary of the Invention

[0005] In one aspect, a method for screening an individual for head and neck cancer is disclosed, the method comprising the steps of collecting a saliva sample from the individual and analyzing the saliva sample to detect at least one of one or more of human papillomavirus (HPV), phenotypic biomarkers, regulatory biomarkers, microbiome-derived biomarkers, and metabolome biomarkers dysregulated by HPV infection, wherein detection of both HPV and at least one of the biomarkers indicates that the individual is at risk of head and neck cancer.

[0006] For example, phenotypic biomarkers may be biomarkers associated with cellular processes that are affected (become dysregulated) by HPV infection. For instance, phenotypic biomarkers can be proteins, mRNA, or miRNAs, while regulatory biomarkers can be miRNAs.

[0007] The step of analyzing a saliva sample to detect HPV may include detecting at least one nucleotide target sequence associated with the HPV genome. In addition, or alternatively, HPV may be detected by detecting mRNA produced by transcription of the HPV genome in infected host cells.

[0008] In various embodiments, at least one protein biomarker may be the p-16 protein and / or the EGF (epidermal growth factor) receptor.

[0009] For example, the detection of HPV associated with increased p-16 and / or EGF protein or mRNA in response to HPV expression in host cells in biological samples taken from an individual (e.g., saliva samples) (e.g., via detection of its nucleic acid sequence or transcript) may indicate that the individual is at risk of HPV-derived cancers, such as head and neck cancer.

[0010] In various embodiments, if an individual is determined to be at risk of developing head and neck cancer, follow-up examinations such as MRI imaging may be recommended for that individual.

[0011] In a relevant embodiment, an assay for detecting a target protein in a biological sample is disclosed, comprising incubating the sample with a first antibody exhibiting specific binding to a first epitope of the target protein and a second antibody exhibiting specific binding to a second epitope of the target protein in the presence of a substrate to form an incubation mixture of the sample and antibodies, wherein the first antibody is further configured to be conjugated to the substrate and the second antibody is conjugated to a nucleotide sequence. As an example, the incubation time may range from about 30 minutes to about 40 minutes. Subsequently, a washing step of the incubation mixture is performed to remove components not bound to the substrate. Subsequently, the oligonucleotide sequence is amplified and detected using an RPA-Exo assay, thereby detecting the target protein.

[0012] In various embodiments, the substrate may be functionalized with streptavidin, and the first antibody may be biotinylated to bind to streptavidin. In some such embodiments, the substrate may correspond to the surface of multiple magnetic beads, which can be collected by applying a magnetic field to the incubation mixture. After collecting the magnetic beads, a washing step may be performed, followed by an RPA-Exo assay to detect the target protein.

[0013] In a relevant embodiment, a method for screening an individual for HPV-mediated cancer is disclosed, which comprises the steps of collecting a biological specimen from the individual and analyzing the biological specimen to detect HPV and at least one of a phenotypic biomarker, regulatory biomarker, metabolic biomarker, and microbiome-derived biomarker that has been dysregulated by HPV infection, thereby indicating that the individual is at risk for HPV-mediated cancer by detecting both HPV and at least one of the biomarkers.

[0014] For example, HPV-mediated cancers can be cervical cancer, head and neck cancer, penile cancer, vaginal cancer, valvular heart cancer, or anal cancer.

[0015] In various embodiments, phenotypic biomarkers may be associated with cellular molecular processes affected (dysregulated) by HPV infection. Phenotypic biomarkers can be either proteins or mRNAs, while regulatory biomarkers can be miRNAs. For example, phenotypic biomarkers may be the p-16 protein and / or the EFG receptor.

[0016] In various embodiments, a biological specimen may be analyzed to detect HPV in the specimen, and at least one target nucleotide sequence associated with the HPV genome may be detected. In addition or alternatively, HPV may be detected in a biological specimen by detecting mRNA associated with the transcription of the HPV genome in infected host cells.

[0017] In various embodiments, once an individual is identified as being at risk of developing HPV-derived cancer, that individual is recommended to undergo follow-up examinations, such as MRI imaging.

[0018] In a relevant embodiment, a method for detecting a disease is disclosed, the method comprising the steps of collecting a biological sample from an individual and analyzing the biological sample to detect at least two orthogonal disease biomarkers present in the biological sample, the detection of the two orthogonal disease biomarkers indicating that the individual is at risk of disease.

[0019] In various embodiments, at least one of the two orthogonal disease biomarkers comprises at least a target protein, and at least one of the two orthogonal disease biomarkers comprises a target nucleotide sequence.

[0020] In various embodiments, either the target protein or the target nucleotide sequence indicates the presence of a pathogen in the biological sample. For example, the pathogen may be a virus.

[0021] The methods described above can be used to detect (diagnose) a variety of different diseases. Examples of such diseases include, but are not limited to, cancer, neurodegenerative diseases, blood disorders, inflammatory diseases, and infectious diseases.

[0022] In a related embodiment, a system for detecting disease biomarkers of at least two classifications in a biological specimen is disclosed, the system including a cartridge frame having a sample well for receiving a biological specimen, a first lysis chamber fluid-communicating with the sample well for receiving a first portion of the biological specimen and preparing a first sample portion for detection of at least one target protein, and a second lysis chamber fluid-communicating with the sample well for receiving a second portion of the sample and preparing a second sample portion for detection of at least one target nucleic acid sequence. The system further includes a wash buffer blister pack for containing wash buffer and a collection well fluid-communicating with the wash buffer blister pack. In addition, the system includes at least one protein detection well fluid-communicating with the first lysis chamber and the collection well and configured to detect at least one target protein, and at least one nucleic acid detection well fluid-communicating with the second lysis chamber for receiving a second specimen portion and configured to detect at least one target nucleic acid sequence. In various embodiments, a first lysis chamber contains at least one lysis agent for preparing a sample for protein detection, and a second lysis chamber contains at least one lysis agent for preparing a sample for nucleic acid detection. Examples of suitable lysis reagents are disclosed in the aforementioned '393 patent. In various embodiments, at least one protein detection well contains a plurality of magnetic particles functionalized with streptavidin, at least one first antibody exhibiting specific binding to a first epitope of a target protein, and at least one second antibody exhibiting binding to a second epitope of a target protein, wherein the first antibody is biotinylated to bind to streptavidin to bind to the magnetic particles, and the second antibody comprises at least one oligonucleotide sequence.

[0023] In various embodiments, at least one protein detection well comprises a plurality of magnetic particles functionalized with streptavidin, at least a first antibody exhibiting specific binding to a first epitope of the target protein, and at least a second antibody exhibiting binding to a second epitope of the target protein, wherein the first antibody is biotinylated to bind to streptavidin and is conjugated to the magnetic particles, and the second antibody comprises at least one oligonucleotide sequence.

[0024] The cartridge may further include a first valve for regulating fluid communication between a first lysis chamber and the at least one protein detection well, and a second valve for regulating fluid communication between a second lysis chamber and the at least one nucleic acid detection well. These valves can be operated under the control of a controller.

[0025] For example, a biological specimen may include specimens derived from biological fluid, fluid biopsy, or tissue biopsy.

[0026] This system can be used to detect (diagnose) a variety of diseases, including cancer, neurodegenerative diseases, blood disorders, and inflammatory diseases. For example, the disease could be cervical cancer, head and neck cancer, penile cancer, or valvular heart cancer.

[0027] In related aspects, a system for detecting disease biomarkers of at least two classifications of diseases in a biological specimen is disclosed. The system includes a sample port configured to receive a biological specimen, a first reservoir containing a first buffer for preparing a sample of the biological specimen for detection of a first classification of cancer biomarkers, a second reservoir containing a second buffer for preparing a sample for detection of a second classification of cancer biomarkers, a first sensor configured to detect a first classification of cancer biomarkers in a portion of the sample processed through the first buffer, and a second sensor configured to detect a second classification of cancer biomarkers in a portion of the sample processed through the second buffer.

[0028] At least one of the first and second buffers comprises a lysing agent for processing the specimen.

[0029] Similar to the foregoing embodiments, the biological specimen comprises any of a biological fluid, a liquid biopsy, and a tissue biopsy-derived specimen. As an example, the system can be used for detection / diagnosis of cancer, neurodegenerative diseases, blood diseases, and inflammatory diseases. As an example, the disease can be any of cervical cancer, head and neck cancer, penile cancer, and valvular cancer.

[0030] A further understanding of various aspects of the present invention can be obtained by reference to the following detailed description in conjunction with the related drawings briefly described below.

Brief Description of the Drawings

[0031] [Figure 1A] It is a flowchart showing each step for analyzing a biological sample obtained from an individual to detect a disease according to an embodiment of the present invention. [Figure 1B] It is a flowchart showing each step for detecting HPV-mediated cancer according to an embodiment of the present invention. [Figure 2] It is a diagram comparing a conventional workflow for cervical cancer detection with a workflow according to an embodiment of the present invention. [Figure 3A] This flowchart shows each step performed in an assay for detecting a target protein in a biological sample, according to an embodiment of the present invention. [Figure 3B] This figure shows the various reactants used in the assay described in relation to Figure 3A, and their interactions. [Figure 4] This figure shows a conventional cartridge that can be modified to carry out various methods based on embodiments of the present invention. [Figure 5] This is a top view of a device used to perform multiple detection of target proteins and nucleotide sequences. [Figure 6] This is a schematic diagram of a cartridge according to an embodiment of the present invention. [Figure 7] This is a schematic diagram showing an example of a controller / fluorescence reader that can be used to control cartridges according to each embodiment of the present invention and to detect fluorescence emission generated in each well of the cartridge. [Modes for carrying out the invention]

[0032] The present invention relates to the detection of orthogonal disease biomarkers in complex diseases such as cancer, neurodegenerative diseases, and hematological disorders. As will be described in more detail below, in various embodiments, the detection of orthogonal disease biomarkers is achieved by introducing a sample (also referred to herein as a specimen) into a cartridge configured to detect two or more orthogonal disease biomarkers. For example, in various embodiments, different sample processing may be used to prepare the specimen for the detection of different biomarkers. However, in many embodiments, the same detection modality, such as fluorescence detection, is used for the detection of different biomarkers. Furthermore, in various embodiments, the preparation and detection of multiple biomarkers in a single specimen are carried out on the same cartridge.

[0033] For example, as will be described in more detail below, some embodiments provide a cartridge comprising at least one sensor for detecting one or more genetic components of the HPV virus, such as high-risk HPVs including DNA and mRNA, such as HPV16, HPV18, HPV31, and HPV11 nucleic acids, and at least another sensor for detecting genes, proteins, mRNA, miRNA, microbiome, metabolome, and other biomarkers related to HPV-mediated cancers such as cervical cancer or head and neck cancer, or host response biomarkers. Furthermore, in some embodiments, cartridges according to the teachings of the present invention may also provide multiple detection of other factors, such as one or more genomic nucleotide sequences, phenotypic biomarkers (e.g., proteins, mRNA, miRNA), and one or more target metabolites and microbiome-derived biomarkers, as described below.

[0034] In various embodiments, a single sample taken from an individual is used for both HPV detection and, in combination with HPV detection, detection of one or more biomarkers indicating that the individual is at risk of HPV-mediated cancers such as head and neck cancer. For example, part of the single sample is used for HPV detection, and another part of the sample is used for biomarker detection. In some embodiments, instead of dividing the sample into multiple parts, the sample is introduced into a single well, and multiple detection of HPV (e.g., via detection of genomic sequences or mRNA associated with HPV transcription in host cells) and one or more biomarkers described herein is performed. In many embodiments, the same detection modality is used for the detection of HPV and one or more biomarkers in a single sample (e.g., saliva) obtained from an individual. Furthermore, in various embodiments, the same statistical analysis algorithm is used for the analysis of signals associated with the detection of HPV and biomarkers, such as fluorescence signals. Detecting not only HPV but also associated biomarkers from a single sample can advantageously improve the accuracy of screening for HPV-derived cancers (such as head and neck cancer). In addition, using the same statistical analysis in various embodiments can further improve screening accuracy.

[0035] More generally, detecting multiple orthogonal disease biomarkers from the same sample using the same detection modality and optionally the same statistical analysis method can improve the accuracy of determining whether an individual is at risk of disease.

[0036] As used herein, the term "orthogonal disease biomarker" refers to a biomarker that can provide complementary information about a disease. For example, such orthogonal biomarkers may belong to different classifications of biomarkers, such as genetic, metabolic, phenotypic, microbiome-derived, and epigenetic biomarkers.

[0037] The term "biomarker" refers to a biomolecule present in biological specimens, such as bodily fluids or tissues, that may indicate a normal or abnormal process, condition, or disease.

[0038] As used herein, "genetic biomarker" refers to a genomic nucleotide sequence that can indicate, for example, the presence of a pathogen (e.g., a virus) in a biological specimen.

[0039] As used herein, "phenotypic biomarker" refers to a protein, regulatory factor, transcription factor, or metabolic factor (compound) whose normal concentration can be regulated (become dysregulated) by the onset and / or progression of a disease. Some examples include proteins, mRNA, and miRNA.

[0040] As used herein, "metabolomic biomarkers" refer to biomarkers that are small molecule substrates, intermediates, or products of cellular metabolism. Metabolomic biomarkers provide a direct "functional readout" of the physiological state of an organism.

[0041] As used herein, “microbiome-derived biomarkers” refer to microbial signatures derived from the composition, diversity, and activity of microorganisms inhabiting a specific environment or organism, particularly the human body. These biomarkers may be based on various aspects of the microbiome, such as the types and abundances of bacteria, archaea, viruses, fungi, and other microorganisms present in each sample.

[0042] As used herein, "epigenetic biomarkers" refer to molecular indicators that reflect changes in gene expression or regulation without altering the underlying DNA sequence. These include a variety of epigenetic modifications, such as DNA methylation, histone modifications, and non-coding RNAs.

[0043] Accurate diagnosis of disease at the point of care enables timely and effective treatment, ultimately saving lives. Over the past 40 years, research into cancer hallmarks has revealed complex features, including genotype, phenotype, metabolic reprogramming, non-mutant epigenetics, polymorphic microbiome, host / immune response, and disruption of extracellular matrix components ("Hallmarks of Cancer: New Dimensions," by Hanahan, Cancer Discovery, vol. 12, Issue 1, 2022, which is referenced here in its entirety). Therefore, simply evaluating a single classification of disease biomarkers is insufficient for early and accurate diagnosis of cancer and other complex diseases such as neurodegenerative diseases.

[0044] Cancer is a multi-stage process that can take many years (up to 15 years in some cases) to develop. By simultaneously detecting two or more biomarkers—genotype, epigenetics, phenotype, immune response, gene mutation, single nucleotide polymorphism, metabolome, DNA methylation, mRNA, miRNA, long non-coding RNA (lncRNA), competitive endogenous RNA (ceRNA), and circular RNA (circ-RNA)—in a multimodal manner from a single sample, early and timely detection of cancer at a curable stage becomes possible. This can reduce unnecessary treatments that many patients are forced to undergo.

[0045] For example, breast cancer is a heterogeneous disease at the molecular level. Some molecular features include activation of human epidermal growth factor receptor 2 (HER2), activation of hormone receptors (estrogen and progesterone receptors), and BRCA mutations. The presence of germline BRCA mutations in a subject may indicate a predisposition to breast, ovarian, pancreatic, or other cancers, but BRCA mutations alone do not indicate that the subject has these cancers at the time of testing. Therefore, rapid, multi-omics, and accurate point-of-care testing for screening and monitoring patients with BRCA mutations can prevent unnecessary mastectomies, oophorectomies, and other procedures performed without evidence of cancer.

[0046] In recent years, additional biomarkers that may be important for the early detection of breast cancer have been discovered. For example, hsa_circRNA_002082 and hsa_circRNA_400031 function as ceRNAs and may play an important role in epithelial-mesenchymal transition in breast cancer (e.g., “Identification of epithelial-mesenchymal transition-related circRNA-miRNA-ceRNA regulatory network in breast cancer,” Sang et al., Pathol Res Pract. 2022 Sep, 216(9), which is referenced here in its entirety). Furthermore, promoter hypermethylation is associated with inactivation of tumor suppressor genes and is an early event of carcinogenesis. In fact, hypermethylated tumor suppressor genes are frequently found in patient-derived breast cancer tissue and peripheral blood specimens (98.doi:10.3892 / ijo.2021.5278).

[0047] More than 95% of cervical cancer cases are caused by human papillomavirus (HPV). The WHO has updated its cervical cancer detection guidelines and recommends HPV testing as the primary screening test for cervical cancer. However, the presence of HPV (high-risk HPV16, HPV18, HPV31, HPV35, etc.) does not indicate that a person has cervical cancer, and many HPV infections can disappear without causing cervical cancer. Rapid and accurate point-of-care, point-of-need molecular testing for HPV nucleic acids can significantly improve cervical cancer screening, but it has a high rate of false positives. Some people who test positive for HPV do not have cervical cancer, and some HPV clears without causing cervical cancer. As a result, many HPV-positive individuals may undergo unnecessary and invasive procedures such as colposcopy, or suffer emotional distress. Combining HPV detection with the detection of other orthogonal biomarkers for cervical cancer can reduce these false positive results. In the United States, HPV-positive testing is flexibly replaced by cytology. However, in many cases, especially among socially disadvantaged groups, follow-up after a positive test can be difficult, and cervical cancer patients may not receive timely treatment.

[0048] Therefore, diagnosing complex diseases requires an innovative diagnostic platform that can detect multiple classifications of disease biomarkers from the same specimen, enabling accurate and early disease diagnosis at the patient's side. As will be described in more detail below, such platforms are provided in various embodiments, utilizing cartridges capable of detecting multiple classifications of disease biomarkers from the same specimen.

[0049] Several DNA biomarkers, namely ASCL1 / LHX8 methylation levels, have also been shown to increase significantly with increasing severity of cervical cancer. These markers can be detected in urine. In one study, analysis of these markers in patient samples resulted in an AUC of 0.84 for urinary CIN3+ detection, corresponding to a specificity of 70% and a sensitivity of 86% ("HPV and DNA methylation testing in urine for cervical intraepithelial neoplasia and cervical cancer detection," van der Held et al., Clin. Cancer Res 2022 May 13:28(10):2061-2068).

[0050] Referring to the flowchart in Figure 1A, a disease detection method according to one embodiment includes the steps of obtaining a biological specimen from an individual and analyzing whether at least two orthogonal biomarkers associated with the disease are present in the biological specimen, the detection of at least two orthogonal biomarkers indicating that the individual is at risk of having the disease. For example, orthogonal biomarkers may include any of the following: genetic biomarkers, phenotypic biomarkers, metabolome biomarkers, and / or microbiome-derived biomarkers. In various embodiments, the disease detection method taught in the present invention may be carried out at the point of care.

[0051] HPV-mediated cancers, such as cervical cancer and head and neck cancer, constitute a category of diseases to which detection methods in various embodiments are applicable. Referring, for example, to the flowchart in Figure 1B, in various embodiments of HPV-mediated cancer detection methods, a biological specimen is collected from an individual. The biological specimen is analyzed to detect HPV and at least one of the following: phenotypic biomarkers, regulatory biomarkers, metabolome biomarkers, and / or microbiome-derived biomarkers that have become dysregulated due to HPV infection. Detecting both HPV and at least one biomarker indicates that the individual is at risk of having HPV-mediated cancer.

[0052] For example, such methods can be used to detect cervical cancer, head and neck cancer, vaginal and vulvar cancer, and anal cancer. In various embodiments, the methods taught in the present invention can be used to screen individuals for these cancers at the point of care.

[0053] As a further example, the systems and methods taught in the present invention can also be used for the detection of head and neck cancer. Head and neck cancer generally refers to the presence of malignant tumors in the head and neck region, particularly in the upper respiratory tract, gastrointestinal tract, and salivary glands. In the United States, head and neck cancer is reported to account for approximately 3% of all cancer cases. More than 70% of head and neck cancer cases are caused by high-risk human papillomavirus (hr-HPV) infection, including but not limited to HPV-16, HPV-18, and HPV-11. While many HPV infections resolve without progressing to cancer, if HPV infection persists in the body for a long period and the HPV genome is integrated into the genome of the affected tissue, it can lead to cancer. Integration of hr-HPV into the genome results in changes to the genes, proteome, epigenetics, metabolome, and microbiota of affected cells and tissues, leading to the initiation and progression of tumorigenesis. Therefore, the presence of the hr-HPV gene or its transcript, and the detection of one or more orthogonal / phenotypic disease biomarkers altered in response to HPV infection, may enable more comprehensive and accurate detection, screening, and diagnosis. For example, when hr-HPV is integrated into the genome of affected tissue, levels of the p16 protein increase. In liquid biopsies (i.e., saliva or blood), detecting the p16 transcript (mRNA) or protein as a phenotypic biomarker before the appearance of a visible tumor indicates that a tumorigenic event has begun and that the patient has cancer or requires close monitoring for early detection of cancer. Definitive diagnosis can be made by imaging studies (e.g., MRI, PET scan). Healthcare providers may recommend more intensive screening for individuals in certain high-risk groups, such as those with a family history of head and neck cancer, those who may be HIV-positive based on age or other behaviors, and those who smoke or drink heavily. In 2023, 23 million Americans were reported to be at high risk of developing head and neck cancer.

[0054] For example, to detect head and neck cancer, an individual's saliva sample may be analyzed using the methods disclosed herein to detect HPV in the sample. This may be done, for example, by detecting one or more target genomic nucleotide sequences of HPV, or by detecting mRNA associated with the transcription of the HPV genome in host cells. For example, in various embodiments, the detection of HPV-related mRNA and mRNA or miRNA biomarkers associated with dysregulated cellular processes due to HPV infection can be used to identify individuals at risk of having head and neck cancer. Alternatively, the simultaneous detection of HPV and protein biomarkers such as the p-16 protein may indicate that an individual is at risk of having head and neck cancer.

[0055] As described above, in various embodiments, the teachings of the present invention can also be used for the detection of cervical cancer. For further explanation, Figure 2 shows an example of a conventional workflow for cervical cancer and a workflow according to one embodiment of the present invention. In the conventional workflow shown on the left, a patient sample is tested for the presence of HPV, and if positive, a test is performed to detect cancer protein biomarkers. If cancer protein markers are detected, the presence of cancer is confirmed and treatment options are considered; however, if they are not detected, no particular action is taken, and future screening is recommended. Furthermore, conventionally, cells can be collected from the surface of the cervix by Pap smear testing. The collected cells can be examined for the detection of cervical cancer or changes that may lead to cervical cancer. The cells can also be examined for the presence of cancer protein biomarkers. However, in only about 10% of cases, the results of biopsies or Pap smear tests performed after a positive HPV test led to positive findings for cancer. In other words, the conventional approach involves many unnecessary tests, resulting in associated costs and potential complications.

[0056] In contrast, the workflow according to one embodiment of the present invention, shown on the right side of Figure 2, allows a single patient sample to be tested for different classifications of cancer biomarkers, such as genetic and protein components, on the same cartridge, for example. A negative result indicates that a treatment protocol is not required, and a positive result indicates the presence of cancer. In other words, practical results can be obtained with a single test.

[0057] In various embodiments, the detection of a target nucleotide sequence, such as a target genome sequence and / or mRNA or miRNA, is achieved by amplifying the target sequence and then detecting the amplicon of the target nucleic acid sequence generated by the amplification. For example, in various embodiments, loop-mediated isothermal amplification (LAMP) is performed, followed by a detection mechanism using a CRISPR (Cas12a) system that can be selectively activated by the target nucleic acid sequence, thereby activating the fluorophore of the fluorophore-quencher probe (via the cleavage of the linker between the fluorophore and the quencher by the activated CRISPR enzyme) and achieving extremely high selectivity.

[0058] Table 1 below shows examples of various types of biomarkers reported for head and neck cancer.

[0059] [Table 1]

[0060] As described above, in various embodiments, amplification techniques such as loop-mediated amplification (LAMP) can be used to amplify various nucleotide sequences, such as the genetic components of viruses in a sample, by the methods described in relation to the embodiments described above. Primers for loop-mediated amplification (LAMP) of HPV16, HPV18, and HPV31 have already been reported (for example, “Method for elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16,” Landaverde et al., Analytical and Bioanalytical Chemistry, volume 412, pages 6199-6209 (2020), the entire literature is incorporated herein by reference. Also, “Detection assay for HPV16 and HPV18 by loop-mediated isothermal amplification with lateral flow dipstick tests,” Kumvongpin et al., Moleclar Medicine Reports, volume 15, Issue 5 (2017), the entire literature is also incorporated herein by reference).

[0061] For example, Table 2 below shows the primers published in “Assessing the performance of a loop-mediated isothermal amplification (LAMP) assay for the detection and subtyping of high-risk subtypes of Human Papilloma Virus (HPV) for Oropharyngeal Squamous Cell Carcinoma (OPSCC) without DNA purification,” Rohtensky et al., BMC Cancer volume 18, Article number: 166 (2018).

[0062] [Table 2]

[0063] Other nucleic acid amplification strategies combining recombinase polymerase amplification (RPA) and exonuclease (Exo) assays (referred to herein as RPA-Exo assays) can also be used for rapid and highly specific amplification and detection of target nucleic acid sequences. For example, RPA-Exo is described in the paper entitled "Rapid detection of SARS-CoV-2 by low volume real-time single tube reverse transcription recombinase polymerase amplification using an exo probe with an internally linked quencher (exo-IQ)" by Ole Berhmann, Iris Bachmann, Martin Spiegen, Marina Schramm, Ahmed Abd El Wahed, Grehrad Dobler, Gregory Dame, and Frank T Hufert, the entire contents of which are incorporated herein by reference.

[0064] In summary, RPA can be used as an isothermal amplification method to replace conventional polymerase chain reaction (PCR). While PCR requires the use of a thermal cycler, RPA functions optimally in the temperature range of 37–42°C. As a result, it can be performed with simpler and less expensive equipment. The RPA process relies on three main enzymes: recombinase, single-strand DNA-binding protein (SSB), and strand-displacing polymerase. Recombinase has the ability to pair oligonucleotide primers with homologous sequences in double-stranded DNA. SSBs bind to substituted DNA strands, preventing primer substitution. Subsequently, strand-displacing polymerase initiates DNA synthesis at the site where the primer has bound to the target DNA. Using forward and reverse primers, exponential DNA amplification is initiated if the target sequence is present. Under optimal temperatures (37–42°C), the reaction proceeds rapidly, specifically amplifying DNA from a few copies of the target to a detectable level, usually within about 10 minutes. This enables rapid detection of DNA, RNA, and even short-chain aptamer DNA. Incorporating exonuclease III allows for the use of exoprobes for real-time fluorescence detection similar to real-time PCR. The fluorescence signal generated by RPA-Exo can be detected with an optical device equipped with a suitable fluorescence filter. Furthermore, lateral flow strip detection can be used when both endonuclease IV and nfo probes are employed. Adding reverse transcriptase operating at 37–42°C allows for the reverse transcription of RNA, and the resulting cDNA can be amplified in a single step. The RPA reaction can be multiplexed by introducing additional primer / probe pairs, enabling the detection of multiple analytes or internal controls within the same reaction mixture.

[0065] In various embodiments, the detection of protein biomarkers can be achieved using a variety of techniques known to those skilled in the art. For example, a target protein can be detected using an antibody labeled with a fluorescent probe that specifically binds to the target protein.

[0066] Furthermore, a novel assay for the detection of target proteins (referred to herein as immunoRPA or its abbreviation iRPA) is described herein and can be used in various embodiments. However, the use of this method for the detection of target proteins is not limited to the uses disclosed herein. Rather, it is broadly applicable to the detection of target proteins in samples, such as biological samples.

[0067] Referring to the flowchart in Figure 3A and the schematic diagram in Figure 3B, such a target protein detection assay involves incubating a sample with a first antibody configured to bind specifically to a first epitope of the target protein in the presence of a substrate, and to bind to a substrate, e.g., a substrate labeled with streptavidin (e.g., see streptavidin-2 functionalized magnetic beads 1), by biotinylation (e.g., see biotinylated antibody 1), and a second antibody conjugated with an oligonucleotide (e.g., see conjugation of antibody 2 with a random RPA sequence of 120-200 nucleotide base pairs). Furthermore, in various embodiments, PBS-Tween supplemented with salmon sperm DNA is added to the reaction mixture.

[0068] The reaction is generally carried out at room temperature for a period of time, for example, in the range of approximately 30 to 40 minutes. If the target protein is present in the sample, the incubation described above will allow the target protein to bind to the first and second antibodies, and the first antibody will become conjugated to the substrate (for example, via the binding of biotin to streptavidin). After the incubation step, a washing step may be performed to remove unbound antibodies, non-target proteins, and other components not bound to the substrate.

[0069] As an example, and referring specifically to Figure 3B, when the capture substrate is magnetic beads, the reaction product is subjected to a magnetic field, and the complex of magnetic beads, associated antibodies, conjugated oligonucleotide sequences, and target proteins is captured. Unbound components are washed with PBS-Tween.

[0070] After the washing step, the RPA-Exo reaction component is added, the oligonucleotide sequence is amplified, and a fluorescent signal is obtained to identify the presence of the target protein.

[0071] In various embodiments, the above immunoRPA (iRPA) method significantly enhances the signal of trace proteins present in the sample.

[0072] As an example, the iRPA protein detection assay described above can be used to detect proteins at femtomole and atomole levels. For example, a target-selective antibody is labeled with biotin and conjugated to a streptavidin-treated tube or plate, or to streptavidin Dynabeads. Another matched antibody for the same target, having a different epitope than the first antibody, is labeled with a 150-200 nucleotide random oligonucleotide designed for use as an RPA primer and labeled exoprobe for the RPA-Exo reaction. In one embodiment, an antibody for p16 is labeled with streptavidin Dynabeads, and another matched p16 antibody is labeled with a random RPA oligonucleotide. The two antibodies and the p16 protein are mixed for 30-45 minutes. Each antibody has a distinct epitope for p16. After incubation, a magnetic field is applied, and a washing step is performed to remove unbound antibodies and other unbound components. The reaction mixture for RPA-Exo is introduced, and the reaction is read with a fluorescence reader after 10 minutes.

[0073] In various embodiments, a system for point-of-care detection of cervical cancer is provided, which includes a sensor, such as an electrochemical or optical sensor, configured for point-of-care detection of at least one of the high-risk HPV16, HPV18, and HPV31 nucleotide acids, as well as at least one cervical cancer protein biomarker, such as the P16 tumor suppressor protein (hereinafter referred to as P16), also known as p16INK4a / CDKN2A, and / or Ki-67. In some embodiments, the system may be configured to detect at least one of the methylated DNA biomarkers ASCL1 or LHX8, in addition to at least one high-risk nucleotide acid and at least one P16 and Ki-67 protein. By simultaneously detecting two or more of the genes, proteins, and methylated DNA markers associated with cervical cancer, clinicians can determine whether an HPV-positive individual is progressing to cervical cancer.

[0074] In various embodiments, cartridges may be used for the multiple detection of orthogonal classification biomarkers in a specimen. For example, the cartridge described in U.S. Patent No. 11,541,393 (hereinafter referred to herein as the "'393 Patent"), “System, Apparatus and Method for Pathogen Detection,” which has been assigned to the assignee of this application and is incorporated herein by reference in its entirety, may be configured for the multiple detection of multiple orthogonal classification biomarkers based on the disclosures of the present invention. For example, the cartridge shown in Figure 19A of the Patent, which is reproduced herein as Figure 4, may be configured for the detection of multiple orthogonal classification biomarkers based on the disclosures of the present invention.

[0075] For example, the cartridge shown in Figure 4 may be configured to include a bank of detection elements configured to detect at least one nucleotide sequence relevant to the detection of the HPV virus, and a bank of other detection elements (also referred to herein as sensors) configured to detect biomarkers derived from phenotypic, regulatory, metabolome, and / or microbiome dysregulation that may occur due to HPV infection. For example, the detection elements may be electrochemical sensors in which the working electrode is functionalized with reagents such as oligonucleotides, antibodies, aptamers, SOMAmers, raptomers, Megastar, or combinations thereof. For example, in some such embodiments, an electrochemical sensor for detecting genetic components of the HPV virus may be functionalized with oligonucleotides that specifically bind to the target genetic components of the virus, and an electrochemical sensor for detecting protein biomarkers may be functionalized with antibodies that specifically bind to proteins. In some embodiments, one detector bank may include a plurality of detectors each configured to detect different genetic biomarkers related to the HPV virus, and the other detector bank may include a plurality of sensors each configured to detect different protein biomarkers related to cervical cancer. In this way, these embodiments not only enable multiple detection of gene and protein biomarkers in a single cartridge, but also provide the ability to detect multiple different members for each category (i.e., gene or protein marker detection), thereby improving the sensitivity and specificity of the instrument.

[0076] More specifically, the disposable cartridge 4000 comprises three layers 4002 (lower layer), 4003 (middle layer), and 4004 (upper layer), which together form a cartridge frame. In this embodiment, the upper and lower layers are made of an optically transparent material, such as glass or an optically transparent polymer material. In some embodiments where the upper layer is made of glass, at least a polymer coating (e.g., PDMS coating) may be applied to the inner surface of the glass in areas that may come into contact with the sample to be analyzed. In some such embodiments, a suitable polymer coating layer may be applied to the entire inner surface of the upper glass layer.

[0077] In this embodiment, the intermediate layer includes a sample receiving well 4005, which can receive a sample for analysis via an inlet port 4007. The cartridge 4000 can be configured for the analysis of various different biological specimens. Generally, the cartridge 4000 can be configured for the diagnostic analysis of liquid biopsy samples. Examples of samples that can be analyzed using the cartridge 4000 include, but are not limited to, blood and saliva.

[0078] Cartridge 4000 further includes two wells (reservoirs) 4008 / 4009, one of which stores a buffer (conveniently referred to herein as "gene buffer") for processing (preparing) a portion of the received sample for the detection of one or more target nucleotide sequences, if present in the received sample; and the other store a buffer (conveniently referred to herein as "protein buffer") for processing (preparing) another portion of the received sample for the detection of one or more target proteins.

[0079] For the sake of explanation, in this embodiment, it is assumed that gene buffer is stored in well 4008 and protein buffer is stored in well 4009. Fluid channel 4011a fluidizes the sample receiving well 4005 and the buffer well 4008, and another fluid channel 4011b fluidizes the sample receiving well and the buffer well 4009. Isolation valve 4013 prevents backflow.

[0080] The specimen portions received in reservoirs 4008 / 4009 come into contact with gene buffer and protein buffer, respectively. The buffer reservoir 4008 may be in the form of a blister pouch 4015 in which the gene buffer is stored. The blister pouch 4015 includes a flexible membrane 4015a that forms an enclosure for storing the buffer internally. A separation membrane 4015b is positioned inside the blister pouch on an internal puncture arm 4015c. The blister pouch can be activated by pressing the flexible membrane 4015a, which increases the liquid pressure in the enclosure inside the blister, and the liquid inside the pouch is released as the internal puncture arm may be crushed via the pressure applied by the separation membrane 4015b.

[0081] In this embodiment, a pneumatic control valve 1 connected to the blister pouch 4015 is controlled by a controller 1a, enabling the transfer of the liquid released from the blister pouch 4015 to a downstream amplification reservoir (well) 4016. In the amplification reservoir 4016, if nucleotide sequences are present in the sample collected from the subject, the nucleotide sequences (e.g., DNA / RNA sequences) in the liquid released from the blister are amplified. As described above, amplification of nucleotide sequences can be achieved using various different amplification modalities. For example, isothermal amplification may be used in some embodiments, and amplification / detection methods such as RPA-Ex may be used in other embodiments.

[0082] For example, in this embodiment, the amplification well 4016 may contain one or more reagents (such as primers) suitable for isothermal amplification of a target nucleotide sequence when a pathogen is present in the sample. For example, the gene buffer contained in the buffer reservoir may, along with other reagents, contain reagents for lysing viral particles and releasing one or more RNA / DNA fragments. Such RNA / DNA fragments can then be isothermal amplified in the amplification well. A heating / cooling element, such as a Peltier element and its associated thermistor, is incorporated into the cartridge to maintain the temperature of the gene buffer at a desired value. After the amplification of the nucleotide sequence is complete, the pneumatic control valve 2, activated via its associated controller 2a, can transfer the amplified sample through the mixing element 4014, implemented as a serpentine channel, to a group of sensors 4020, each configured to detect a different nucleotide sequence. The passage of the sample through the mixing element 4014 further facilitates sample preparation for detection of the target nucleotide sequence in the sample. For convenience of explanation, the liquid exiting the mixing element is referred to herein as the processed sample.

[0083] More specifically, in this embodiment, the detection element group 4020 includes four sensors 4020a, 4020b, 4020c, and 4020d, each sensor configured to detect a different target nucleic acid sequence (e.g., RNA and / or DNA strands). For example, in some embodiments, each detection element 4020 may be implemented as an electrochemical sensor functionalized for the detection of a target nucleotide sequence.

[0084] Continuing to refer to Figure 4, the protein buffer reservoir 4009 is fluidically connected to the sample receiving reservoir 4005 via a fluid channel 4011b to receive a portion of the sample collected from the individual. The interaction between the received sample portion and the protein buffer can generate a processed sample suitable for introduction into the detection element group 4021 for detecting multiple proteins associated with the pathogen, if the target pathogen is present in the sample collected from the subject. More specifically, in this embodiment, the detection element group 4021 includes four sensors 4021a, 4021b, 4021c, and 4021d, each sensor configured to detect a different target protein. In some embodiments, there may be only one sensor for detecting a single target protein, or there may be multiple sensors for detecting additional target proteins. For example, in this embodiment, each detection element may be implemented as an electrochemical sensor functionalized for the detection of different target proteins by functionalizing the working electrode of each electrochemical sensor with different antibodies that specifically bind to different target proteins.

[0085] While this cartridge does not implement amplification of signals related to target proteins, other cartridges can amplify signals related to target protein detection using various methods, such as the iRPA technology described here.

[0086] In other embodiments, in addition to or instead of electrochemical detection, one or more detection elements may be configured for the optical detection of target proteins and / or target nucleotide sequences. For example, a fluorescent dye-quencher probe may be used to detect target nucleotide sequences using an RPA-Exo assay or the like.

[0087] The reader unit (also referred to herein as an analyzer) may be one described in the '393 patent or one modified based on the teachings of the present invention, and may be capable of receiving detection signals from a cartridge and processing these signals to detect various desired biomarkers.

[0088] In some embodiments, electrochemical detection of nucleic acid sequences (e.g., those associated with viral particles such as HPV) can be performed using the technique described by Zamani et al., “Electrochemical strategy for low-cost viral detection,” (ASC Cent.Sci.2021, 7, 963-972, incorporated here in its entirety by reference). Briefly, this method monitors HPV DNA-activated Cas12a endonuclease activity using a DNA-modified gold foil electrode. The gold foil electrode is functionalized with methylene blue-labeled oligonucleotides, and Cas12a is used to detect plasmids containing the HPV gene sequence. The guide RNA (gRNA) of the Cas12a enzyme is designed to recognize the p24 locus of the HPV gag gene. The activating enzyme cleaves the oligonucleotide bound to the gold surface, releasing methylene blue, a well-known redox mediator, which causes a change in the electrochemical signal obtained by cyclic voltammetry. This strategy cannot be multiplexed within the same well, but spatial multiplexing may be possible.

[0089] Furthermore, in some embodiments, one or more sensors in the cartridge according to various embodiments may be functionalized with agents that specifically bind to protein biomarkers for the detection of proteins in a sample. For example, antibodies that specifically bind to the p16 protein are commercially available and can be used to functionalize the working electrodes of the electrochemical sensors according to various embodiments. For example, Abcam's Human CDKN2A / p16INK4a (ab227903) antibody can be used for this purpose.

[0090] Furthermore, in some embodiments, one or more sensors in the cartridge according to the teachings of the present invention may be configured for the detection of one or more disease-related epigenetic changes in conjunction with point mutations and / or protein expression. For example, such epigenetic changes may correspond to methylation of genes such as RUNX3, SFRP1, WIF1, PCDH10, DKK2, DKK3, TMEFF2, OPCML, and SFRP2, which exhibit higher methylation levels in tumors compared to normal tissue. Additionally, K-Ras mutations are found in over 40% of colorectal cancers. https: / / doi.org / 10.3389 / fonc.2021.697409.

[0091] As a further example, Figure 5 shows an example of an apparatus suitable for carrying out the methods according to various embodiments. Figure 5 is a schematic top view of system 500 in which six emitter / detector pairs 504a, 504b, 504c, 504d, 504e, and 504f are arranged in a housing 502, distributed around an opening 506. A vial 508 can be inserted into the opening 506, and the specimen in the vial can be exposed to radiation emitted from the emitters of the emitter / detector pairs. The detector of each emitter / detector pair can detect the fluorescence emission emitted from the specimen in the vial, as will be described later. For example, in this embodiment, the emitter / detector pairs enable multiple detection of six different target nucleotide sequences using six different fluorescent dye / quencher probe pairs. In various embodiments, optical filters placed in front of the detectors of the emitter / detector pairs can be used to ensure that each detector detects fluorescence emission at different wavelengths.

[0092] More specifically, in this embodiment, each emitter / detector pair can be used to excite and detect fluorescence emission from different nucleotide sequences, but the system can also be used to detect different proteins (e.g., using fluorescently labeled antibodies) or combinations of one or more proteins and one or more nucleotide sequences. In some embodiments, one or more emitter / detector pairs can be used to detect one or more target proteins, and one or more other emitter / detector pairs can be used to detect one or more target genomic sequences, one or more mRNAs or miRNAs, etc.

[0093] Multiplexing of the detection of six target biomarkers (e.g., proteins) can be achieved by using the following fluorescent dyes for the detection of each target protein: FAM (excitation: 430 nm, detection: 480 nm), HEX / VIC (excitation: 470 nm, detection: 515 nm), ROX (excitation: 545 nm, detection: 565 nm), Cy5 (excitation: 575 nm, detection: 610 nm), Cy5.5 (excitation: 628 nm, detection: 670 nm), and Ato425 (excitation: 682 nm, detection: 725 nm).

[0094] The fluorescence signal generated by the emitter / detector pair can be processed, for example, using an onboard process, to identify whether or not a target nucleic acid sequence is present in the sample.

[0095] As a further example, Figure 6 is a schematic diagram of a cartridge 600 according to several embodiments, including a frame 602 having a sample well capable of receiving a specimen. A pneumatic port P1 can be operated (for example, under the control of a controller not shown in this figure) to apply positive pressure to the sample in the sample well, housing a nucleic acid lysis chamber 606a (also referred to herein as the nucleic acid lysis chamber) containing lyophilized reagents and capable of distributing the sample for processing for nucleic acid detection, and a protein lysis chamber 606b (also referred to herein as the protein lysis chamber) capable of distributing the sample for processing for the detection of one or more target proteins.

[0096] Cartridge 600 further includes multiple protein detection wells (protein 1, protein 2, protein 3, protein 4, protein 5). Two valves V1 and V5 regulate the flow of sample from the protein lysis chamber 606b to the multiple protein detection wells. The cartridge further includes multiple nucleic acid (NA) detection wells (NA1, NA2, NA3, NA4, NA5, NA6, NA7, NA8, NA9, NA10, NA11, NA12). The flow of sample between the nucleic acid lysis chambers is regulated by valve V2.

[0097] As an example of the operating mode, when the sample is transferred from the sample well 604 to the nucleic acid and protein lysis chambers 604a and 604b under positive pressure (via the pneumatic port P1), valves V1, V5, V2, and V4 are closed. Subsequently, valves V5 and V1 are opened, while valves V2, V4, and V6 remain closed, and the sample portion in the protein lysis chamber is transferred to the protein detection wells (proteins 1 to 5). In this embodiment, the protein detection wells contain multiple magnetic beads functionalized with streptavidin and two antibodies that specifically bind to one epitope of the target protein, one of which is biotinylated to bind to the streptavidin on the magnetic beads, and the other antibody is conjugated with one or more random nucleotide sequences of 120-200 bp.

[0098] After a predetermined incubation period, the wash buffer blister pack 608 located on the cartridge frame may be punctured (by the controller unit into which the cartridge is introduced after sample introduction), thereby allowing the wash buffer inside to flow into the wash buffer collection well 610. Multiple valves V3 located between each protein detection well and the wash buffer collection well 610 may be opened, and the pneumatic port P5 may be activated, pressurizing the wash buffer in the collection well and allowing it to flow into the protein detection well. Subsequently, valve V1 is closed and valve V4 is opened, and the wash buffer is transferred from the protein detection well to the empty sample reservoir (which functions as a waste well at this stage) by the positive pressure from the pneumatic port P4. During the washing step, a magnetic field is applied to the protein detection well (by a controller not shown in this figure) to collect the magnetic beads at the bottom of the well, thereby preventing the beads from flowing out of the protein detection well.

[0099] Multiple hydrophobic vents 613 prevent interference from bubbles that may form as liquid enters and exits the protein detection well.

[0100] After the washing step is complete, valve V4 is closed, puncturing the blister pack 612 containing water, and the pneumatic port P6 is activated to apply positive pressure to the water released from the blister pack, transferring the water to well 614, which stores the lyophilized reagent for performing the RPA-Exo assay. Here, the water reconstitutes the lyophilized reagent. Subsequently, valves V6 and V1 are opened, transferring the RPA-Exo reagent to the protein detection wells. Each protein detection well may be configured for the detection of different target proteins, depending on the selection of antibodies. The fluorescence emitted from the fluorescent dye of the RPA probe may be detected by a detector provided in the reader unit, and the signal may be analyzed via firmware on the reader unit or remotely to determine whether one or more target proteins are present in the sample being analyzed. For example, the fluorescence emission from each protein detection well may be detected periodically, for example every minute, generating multiple fluorescence detection signals that can be analyzed for the detection of target proteins.

[0101] In some embodiments, at least one protein detection well may be configured for the multiple detection of two or more proteins, which can be achieved, for example, by storing fluorescently labeled antibodies that specifically bind to two or more target proteins and having fluorescent dye probes that emit light at different wavelengths when excited.

[0102] Next, during the fluorescence detection process from the protein detection well, valve V2 is opened and valve V5 remains closed, and the sample portion in the lysis chamber 606a is transferred to several nucleic acid detection wells NA1 to NA12. Each of the wells NA1 to NA12 may contain lyophilized reagents for amplifying different target nucleic acid sequences, such as isothermal amplification reagents, or lyophilized reagents such as the CAS12a system and fluorescent dye-quencher probes. In various embodiments, each nucleic acid well may be used for the detection of up to six nucleic acid sequence targets using probes with six different fluorescent dye and quencher pairs (e.g., FAM, HEX / VIC, ROX, Cy5, Cy5.5, Ato425). The fluorescence emission generated in each nucleic acid well is detected by multiple detectors provided on a reader device configured to accept cartridges and can be analyzed for the identification of target nucleic acid sequences of interest.

[0103] Similar to the protein detection wells, each nucleic acid detection well is connected to one of several hydrophobic vents 615.

[0104] Figure 7 is a schematic diagram showing the components of a controller / fluorescence reader 700 that can be used to control the cartridge 600 and detect fluorescence emission generated in each well of the cartridge. The controller 700 includes a digital data processor 702, a random access memory module (RAM) 704, and a read-only memory (ROM) module 706, which operate under the control of the processor 702. The controller 700 further includes multiple valve actuators 708, multiple fluorescence detectors 710, a magnetic field generator 712, a pneumatic port controller 714, and multiple blister pack puncture elements 716, which also operate under the control of the processor 702.

[0105] Various commercially available valve actuators, fluorescence detectors, magnetic field generators, and pneumatic port controllers can be used in various implementations of the controller. For example, the valve actuator may be in the form of a spring-loaded pogo pin that can be operated to block the flow path and prevent the passage of liquid. Furthermore, the magnetic field generator may be an electromagnet operating under the control of the controller, which can be operated to hold magnetic beads in the protein detection well within the well.

[0106] Instruction sets for operating the cartridge according to various embodiments may be stored in a ROM module and transferred to a RAM module at runtime for execution by a processor. For example, instruction sets may include instructions for opening and closing various valves, operating various pneumatic ports, puncturing various blister packs, applying a magnetic field to protein detection wells, and detecting radiation using a fluorescence detector. Furthermore, in some embodiments, the ROM module may also include instructions for analyzing the fluorescence signal generated by the fluorescence detector to identify pathogens and / or biomarkers of interest.

[0107] By enabling multiplexing within each chamber and spatial multiplexing, this cartridge allows for high-performance detection of not only biomarkers of the same classification but also multiple biomarkers of different classifications, all within a sealed, easy-to-use cartridge, simply by the user placing the specimens on it.

[0108] As described above, the fully sealed cartridge has spatially separated chambers, enabling the multiplexing of multiple gene markers and multiple protein, genome, metabolomics, and other markers. For example, HPV16, HPV18, HPV31, and HPV35 can each be detected in separate nucleic acid wells, and each chamber contains lyophilized reagents for amplification of specific targets. Alternatively, multiple detection of the above HPV strains can be performed using the same nucleic acid well, with probes and lyophilized reagents having different fluorescent dyes and quenchers for each target. For example, FAM, HEX, CY5, ROX, and quenchers specific to each target can be used for multiplexing.

[0109] Multiplexing of 120 biomarkers can be easily achieved with a chip having 20 reaction chambers. Such multiplexing is performed by combining RPA (recombinant polymerase amplification) at 37-40°C with exonuclease assays. Exonuclease probes with up to six different fluorescent dyes and quenchers for different wavelengths, such as FAM, HEX / VIC, ROX, CY5, CY5.5, and Ato425, can be incorporated, excited at 430nm, 470nm, 535nm, 575nm, 628nm, and 682nm, and detected at 480nm, 515nm, 565nm, 610nm, 67nm, and 725nm using LED light and photodiode detectors.

[0110] In some embodiments, innovative immuno-RPA (iRPA) may be used in a cartridge to amplify and detect target proteins. For example, a p16-positive sample may be introduced into a cartridge and sent to a protein lysis chamber. After incubation and mixing in the cartridge for 1–5 minutes, the lysis solution is sent to the i-RPA chamber, which contains streptavidin-Dynabeads-labeled antibody and oligo-labeled antibody against p16. A mixing step is performed in the cartridge, such as a push-pull, and after a reaction of 30–40 minutes, a magnetic field is activated. The blister pack in the cartridge is punctured, and the washing buffer is sent to the protein reaction chamber. The used washing buffer can be pushed back into the now-empty sample chamber and used as a waste chamber to save space. Another chamber containing reaction reagents for the RPA-Exo assay is activated and the reagents are sent to the protein chamber. After 10 minutes, the presence or absence of the target protein is measured based on an exoprobe incorporating a fluorescent dye and quencher used for the specific target.

[0111] The teachings of the present invention may provide an orthogonal classification biomarker detection platform for the diagnosis of not only cancer but also other diseases associated with multiple classifications of biomarkers, such as neurodegenerative diseases and infectious diseases.

[0112] For example, in ALS patients, novel differentially methylated RHBDF2 genes have been discovered using cell-free DNA (cfDNA) isolated from whole blood samples compared to a control group (e.g., “Liquid biopsy: a new source of candidate biomarkers in amyotrophic lateral sclerosis,” Mendioroz et al., Ann Clin Transl Neurol., 2018 Apr, 16:5(6):763-768, incorporated herein by reference in its entirety).

[0113] Furthermore, serum NfL is a clinically validated prognostic biomarker for ALS (e.g., “Validation of serum neurofilaments as prognostic and potential pharmacodynamic biomarkers for ALS,” Benatar et al., Neurology, 2020 Jul 7:95(1):e59-e69, incorporated herein by reference in its entirety). Mutations in ALS-predisposing genes such as A4V and SOD1, and hexanucleotide repeat elongation of the C9ORF72 gene are also genotype biomarkers for ALS (“ALS biomarkers for therapy development: State of the field and future directions,” Benatar et al., Muscle & Nerve, vol.53, issue 2, pp.169-182, incorporated herein by reference in its entirety). Numerous increased or decreased snRNAs and muscle snRNAs have been identified in ALS and are being evaluated as diagnostic biomarkers for the disease. For example, miR-1825 and miR-1234-3p were consistently downregulated in the serum of patients with sporadic ALS. Table 2 lists the diagnostic biomarkers for ALS ("Serum microRNA in sporadic amyotrophic lateral sclerosis," Freischmidt et al., Neurobiol Aging, 2015 Sep:36(9):2660.e15-20, incorporated herein by reference in its entirety). Simultaneous detection of two or more of these genotype, phenotype, and epigenetic ALS biomarkers can enable faster and more accurate treatment decisions, potentially improving or saving patients' lives.

[0114] [Table 3-1] [Table 3-2]

[0115] As another example, Alzheimer's disease (AD) is characterized by the accumulation of amyloid-beta (Aβ) peptide precursor protein (APP). miR-346 has been found to regulate Aβ production. (See, for example, "Novel upregulation of amyloid-β precursor protein (APP) by microRNA-346 via targeting of APP mRNA 5'-untranslated region: Implications in Alzheimer's disease" (Long et al., Mol Psychiatry. 2019 Mar:24(3):345-363, the entire work is incorporated herein by reference)). Other studies have shown that 10 miRNAs (hsa-mir-107, hsa-mir-26b, hsa-mir-30e, hsa-mir-34a, hsa-mir-485, hsa-mir-200c, hsa-mir-210, hsa-mir-146a, hsa-mir-34c, and hsa-mir-125b) are dysregulated in the early stages of AD. (See, for example, "Systemic review of miRNA as biomarkers in Alzheimer's disease" (Swarbrick et al., Mol Neurobiol, February 8, 2019, incorporated herein by reference in its entirety)).

[0116] Furthermore, several blood-based biomarkers have been identified that alter gene expression in the early stages of AD. These include RAB7A, NPC2, TGFB1, GAP43, ARSB, PER1, GUSB, MAPT, GSK3B, PTGS2, APOE, BACE1, PSEN1, and TREM2 in the blood. (See, for example, "Blood biomarkers for memory: toward early detection of risk for Alzheimer disease, pharmacogenomics, and repurposed drugs" (Niculescu, Mol Psychiatry, 25(8):1651-1672, 2020)). Metabolite signatures in the blood have also been identified as promising biomarkers for the early detection of AD. In particular, sphingolipids are considered to be biomarkers that are biologically relevant to the early detection of AD. The present invention's approach of detecting two or more of the altered gene or protein expression, miRNAs, or metabolites in AD may provide early and accurate detection of this devastating disease.

[0117] As another example, RT-PCR, isothermal PCR, and other nucleic acid (NA) amplification tests have been developed for the detection of SARS-CoV-2. Antigen detection assays such as lateral flow assays (LFA) have also been developed. Lab-based and some point-of-care (POCT) tests for SARS-CoV-2 detection are highly sensitive and can detect the pathogen with low detection limits. However, clinical and public health challenges arise because some patients continue to test positive for SARS-CoV-2 weeks or months after infection. Therefore, NA testing alone cannot distinguish between infection and infectivity. On the other hand, protein assays that detect nucleocapsid proteins alone generally have low sensitivity and high false-negative rates. Therefore, simultaneously detecting both proteins and nucleic acids can enable early and accurate detection of SARS-CoV-2 and provide information about the patient's infection status and infectivity. Furthermore, understanding the host immune response to the pathogen is also important. After SARS-CoV-2 or COVID-19 vaccination, host response antibodies are produced within 2-3 weeks. These antibodies can persist in the blood for several months.

[0118] Simultaneous and orthogonal detection of antibody responses to infection or vaccination, combined with assessment of the presence of infectious agents, can provide comprehensive, timely, and accurate results regarding infection and infectious status.

[0119] Those skilled in the art will understand that various modifications can be made to the above embodiments without departing from the scope of the present invention.

Claims

1. A method for screening individuals for head and neck cancer, The steps include: collecting a saliva sample from the aforementioned individual; The procedure includes the step of analyzing the aforementioned saliva sample to detect one or more HPV genomes and at least one of the phenotypic biomarkers, regulatory biomarkers, microbiome-derived biomarkers, and metabolome biomarkers that have become dysregulated by HPV infection. A method for screening an individual for head and neck cancer, which indicates that the individual is at risk of head and neck cancer by detecting both HPV and at least one of the biomarkers.

2. The method according to claim 1, wherein the phenotypic biomarker comprises either a protein or mRNA.

3. The method according to claim 1, wherein the regulatory biomarker comprises miRNA.

4. The method according to claim 1, wherein the step of analyzing the saliva sample to detect HPV includes detecting at least one nucleotide target sequence associated with the HPV genome.

5. The method according to claim 1, wherein the step of analyzing the saliva sample to detect HPV includes detecting at least mRNA associated with the transcription of the HPV genome in infected cells.

6. The method according to claim 1, wherein any of the at least one phenotypic biomarkers is associated with a cellular molecular process affected by HPV infection.

7. The method according to claim 1, wherein the at least one protein biomarker comprises a p-16 protein and an EGF receptor.

8. The method according to claim 1, further comprising the step of triaging the individual for further confirmatory diagnosis, such as MRI imaging, if the individual is found to be at risk of head and neck cancer.

9. An assay for detecting a target protein in a sample, The steps include: incubating a sample in the presence of a substrate with a first antibody that specifically binds to a first epitope of a target protein and a second antibody that specifically binds to a second epitope of the target protein to form an incubation mixture of the sample and the antibodies, wherein the first antibody is further configured to be conjugated to the substrate and the second antibody is conjugated to a nucleotide sequence; Next, the incubation mixture is washed to remove components that are not bound to the substrate. An assay for detecting a target protein in a sample, comprising the steps of: amplifying an oligonucleotide sequence using an RPA-Exo assay and detecting the oligonucleotide sequence to detect the target protein.

10. The assay according to claim 9, wherein the substrate is functionalized with streptavidin, and the first antibody is biotinylated to bind to streptavidin.

11. The assay according to claim 9, wherein the substrate comprises the surfaces of a plurality of magnetic beads.

12. The assay according to claim 11, further comprising the step of collecting the magnetic beads using a magnetic field, following the incubation step.

13. The assay according to claim 12, wherein the incubation step is performed for a time range of approximately 30 to 40 minutes.

14. A method for screening individuals for cancer transmitted by HPV infection, The steps include: collecting a biological specimen from the aforementioned individual, The procedure includes the step of analyzing the biological specimen to detect HPV and at least one of a phenotypic biomarker, regulatory biomarker, metabolome biomarker, and microbiome-derived biomarker that has been dysregulated by HPV injection, By detecting both HPV and at least one of the biomarkers, it is indicated that the individual is at risk of the cancer. A method for screening individuals for cancer transmitted by HPV infection.

15. The method according to claim 14, wherein the cancer is any one of cervical cancer, head and neck cancer, penile cancer, vaginal cancer, valvular cancer, and anal cancer.

16. The method according to claim 14 or 15, wherein the phenotypic biomarker comprises either a protein or mRNA.

17. The method according to claim 16, wherein the regulatory biomarker comprises miRNA.

18. The method according to claim 16, wherein the step of analyzing the biological sample to detect HPV includes detecting at least one nucleotide target sequence associated with the HPV genome.

19. The method according to claim 16, wherein the step of analyzing the biological sample to detect HPV includes detecting at least mRNA associated with the transcription of the HPV genome in infected cells.

20. The method according to claim 16, wherein any of the at least one phenotypic biomarkers is associated with a cellular molecular process affected by the HPV infection.

21. The method according to claim 16, wherein the at least one protein biomarker comprises a p-16 protein and an EGF receptor.

22. The method according to claim 16, further comprising the step of triaging the individual for MRI imaging if the individual is found to be at risk of head and neck cancer.

23. The steps of collecting biological samples from individuals, The steps include analyzing the biological sample and detecting at least two orthogonal disease biomarkers if present in the biological sample, A method for detecting disease, which indicates that an individual is at risk of having a disease by detecting the two orthogonal disease biomarkers mentioned above.

24. The method according to claim 23, wherein one of the at least two orthogonal disease biomarkers comprises at least a target protein, and another of the at least two orthogonal disease biomarkers comprises a target nucleotide sequence.

25. The method according to claim 24, wherein either the target protein or the target nucleotide sequence indicates the presence of a pathogen in a biological sample.

26. The method according to claim 25, wherein the pathogen comprises a virus.

27. The method according to claim 25, wherein the disease comprises any one of cancer, neurodegenerative disease, hematological disease, or inflammatory disease.

28. A system for detecting disease biomarkers for at least two classifications of diseases in a biological specimen, It is a cartridge frame, Sample wells for receiving biological specimens, A first lysis chamber, which is in fluid communication with the sample well, for receiving the first portion of the biological specimen and for preparing the first sample portion for detection of at least one target protein, A second lysis chamber, which is in fluid communication with the sample well, for receiving a second portion of the sample and for preparing a second sample portion for detection of at least one target nucleic acid sequence, A washing buffer blister containing washing buffer, A collection well that is in fluid communication with the aforementioned washing buffer blister, At least one protein detection well, which is in fluid communication with the first lysis chamber and the collection well and configured to detect at least one target protein, The at least one protein detection well is Multiple magnetic particles functionalized with streptavidin, A first antibody that specifically binds to a first epitope of the target protein, The preparation contains at least a second antibody that binds to a second epitope of the target protein, The first antibody is biotinylated to bind to the streptavidin so as to be conjugated to the magnetic particles, and the second antibody comprises at least one protein detection well containing at least one oligonucleotide sequence, The apparatus comprises, a second lysis chamber and a second nucleic acid detection well configured to receive the second sample portion and to detect at least one target nucleic acid sequence, Equipped with a cartridge frame, A system for detecting disease biomarkers for at least two classifications of diseases in a biological specimen.

29. The system according to claim 29, wherein the cartridge comprises a first valve for regulating fluid communication between the first lysis chamber and the at least one protein detection well, and a second valve for regulating fluid communication between the second lysis chamber and the at least one nucleic acid detection well.

30. The system according to claim 29, wherein the biological specimen comprises any of a biological fluid, a liquid biopsy, or a tissue biopsy-derived specimen.

31. The system according to claim 29, wherein each of the first and second dissolution chambers contains a dissolving agent for processing the specimen.

32. The system according to claim 29, wherein the disease is any one of cancer, neurodegenerative disease, hematological disease, or inflammatory disease.

33. The system according to claim 33, wherein the disease is any one of cervical cancer, head and neck cancer, penile cancer, and valvular heart cancer.

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