Plasma and saliva liquid biopsy platform

Abstract

A liquid biopsy system and method for detecting biomarkers in bodily fluids is described. In particular, the system is suitable for detecting lung cancer biomarkers in a subject.

Description

[Technical Field]

[0001] Cross-reference with related applications This application claims priority to U.S. Provisional Application No. 63 / 021,172 filed 7 May 2020, which is incorporated herein by reference in its entirety.

[0002] Description of research and development funded by the federal government. This invention was made with government support under grant numbers DE017790, DE017170, CA206126, and CA233370 granted by the National Institutes of Health. The government reserves certain rights with respect to this invention.

[0003] Background of the Invention Next-generation sequencing (NGS) has uncovered a wealth of new biological information, including characteristic mutations associated with numerous solid tumors, including lung cancer, colon cancer, breast cancer, and prostate cancer (Almodovar et al., 2018, J Thorac Oncol, 13: 112-123; Schwaederle et al., 2016, Clin Cancer Res, 22: 5497-5505; Rolfo et al., 2018, J Thorac Oncol, 13: 1248-1268). Previously, tissue samples obtained by biopsy or during surgery were the only available source of specimens. Therefore, the amount of available tissue was often limited. Recently, technology has made it possible to detect and measure extracellular free DNA (cfDNA) in blood (Wei et al., 2018, J Mol Diagn, 20: 738-742) and saliva (Wei et al., 2014, Am J Respir Crit Care Med, 190: 1117-1126; Pu et al., 2016, Thoracic Cancer, 7: 428-436; Elazezy et al., 2018, Comput Struct Biotechnol J, 2018, 16: 370-378), making it possible to immediately utilize an unlimited supply of samples. This sample collection method is known as "liquid biopsy."

[0004] While liquid biopsy is still in its early stages as a clinical tool, its value in detecting epidermal growth factor receptor (EGFR) mutations at the onset and relapse of non-small cell lung cancer (NSCLC) has already been demonstrated, prompting Roche Molecular Systems to commercially launch the Cobas EGFR Mutation Test in 2017. Furthermore, private laboratories now offer single or panel mutation tests using cfDNA obtained by liquid biopsy for NSCLC patients.

[0005] To date, the widespread use of liquid biopsies has been negatively impacted by challenges related to the sensitivity and specificity of assays, and by their significant costs, often exceeding $1,000 per single mutation. Furthermore, CMS-Medicare and other third-party payers are hesitant to bear the burden of such tests, with the exception of the FDA-approved Cobas assay.

[0006] The complexity of current liquid biopsy tests necessitates the introduction of numerous pre-analysis and analytical variables, which stem from the sample collection and handling required for multiplex PCR testing. Furthermore, the complexity of analysis in next-generation sequencing (NGS) based tests requires a significant amount of time, resulting in an assay turnaround time (TAT) of typically 10–14 days.

[0007] Therefore, there is a need in the art for a high-throughput liquid biopsy system that is non-invasive, readily available, involves minimal or no sample preparation, and provides immediate information about the mutational state. The present invention satisfies this need. [Overview of the project]

[0008] In one embodiment, the present invention relates to a system for detecting a target nucleic acid molecule in a sample, comprising: a) a multiwell plate including an array of sensors, each well including an electrode tip comprising a working electrode, a counter electrode, and a reference electrode, wherein at least one unit of the working electrode is coated with a conductive polymer; b) at least one probe set including a pair of capture probes and detection probes, wherein the capture probes are embedded or functionalized in a conductive polymer, and the detection probes are further biotin-labeled at their 3' terminal nucleotides; c) a multiwell plate washer; and d) a multichannel electrochemical reader that controls the electric field applied to the array sensors and simultaneously reports the measured current.

[0009] In one embodiment, at least one of the capture probe and the detection probe hybridizes with a nucleic acid molecule containing a lung cancer marker.

[0010] In one embodiment, the capture probe includes a poly-A region.

[0011] In one embodiment, the lung cancer marker is a variant of the epidermal growth factor receptor (EGFR). In one embodiment, the lung cancer marker is exon 19 deletion, T790M, L858R, c.2235_2249 del18, c.2236_2250 del18, c.2240_2257 del18, c.2239_2248TTAAGAGAAG>C, c.2239_2247delTTAAGAGAA, or c.2239_2248 del11.

[0012] In one embodiment, the marker for lung cancer is exon 19 deletion, T790M, or L858R.

[0013] In one embodiment, a pair of capture probes and detection probes may be: a) a probe pair for detecting exon 19 deletion, where the capture probe contains the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and the detection probe contains SEQ ID NO: 6; b) a probe pair for detecting L858R, where the capture probe contains the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 14, and the detection probe contains SEQ ID NO: 2; or c) a probe pair for detecting T790M, where the capture probe contains the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 15, and the detection probe contains SEQ ID NO: 10.

[0014] In one embodiment, the present invention relates to a method for detecting lung cancer in a subject, comprising the steps of: obtaining at least one sample of the subject; mixing a first portion of at least one sample with a solution containing a labeled detection probe; and adding the mixture to a single well of a multiwell plate for use in a system for detecting a target nucleic acid molecule in the sample, wherein the system is: a) a multiwell plate comprising an array of sensors, each well comprising an electrode tip comprising a working electrode, a counter electrode, and a reference electrode, and at least one unit of the working electrode being coated with a conductive polymer; and b) at least one probe set comprising a pair of capture probe and detection probe, wherein the capture probe is embedded in a conductive polymer. The present invention relates to a method comprising the steps of: a) a probe set, wherein the detection probe is embedded or functionalized and further, the detection probe is biotin-labeled at its 3' terminal nucleotide; c) a multi-well plate washer; and d) a multi-channel electrochemical reader for controlling an electric field applied to an array sensor and simultaneously reporting a measured current, wherein each well of the multi-well plate includes an electrode tip comprising a working electrode, a counter electrode, and a reference electrode, the working electrode being coated with a conductive polymer in which a capture probe is embedded; a step of applying a periodic square wave electric field to the electrode tip; and a step of measuring the current in the electrode tip, wherein the change in current correlates with the presence of a marker associated with lung cancer in the sample.

[0015] In one embodiment, the method further includes at least one washing step in which a multiwell plate is washed using an automated plate washer.

[0016] In one embodiment, the method further includes the step of amplifying the signal. In one embodiment, the method includes the steps of: a) mixing a first portion of at least one sample with a solution containing a biotin-labeled detection probe; b) adding the mixture to a single well of a multiwell plate for use in a system for detecting nucleic acid molecules of interest in the sample, wherein the system is: a) a multiwell plate containing an array of sensors, each well containing an electrode tip comprising a working electrode, a counter electrode, and a reference electrode, and at least one unit of the working electrode being coated with a conductive polymer; b) a probe set comprising a pair of capture probes and detection probes, wherein the capture probes are embedded or functionalized in a conductive polymer, and the detection probes are further biotin-labeled at their 3' terminal nucleotides; c) a multiwell plate washer; The present invention comprises the steps of: d) a multichannel electrochemical reader for controlling an electric field applied to an array sensor and simultaneously reporting a measured current, wherein each well of a multi-well plate includes an electrode tip comprising a working electrode, a counter electrode, and a reference electrode, the working electrode being coated with a conductive polymer in which a capture probe is embedded; c) applying a periodic square wave electric field to the electrode tip; d) adding a first round of streptavidin-conjugated horseradish peroxidase (HRP) to the well; e) adding a biotin-labeled anti-HRP antibody to the well; f) adding a second round of streptavidin-conjugated HRP to the well; and g) measuring the current in the electrode tip, wherein a change in current correlates with the presence of a lung cancer-related marker in the sample.

[0017] In one embodiment, at least one reagent is maintained at 4°C before use.

[0018] In one embodiment, at least one of the capture probe and the detection probe hybridizes with a nucleic acid molecule containing a lung cancer marker. In one embodiment, the nucleic acid molecule is a circulating tumor DNA (ctDNA) molecule.

[0019] In one embodiment, the capture probe includes a poly-A region.

[0020] In one embodiment, the lung cancer marker is an EGFR variant. In one embodiment, the lung cancer marker is exon 19 deletion, T790M, L858R, c.2235_2249 del18, c.2236_2250 del18, c.2240_2257 del18, c.2239_2248TTAAGAGAAG>C, c.2239_2247delTTAAGAGAA, or c.2239_2248del11. In one embodiment, the lung cancer marker is exon 19E deletion, T790M, or L858R.

[0021] In one embodiment, a pair of capture probes and detection probes may be: a) a probe pair for detecting exon 19 deletion, where the capture probe contains the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 13, and the detection probe contains SEQ ID NO: 6; b) a probe pair for detecting L858R, where the capture probe contains the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 14, and the detection probe contains SEQ ID NO: 2; or c) a probe pair for detecting T790M, where the capture probe contains the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 15, and the detection probe contains SEQ ID NO: 10.

[0022] In one embodiment, at least one sample is a saliva sample, a blood sample, a plasma sample, or a serum sample.

[0023] In one embodiment, a saliva sample from a subject is added to a first well of a multi-well plate, and a plasma sample from the same subject is added to a second well of the multi-well plate. In one embodiment, if a marker for lung cancer is detected in both the saliva sample from the subject and the plasma sample from the same subject, the subject is diagnosed as having lung cancer or being at risk of lung cancer.

[0024] In one embodiment, the method further includes the step of subjecting the subject to treatment for lung cancer if a marker for lung cancer is detected.

[0025] The following detailed description of exemplary embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, the drawings show presently preferred embodiments. However, it should be understood that the present invention is not limited to the exact arrangements and instrumentalities of the embodiments shown in the drawings.

Brief Description of the Drawings

[0026] [Figure 1A] It is a graph showing an exemplary eLB assay experiment using Rnase treatment to evaluate whether the target detected by eLB is DNA or RNA. It shows the RNAse treatment experiment applied to the RNA-based assay. [Figure 1B] It is a graph showing an exemplary eLB assay experiment using Rnase treatment to evaluate whether the target detected by eLB is DNA or RNA. It shows the RNAse treatment applied to healthy controls and subjects (UCLA140) with exon 19 deletions. [Figure 2] It is a graph showing an exemplary eLB assay experiment using exonuclease VII treatment to evaluate whether the target detected by eLB is DNA or RNA. Exonuclease VII treatment was applied to healthy samples; healthy plasma samples spiked with synthetic double-stranded T790M oligonucleotides; healthy plasma spiked with synthetic single-stranded T790M; or subjects carrying p.T790M. [Figure 3]This graph shows an exemplary experiment demonstrating a reference range test for 100 healthy saliva samples tested using the eLB assay. Two standard deviation lines are shown as dotted lines. The eLB assay for exon 19 deletion mutations is shown. [Figure 4] This graph shows an exemplary experiment demonstrating a reference range test for 100 healthy plasma samples tested using the eLB assay. Two standard deviation lines are shown as red dotted lines. The eLB assay for exon 19 deletion mutations is shown. [Figure 5A-B] This figure graph illustrates an exemplary experiment demonstrating the eLB assay results of serially diluted genomic DNA for the top four EGFR exon 19 variants. It shows the linear range for c2235_2249 del18. [Figure 5C-D] This figure graph illustrates an exemplary experiment demonstrating the eLB assay results of serially diluted genomic DNA for the top four EGFR exon 19 variants. The plot shows the measurement results for c.2239_2248 del11. [Figure 5E-F] This figure graph illustrates an exemplary experiment demonstrating the eLB assay results of serially diluted genomic DNA for the top four EGFR exon 19 variants. It shows the linear range for c.2236_2250 del18. [Figure 5G-H] This figure graph illustrates an exemplary experiment demonstrating the eLB assay results of serially diluted genomic DNA for the top four EGFR exon 19 variants. It shows the linear range for del11 (c.2239-2248). [Figure 6] This graph shows exemplary eLB assay results for serially diluted genomic DNA for the p.T790M mutation. Scatter plots of diluted genomic DNA and EFIRM measurements are shown. [Figure 7] This graph shows exemplary eLB assay results for serially diluted genomic DNA for the p.L858R mutation. Scatter plots of diluted genomic DNA and EFIRM measurements are also shown. [Figure 8A]This graph shows exemplary eLB assay results for a standard substance derived from Horizon Diagnostics standards. The eLB for p.T790M is shown. [Figure 8B] This graph shows exemplary eLB assay results for standard materials derived from Horizon Diagnostics standards. It shows eLB for exon 19 deletion. [Figure 8C] This graph shows exemplary eLB assay results for a standard substance derived from a Horizon Diagnostics standard. The eLB for p.L858R is shown. [Figure 9] This is a schematic diagram of the EFIRM assay system.

[0027] Detailed explanation The figures and description of this invention are simplified to illustrate relevant elements for a clear understanding of the invention, and it should be understood that, for clarity, many other elements found in typical biomarker detection systems and methods have been excluded. Those skilled in the art will be able to understand other elements and / or steps that are desirable and / or necessary in carrying out the invention. However, since such elements and steps are well known in the art and would not facilitate a better understanding of the invention, no consideration of such elements and steps is included herein. This disclosure covers all such variations and modifications of such elements and methods known to those skilled in the art.

[0028] definition Unless otherwise defined, all scientific and technical terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but only exemplary methods and materials are described.

[0029] As used herein, the following terms have the meanings associated with them in this section.

[0030] The articles “a” and “an” are used herein to refer to one or more grammatical objects of that article (i.e., at least one grammatical object of that article). For example, “an element” means one or more elements.

[0031] When used herein, "approximately" refers to measurable values ​​such as quantity or duration, and includes a range of ±20%, ±10%, ±5%, ±1%, and ±0.1% from the specified value, with such variation being considered reasonable.

[0032] When used in reference to an organism, tissue, cell, or component of it, the term “abnormal” refers to an organism, tissue, cell, or component of it that differs from the “normal” (expected) respective characteristic (e.g., age, treatment, time of administration) of that organism, tissue, cell, or component of it. A cell or tissue type may be abnormal if the normal or expected characteristics for one cell or tissue type differ.

[0033] As used herein, the terms “modification,” “deletion,” “mutation,” or “spontaneous mutation” refer to a mutation that affects the function, activity, expression (transcription or translation), or conformation of an intracellular gene of the polypeptide it encodes. Mutations as incorporated herein may be any mutation in an intracellular gene that results in enhancement or disruption of the function, activity, expression, or conformation of the encoded polypeptide, and may include, for example, the complete absence of expression of the encoded protein, and may include missense and nonsense mutations, insertions, deletions, frameshifts, and premature terminations. Mutations as incorporated herein may also include those that alter the splicing of mRNA (splice site mutations) or those that cause a shift in the reading frame (frameshifts).

[0034] The term "amplification" refers to the process of increasing the number of copies of a target nucleotide sequence present in a sample.

[0035] As used herein, the term “antibody” refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies may be intact immunoglobulins derived from natural or recombinant sources, or they may be the immunoreactive portion of intact immunoglobulins. Antibodies are generally tetramers of immunoglobulin molecules. The antibodies in this invention may exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Bird et al., 1988, Science 242: 423-426).

[0036] As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0037] As used herein, "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. κ and λ light chains refer to the two main antibody light chain isotypes.

[0038] As used herein, the term “synthetic antibody” means an antibody produced using recombinant DNA technology, for example, an antibody expressed by a bacteriophage as described herein. The term should also be interpreted to mean an antibody produced by the synthesis of an antibody-encoding DNA molecule, the DNA molecule expressing an antibody protein or an amino acid sequence that identifies the antibody, where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

[0039] As used herein, the term "specifically binding" refers to an antibody that recognizes a particular antigen in a sample but substantially does not recognize or bind to other molecules. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. However, such interspecies reactivity does not in itself alter the antibody's specificity classification. In another example, an antibody that specifically binds to an antigen may also bind to antigens of different alleles. However, such cross-reactivity does not in itself alter the antibody's specificity classification. In some cases, the term "specific binding" or "specifically binding" can be used in relation to the interaction between an antibody, protein, or peptide and a second chemical species, meaning that the interaction depends on the presence of a specific structure on the chemical species (e.g., an antigenicity determinant or epitope); for example, an antibody recognizes and binds to a specific protein structure rather than broadly recognizing and binding to a protein. If the antibody is specific to epitope "A", the presence of labeled "A" and molecules containing epitope A (or free, unlabeled A) in the reaction containing the antibody reduces the amount of labeled A that binds to the antibody.

[0040] As used herein, the terms “marker” or “biomarker” shall encompass parameters useful in accordance with the present invention for determining the presence and / or severity of lung cancer.

[0041] A marker or biomarker level is considered "significantly" different if the level of the marker or biomarker in the reference sample differs from the level of the marker in the patient-derived sample by an amount greater than the standard error of the assay used to evaluate the marker, e.g., by at least 5%, 10%, 25%, 50%, 75%, or 100%, compared to the level in the sample from the reference subject.

[0042] The term "control or standard" refers to a material that does not contain, or contains, one or more marker (or biomarker) expression products of one or more markers (or biomarkers) of the present invention at normal, low, or high levels, and therefore, a control or standard can serve as a comparative object that can be used to compare a sample.

[0043] The phrase "determining the level of marker (or biomarker) expression" means assessing the degree of marker expression in a sample at the nucleic acid level or protein level, using techniques available to those skilled in the art to detect a sufficient portion of any marker expression product.

[0044] "Differentially increased expression" or "upregulation" means that the level of a biomarker product is at least 10% higher than the control, e.g., 20%, 30%, 40%, or 50%, 60%, 70%, 80%, or 90% higher or more, and / or 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times, or 2.0 times higher or more, and any overall or partial increase in between.

[0045] "Differentially reduced expression" or "downregulation" means that the level of a biomarker product is at least 10% lower than the control, e.g., 20%, 30%, 40%, or 50%, 60%, 70%, 80%, or 90% lower, and / or less than 2.0 times, 1.8 times, 1.6 times, 1.4 times, 1.2 times, or 1.1 times lower, and any overall or partial reduction in between.

[0046] "Disease" refers to the state of an animal's health. If the animal is unable to maintain homeostasis and the disease does not improve, the animal's health will continue to deteriorate.

[0047] Where used herein, “informative materials” include publications, records, charts, or any other medium of expression that can be used to convey the usefulness of the components of the present invention in a kit for detecting the biomarkers disclosed herein. The explanatory materials for a kit of the present invention may, for example, be attached to the container containing the components of the present invention, or shipped together with the container containing the components. Alternatively, the explanatory materials may be shipped separately from the container, with the intention that the explanatory materials and the components be used collaboratively by the recipient.

[0048] The term “labeled,” as used herein, refers to a detectable compound or composition that is directly or indirectly conjugated with a probe to produce a “labeled” probe. The label may be detectable on its own (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, may catalyze the chemical alteration of a detectable substrate compound or composition (e.g., avidin-biotin). In some cases, primer labeling may be used to detect PCR products.

[0049] The “level” of one or more biomarkers refers to the absolute or relative amount or concentration of the biomarker in the sample.

[0050] The term “marker (or biomarker) expression,” as used herein, encompasses the transcription, translation, post-translational modification, and phenotypic manifestation of genes, and includes all aspects of the conversion of information encoded by a gene into RNA or protein. In non-limiting examples, marker expression includes transcription to messenger RNA (mRNA) and translation to protein, as well as transcription to other types of RNA, such as transfer RNA (tRNA) and ribosomal RNA (rRNA), which are not translated into protein.

[0051] "Measuring" or "detecting" means assessing the presence, absence, quantity or amount (which may be an effective amount) of a given substance in a clinical or subject sample, including deriving a qualitative or quantitative concentration level of such substance, or otherwise assessing the value or categorization of the subject's clinical parameters.

[0052] The terms “patient,” “subject,” and “individual” are used interchangeably herein and refer to any animal or its cells suitable for the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0053] As used herein, the term “to provide prognosis” means to provide a prediction of the expected course and outcome of lung cancer, the prediction including, but not limited to, severity, duration, and likelihood of recovery. Using the method, for example, an appropriate treatment plan can be devised by indicating whether the condition is still in an early stage or whether it has progressed to a stage where invasive therapy is ineffective.

[0054] The "reference level" of a biomarker refers to the level of the biomarker that indicates a specific condition, phenotype, or lack thereof, or a combination of such conditions, phenotypes, or lack thereof. The "positive" reference level of a biomarker refers to the level that indicates a specific condition or phenotype. The "negative" reference level of a biomarker refers to the level that indicates a lack of a specific condition or phenotype.

[0055] As used herein, “sample” or “biological sample” means biological material isolated from an organism. A biological sample may include any biological material suitable for detecting a desired biomarker, and may include cellular material and / or non-cellular material obtained from an organism.

[0056] When used herein, "standard control value" refers to a predetermined amount of a specific, detectable protein or nucleic acid in a sample, for example, in a saliva sample, whole saliva, or salivary supernatant. The standard control value is suitable for use in the methods of the present invention to compare the amount of the target protein or nucleic acid present in a saliva sample. A verified sample serving as a standard control provides an average amount of the target protein or nucleic acid in saliva that is typical of an average healthy person, reasonably adjusted for background, e.g., sex, age, ethnicity, and medical history. The standard control value may vary depending on the protein or nucleic acid of interest and the nature of the sample (e.g., whole saliva or supernatant).

[0057] Throughout this disclosure, various aspects of the invention may be described in range form. It should be understood that descriptions in range form are for convenience and brevity only and should not be interpreted as inflexible limitations on the scope of the invention. Therefore, descriptions of ranges should be considered as specifically disclosing all possible subranges and the individual values ​​that fall within those ranges. For example, a range description such as 1 to 6 should be considered as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, and the individual numbers that fall within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, 6 and any overall and partial increments in between. This applies regardless of the breadth of the range.

[0058] explanation The present invention relates to a rapid and precise polymer-based electrochemical platform array for detecting single or multiple biomarkers indicating a disease or disorder, such as lung cancer, from at least one biological sample, such as a saliva sample or a blood sample. Although the present invention generally describes the testing of saliva or blood samples, it should be understood that any biological fluid sample, or even other tissue type, can be used, provided that such alternative sample type has the targeted marker to be analyzed. Non-limiting examples of such markers include all saliva and serum-based markers for disease or disorder. In some embodiments, the markers are, but are not limited to, markers for lung cancer, but include nucleic acid molecules having gene mutations, such as one or more mutations in the epidermal growth factor receptor (EGFR), or any other markers associated with or indicating lung cancer. For example, markers indicating lung cancer, or detectable gene mutations, include, but are not limited to, EGFR exon 19 deletions, T790M, L858R, c.2235_2249 del18, c.2236_2250 del18, c.2240_2257 del18, c.2239_2248TTAAGAGAAG>C, c.2239_2247delTTAAGAGAA, and c.2239_2248 del11 variants. Any number of biomarkers can be incorporated into the assay platform, and it should be understood that, without limitation, each array may contain 1, 2, 4, 8, 16, 32, or 64 biomarkers.

[0059] The non-invasive detection of lung cancer in subjects according to the present invention enables clinicians to identify the presence of lung cancer in a fast, economical, and non-invasive manner.

[0060] As intended herein, the present invention includes a multiplexed electrochemical sensor for detecting biomarkers. This device utilizes small sample volumes and offers high accuracy. Furthermore, it can simultaneously measure multiple markers with a single sample loading into the device. This device can significantly reduce costs to the healthcare system by reducing the burden on patients to return to clinics and laboratories.

[0061] In one embodiment, the electrochemical sensor is an array of electrode tips (EZ Life Bio, USA). In one embodiment, each unit of the array has a working electrode, a counter electrode, and a reference electrode. The three electrodes can be constructed of bare gold or other conductive material before the reaction, so that the sample can be immobilized on the working electrode. The electrochemical current between the working electrode and the counter electrode can be measured in the presence of a potential between the working electrode and the reference electrode. The potential profile may be, for example, a constant value, a linear sweep, or a periodic square wave. Each set of three electrodes can be separated using an array of plastic wells, which helps to avoid cross-contamination between different sensors. In one embodiment, the set of three electrodes in each well is placed in a 96-well gold electrode plate. A conductive polymer can also be placed on the working electrode as a support film, and in some embodiments as a surface for functionalizing the working electrode. Any conductive polymer, such as polypyrrole, polyaniline, polyacetylene, polyphenylenevinylene, polythiophene, etc., can be used as intended herein.

[0062] In one embodiment, a periodic square wave electric field is generated across the electrodes in the sample well. In a particular embodiment, the square wave electric field is generated to assist the polymerization of polymers of one or more capture probes and sensors. In a particular embodiment, the square wave electric field is generated to assist the hybridization of the capture probe and the marker and / or detection probe. The positive potential in the csw E-field helps in the accumulation of molecules on the working electrode, while the negative potential removes weak nonspecific binding, resulting in enhanced specificity. Furthermore, the alternation of positive and negative potentials across the periodic square wave also results in excellent mixing during incubation without disrupting desired specific binding, thereby accelerating the binding process and resulting in faster inspection or assay times. In one embodiment, the square wave cycle may consist of longer low-voltage cycles and shorter high-voltage cycles to enhance the hybridization of binding partners in the sample. There is no limit to the actual period selected, but examples include low-voltage cycles of 0.15 to 60 seconds and high-voltage cycles of 0.1 to 60 seconds. In one embodiment, each square wave cycle consists of 1 second at low voltage and 1 second at high voltage. For hybridization, the low voltage can be approximately -200mV and the high voltage can be approximately +500mV. In some embodiments, the total number of square wave cycles is 2 to 50. In one embodiment, 5 periodic square waves are applied to each surface reaction. Using the csw E-field, both polymerization and hybridization are completed on the same chip within minutes. In some embodiments, the total detection time from sample loading is less than 30 minutes. In other embodiments, the total detection time from sample loading is less than 20 minutes. In other embodiments, the total detection time from sample loading is less than 10 minutes. In other embodiments, the total detection time from sample loading is less than 5 minutes. In other embodiments, the total detection time from sample loading is less than 2 minutes. In other embodiments, the total detection time from sample loading is less than 1 minute.

[0063] A multi-channel electrochemical reader (EZ Life Bio) controls the electric field applied to the array sensor, and the measured current is reported simultaneously. In practice, the solution can be loaded into the entire 3-electrode region, which includes a working electrode, a counter electrode, and a reference electrode, and these electrodes are localized and separated by an array of plastic wells. After each step, the electrochemical sensor can be rinsed with ultrapure water or another washing solution and then dried, for example, under pure N2. In some embodiments, the sensor is a single-use, disposable sensor. In other embodiments, the sensor is reusable.

[0064] In one embodiment, the present invention is based on the affinity between a capture probe and a detection probe, as shown in Figure 9. As intended herein, the assay platform can be organized as any type of affinity-binding assay or immunoassay, which will be understood by those skilled in the art. In another embodiment, the present invention includes a single platform for measuring multiple lung cancer biomarkers rather than a single marker. Currently, no such technology or device is available for this purpose. In yet another embodiment, the present invention offers extremely high efficiency in that it is simple, rapid, and robust. For example, only a small sample volume is required (e.g., 10 μl), and the required run time is less than 10 minutes. Multiple marker levels can be provided by this device. By providing statistical analysis, users can have an estimate of their own risk, and by utilizing the available network system, results can be rapidly communicated for further examination by clinicians for evaluation.

[0065] For example, probe pairs (capture and detector) specific to mutations such as deletion mutations or point mutations can be designed. In one embodiment, the probe pair set of the present invention for detecting exon 19del EGFR variants includes a capture probe containing SEQ ID NO: 13 and a detection probe containing SEQ ID NO: 6. In one embodiment, the probe pair set of the present invention for detecting L858R EGFR variants includes a capture probe containing SEQ ID NO: 14 and a detection probe containing SEQ ID NO: 2. In one embodiment, the probe pair set of the present invention for detecting T790M EGFR variants includes a capture probe containing SEQ ID NO: 15 and a detection probe containing SEQ ID NO: 10.

[0066] In some embodiments, at least one of the capture probe and the detection probe includes a polyA tail. In various embodiments, the polyA tail includes at least 40, 45, 50, 55, 60, 65, 70, 75, 80, or 80 or more adenine nucleotides at the 5' or 3' end of the probe. In some embodiments, the polyA tail includes a sequence containing at least 65 nucleotides, where at least 80% of the nucleotides are adenine, and the polyA sequence is interrupted by non-adenine nucleotides. In one embodiment, the probe pair set of the present invention for detecting the L858R EGFR variant includes a capture probe containing SEQ ID NO: 1 and a detection probe containing SEQ ID NO: 2. In one embodiment, the probe pair set of the present invention for detecting the exon 19del EGFR variant includes a capture probe containing SEQ ID NO: 5 and a detection probe containing SEQ ID NO: 6. In one embodiment, the probe pair set of the present invention for detecting the T790M EGFR variant includes a capture probe containing SEQ ID NO: 9 and a detection probe containing SEQ ID NO: 10.

[0067] The detection probe can be labeled with, for example, fluorescein isothiocyanate or any other label known in the art. In one embodiment, the detection probe contains a biotinylated nucleotide to enable streptavidin binding. The capture probe is first copolymerized onto a bare gold electrode by applying a periodic square wave electric field. For example, for each cycle during copolymerization, the electric field can be set to +350 mV for 1 second and +950 mV for 1 second. In total, polymerization can be carried out over 5 cycles, 10 seconds, although longer periods may be required.

[0068] After polymerization, the sensor tip can be rinsed and dried for subsequent sample measurement. The sample, such as cell culture medium, blood sample, or saliva sample, can be mixed with the detection probe and transferred to the electrode. Hybridization is then performed using low-voltage and high-voltage cycles, for example, -200mV for 1 second and +500mV for 1 second. The total hybridization time may be, for example, 5 cycles, or 10 seconds. Next, the label is detected based on the label type. For example, an anti-fluorescein antibody conjugated with horseradish peroxidase in casein-phosphate buffered saline can be used, loading the horseradish peroxidase with a 3,3',5,5'-tetramethylbenzidine substrate and measuring the current measurement signal.

[0069] A capture probe, embedded in a conductive polymer or otherwise used to functionalize the surface of a working electrode, and a detection probe, which is mixed with a sample, can be constructed according to any protocol known in the art for the production of probes.

[0070] In one embodiment, the capture probe is immobilized in a conductive polymer gel at the bottom of a 96-well gold electrode plate.

[0071] The sensor's capture probe or detection probe may be any nucleic acid, protein, small molecule, etc., that specifically binds to one or more of the target markers. For example, in certain embodiments, the capture probe and detection probe are oligonucleotides or polynucleotides containing a region substantially complementary to one or more nucleic acid markers of the present invention. In one embodiment, the capture probe and detection probe contain regions substantially complementary to each other; that is, in one embodiment, the capture probe contains a region substantially complementary to a region of the detection probe. Methods for designing and assembling oligonucleotide probes are well known in the art.

[0072] In one embodiment, the marker includes a variable region, which may include disease-related mutations, such as deletions, substitutions, or point mutations. In one embodiment, a capture probe is designed to hybridize with a conserved region of the nucleic acid marker (i.e., present in both the wild-type and mutant isoforms). In one embodiment, a detection probe is designed to hybridize with the nucleic acid sequence of the variable region having the disease-related mutation of the marker, thereby detecting the mutation in the sample. In another embodiment, a detection probe is designed to hybridize with the nucleic acid sequence of the variable region having either the wild-type or unmutated sequence, thereby detecting the presence of the wild-type marker in the sample. In one embodiment, a capture probe is designed to hybridize with either the wild-type or mutated variable region, while a detection probe is designed to hybridize with the conserved region.

[0073] For example, in one embodiment, the capture probe includes a nucleotide sequence substantially complementary to a conserved region of the nucleic acid encoding EGFR. In one embodiment, the detection probe includes a nucleotide sequence substantially complementary to a variable region of the nucleic acid encoding EGFR, where the variable region encodes the wild-type amino acid sequence of EGFR. In one embodiment, the detection probe includes a nucleotide sequence substantially complementary to a variable region of the nucleic acid encoding EGFR, where the variable region encodes a disease-related mutant sequence of EGFR. For example, in a particular embodiment, the detection probe includes a nucleotide sequence substantially complementary to a variable region of the nucleic acid encoding EGFR, where the variable region encodes a disease-related deletion mutant or point mutation of EGFR.

[0074] In one embodiment, when the present invention is used to detect nucleic acids encoding the EGFR L858R mutant, the capture probe comprises a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 14. In another embodiment, when the present invention is used to detect nucleic acids encoding the EGFR L858R mutant, the detection probe comprises a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 2.

[0075] In one embodiment, when the present invention is used to detect nucleic acids encoding an exon 19 del mutant of EGFR, the capture probe comprises a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 13. In another embodiment, when the present invention is used to detect nucleic acids encoding an exon 19 del mutant of EGFR, the detection probe comprises a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 6.

[0076] In one embodiment, when the present invention is used to detect nucleic acids encoding the EGFR T790M mutant, the capture probe comprises a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 9 or SEQ ID NO: 15. In one embodiment, when the present invention is used to detect nucleic acids encoding the EGFR T790M mutant, the detection probe comprises a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 10.

[0077] In one embodiment, the detection probe includes a detectable label that induces a change in the sensor current, thereby indicating hybridization of the detection probe and an associated marker with the capture probe. In a particular embodiment, the detectable label itself may be sufficient to alter the sensor current. In a particular embodiment, the detectable label induces a change in current upon contact with an exogenous reactant. For example, the detectable label may react with the reactant to produce a local change, which the sensor electrodes detect to generate a current measurement signal. Thus, in a particular embodiment, the reactant is added to the sensor before, during, or after applying the sample to the sensor.

[0078] In certain embodiments, the detectable label is directly conjugated to the detection probe. In other embodiments, the detectable label is conjugated to the detection probe via an intermediate tag or a label on the probe. In some embodiments, the detectable label is a modified nucleotide containing biotin that is incorporated into the detection probe during synthesis. For example, in one embodiment, the detection probe comprises a tag, label, or epitope, which can be used to conjugate an antibody or other conjugate compound having the above-mentioned detectable label.

[0079] Examples of detectable labels and reactants for inducing localized changes in electrochemical sensors are well known in the art. In one embodiment, the detectable label comprises HRP and the reactant is TMB, which react to produce a current measurement signal. In another embodiment, the detectable label comprises urease and the reactant comprises urea.

[0080] In one embodiment, the signal is amplified using multiple rounds of HRP. In this embodiment, 1) a biotin-labeled detector molecule is contacted with a first round of HRP in the form of streptavidin-bound HRP, 2) the complexed HRP molecule is contacted with a biotin-labeled anti-HRP antibody, and 3) a second round of streptavidin-bound HRP is added to amplify the signal. In an exemplary embodiment, the detection probe is mixed with casein-phosphate buffered saline at a dilution of 1:100 and transferred to the electrode. Hybridization is performed at room temperature for 150 cycles of 1 second at 300 mV and 1 second at 500 mV. Subsequently, streptavidin polyHRP is mixed with casein-phosphate buffered saline at a ratio of 1:1000 and incubated on the electrode at room temperature for 30 minutes. After adding HRP, the anti-HRP antibody is added together with casein-phosphate buffered saline, then incubated at room temperature for 30 minutes and washed with PBS-T buffer. Next, streptavidin polyHRP80 conjugate is added in a mixture with casein-phosphate buffered saline and incubated for 30 minutes to increase the amount of available HRP molecules. This method results in increased signal amplification, thereby increasing the sensitivity and specificity of the eLB system. In some embodiments, one or more washing steps are performed. In some embodiments, the plate is washed in an automated 96-well plate washer. This involves aspirating the existing liquid from each well of the microtiter plate and then distributing the washing buffer into each well. In one embodiment, the washing buffer is then aspirated and this is repeated for at least one additional cycle.

[0081] Due to the enhanced sensitivity of the present invention, the desired assay can be performed using a very small volume. For example, the size of the biological sample from the subject may be 5 to 100 microliters. In one embodiment, the required sample size is only about 40 microliters. There is no limit to the actual or final sample size being tested.

[0082] The present invention also relates to a method for detecting one or more markers associated with or indicating lung cancer in a subject. In one embodiment, the method may be carried out as a hybridization assay and includes the steps of: obtaining a sample from a subject; adding a detection probe labeled with a detectable portion targeting a marker of lung cancer to the sample; applying the sample to an electrode tip coated with a conductive polymer pre-embedded with or functionalized with a capture probe; and measuring the current in the electrode tip. The detectable portion can be measured or the magnitude of the current in the sample can be determined to determine the presence or absence of the marker in the sample. In certain embodiments, hybridization of the marker and the sensor electrode results in an increase or a negative current. For example, in one embodiment, hybridization results in a current in the range of about -10 nA to about -1000 nA.

[0083] In one embodiment, the liquid and enzyme reagents used in the method of the present invention are refrigerated at 4°C when not in use. In one embodiment, the sample and reagents used in the method of the present invention are kept on ice before use. In one embodiment, the sample is processed within 30 minutes of collection. The present invention provides a method for diagnosing lung cancer in subjects. Accordingly, the present invention features a method for identifying subjects at risk of developing lung cancer, including subjects that are asymptomatic or that show only indicators that are not specific to lung cancer upon detection of the biomarkers disclosed herein. These biomarkers are also useful for monitoring subjects undergoing treatment and therapy for lung cancer, and for selecting or modifying treatments and procedures that appear to be effective in subjects with lung cancer, where the selection and use of such treatments and procedures slows the progression of lung cancer or prevents its development.

[0084] In certain embodiments, biomarkers detected by the systems and methods of the present invention include, but are not limited to, nucleic acids or detectable gene mutations, including those associated with EGFR, KRAS, BRAF, CCNI, FGF19, FRS2, GREB1, and LZTS1. In certain embodiments, nucleic acid biomarkers include one or more disease-associated mutations, including insertions, deletions, substitutions, translocations, point mutations, single nucleotide variants (SNVs), and the like. The present invention can be used to detect any disease-associated biomarkers or disease-associated mutations known or hereafter discovered in the art. Exemplary mutations present in various forms of cancer can be found, for example, in the Catalogue of Somatic Mutations in Cancer (COSMIC) (Wellcome Trust Sanger Institute).

[0085] In certain embodiments, the biomarkers detected by the systems and methods of the present invention include mutant EGFR and nucleic acid molecules encoding mutant EGFR. For example, various EGFR mutants have been associated with lung cancer, and these EGFR mutants include, but are not limited to, EGFR exon 19 deletion, T790M, L858R, c.2235_2249 del18, c.2236_2250 del18, c.2240_2257 del18, c.2239_2248TTAAGAGAAG>C, c.2239_2247delTTAAGAGAA, and c.2239_2248 del11 variants.

[0086] The present invention can be used to detect any such mutation, because the capture probe and / or detection probe can be specifically designed to hybridize with the nucleic acid molecule encoding the mutant protein of interest.

[0087] The present invention provides improved diagnosis, therapeutic monitoring, recurrence detection, and prognosis for lung cancer. The risk of developing lung cancer can be assessed by measuring one or more of the biomarkers described herein and comparing the measured values ​​to reference or index values. Such comparisons are performed using mathematical algorithms or formulas to combine information from the results of multiple individual biomarkers and other parameters into a single measurement or index. Subjects identified as having an increased risk of lung cancer may, if applicable, be selected to receive treatment regimens, such as the administration of prophylactic or therapeutic compounds or measures to prevent, treat, or delay the onset of lung cancer.

[0088] Identifying subjects before lung cancer develops allows for the selection and initiation of various therapeutic interventions or treatment regimens to delay, reduce, or prevent the onset or severity of cancer. In certain cases, monitoring the level of at least one biomarker allows for monitoring the course of lung cancer treatment. For example, samples may be provided from subjects undergoing treatment regimens or therapeutic interventions for lung cancer, such as drug therapy, radiation therapy, or chemotherapy. Samples can be obtained from subjects at various time points before, during, or after treatment.

[0089] Therefore, the biomarkers of the present invention can be used to generate a biomarker profile or signature for subjects: (i) subjects who do not have lung cancer and are not expected to develop lung cancer, and / or (ii) subjects who have lung cancer or are expected to develop lung cancer. The biomarker profile of a subject can be compared to a predetermined or reference biomarker profile in order to diagnose or identify subjects at risk of developing lung cancer, to monitor disease progression and the rate of disease progression, and to monitor the effectiveness of lung cancer treatment. Data relating to the biomarkers of the present invention can also be combined with or correlated with other data or test results relating to lung cancer, including, but not limited to, imaging data, medical history, smoking status, and any relevant family history.

[0090] The present invention also provides a method for identifying agents for treating lung cancer that are suitable for a specific subject or otherwise customized. In this regard, test samples can be obtained from subjects exposed to a therapeutic agent, drug, or other treatment regimen, and the levels of one or more biomarkers can be determined. The levels of one or more biomarkers can be compared to samples from subjects before and after treatment, or to samples from one or more subjects that showed improvement in risk factors as a result of such treatment or exposure.

[0091] In one embodiment, the present invention is a method for diagnosing lung cancer. In one embodiment, the method includes determining the stage or severity of lung cancer. In some embodiments, these methods may utilize at least one biological sample (e.g., urine, saliva, blood, serum, plasma, amniotic fluid, or tears) to detect one or more markers of the present invention in the sample. Frequently, the sample is a “clinical sample,” which is a sample derived from a patient. In one embodiment, the biological sample is a blood sample. In certain embodiments, the biological sample is a serum or plasma sample derived from the blood sample of interest.

[0092] In some embodiments, the sample comprises at least one very small single-stranded DNA molecule. In one embodiment, the sample comprises at least a circulating tumor DNA (ctDNA) molecule. In one embodiment, the ctDNA comprises a cancer-related biomarker.

[0093] In one embodiment, the method includes detecting one or more markers in at least one biological sample of the subject. In various embodiments, one or more levels of the markers of the present invention in the biological sample of the subject are compared to levels of corresponding biomarkers in a comparison subject. Non-limiting examples of comparison subjects include, but are not limited to, negative controls, positive controls, predicted normal background values ​​of the subject, normal background values ​​based on the subject's past history, predicted normal background values ​​of the population to which the subject is a member, or normal background values ​​based on the past history of the population to which the subject is a member.

[0094] In one embodiment, the method includes simultaneously detecting one or more markers in two or more different biological samples of a subject. In one embodiment, the method includes simultaneously detecting one or more markers in a saliva sample of a subject and in a blood, plasma, or serum sample of a subject.

[0095] In one embodiment, the method includes sequentially detecting one or more markers in two or more different biological samples of a subject. In one embodiment, the method includes detecting one or more markers in a saliva sample of a subject before or after detecting one or more markers in a blood, plasma, or serum sample of a subject.

[0096] In various embodiments, the level of one or more markers of the present invention in each subject's biological sample is compared to the level of the corresponding biomarker in a suitable comparison subject. In one embodiment, a subject is diagnosed with having or being at risk of having a disease or disorder if the level of the biomarker in each of two or more biological samples is elevated or decreased compared to a suitable comparison subject. For example, in one embodiment, a subject is diagnosed with having or being at risk of having lung cancer if 1) the level of at least one DNA molecule containing a lung cancer-related mutation is elevated in the subject's saliva sample compared to the predicted normal background level of the biomarker in the saliva of a healthy subject or healthy population, and 2) the level of at least one DNA molecule containing a lung cancer-related mutation is elevated in the subject's plasma sample compared to the predicted normal background level of the biomarker in the serum of a healthy subject or healthy population.

[0097] In another embodiment, the present invention is a method for monitoring lung cancer progression in a subject by evaluating the level of one or more markers of the present invention in a biological sample of at least one subject.

[0098] In various embodiments, the subject is a human subject, which may be of any race, sex, and age.

[0099] Information obtained from the methods of the present invention described herein can be used alone or in combination with other information from a subject or a biological sample obtained from a subject (e.g., medical condition, medical history, vital signs, blood chemistry, etc.).

[0100] In various other embodiments of the method of the present invention, the level of one or more markers of the present invention is determined to be elevated if the level of one or more markers of the present invention is elevated by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to a control.

[0101] In the method of the present invention, at least one biological sample from a subject is evaluated for one or more levels of the marker of the present invention in the biological sample obtained from a patient. The one or more levels of the marker of the present invention in the biological sample can be determined by evaluating the amount of one or more polypeptides of the biomarker of the present invention in the biological sample, the amount of one or more mRNAs of the biomarker of the present invention in the biological sample, the amount of one or more DNAs of the biomarker of the present invention in the biological sample, the amount of one or more enzyme activities of the biomarker of the present invention in the biological sample, or a combination thereof.

[0102] The present invention further includes an assay kit containing an electrochemical sensor array and instructions for setting up, performing, monitoring, and interpreting the assay of the present invention. Optionally, the kit may include reagents for detecting at least one of a set of biomarkers. The kit may also optionally include a sensor reader. [Examples]

[0103] The present invention will be described in further detail by reference to the following embodiments. These embodiments are presented for illustrative purposes only and are not limiting unless explicitly stated otherwise. Accordingly, the present invention should not be construed as being limited to the following embodiments, but rather as encompassing all possible variations that become apparent as a result of the teachings presented herein.

[0104] Without further explanation, those skilled in the art will likely be able to construct and utilize the present invention and carry out the claimed method by using the foregoing description and the following examples. Accordingly, the following examples specifically point to exemplary embodiments of the present invention and should not be construed as limiting the remainder of this disclosure in any way. [Example 1]

[0105] Non-invasive detection of EGFR gene mutations in lung cancer patients Currently, there are two common liquid biopsy platforms: one based on next-generation sequencing (NGS) and the other on droplet digital PCR (ddPCR) (Zhang et al., 2017, J Hematol Oncol, 10: 167; Olmedillas-Lopez et al., 2017, Mol Diagn Ther, 21: 493-510). Both of these platforms require 10-20 mL of blood to perform the analysis. This report describes the technical validation of four separate assays using a novel plate-based electrochemical signal amplification method for liquid biopsy, referred to herein as eLB. Early reported data have demonstrated that eLB can detect circulating EGFR variant DNA in patients with early-stage lung cancer. This is the only platform capable of this level of identification for stage I and II NSCLC patients. eLB is performed using only 20 μL of plasma for each variant. This is less than 1 / 100th of the sample required for a liquid biopsy based on either NGS or ddPCR.

[0106] eLB is performed on untreated, unpurified plasma or saliva. Both ddPCR and NGS require a DNA isolation step before the assay. A large portion of circulating tumor DNA fragments is likely to be lost during the DNA isolation procedure or subsequent operations. NGS requires several enzymatic treatments and purifications between steps, and small fragments of DNA are likely to be lost during these steps. Furthermore, the enzymatic steps before library formation are not 100% efficient, and therefore, material is lost at each step due to incomplete reactions.

[0107] The usual assumption is that ctDNA fragments are similar in size to fetal sequences found circulating in maternal circulation during non-invasive prenatal screening processes. These 150 bp double-stranded fragments are a result of nucleosome protection of apoptotic DNA. ctDNA may not arise from ordered apoptosis, or is even more likely to not. In fact, the vast majority of cancer cells lack programmed cell death that produces apoptotic DNA fragments. Unlike fetal DNA in maternal circulation, ctDNA is more likely to be a result of necrosis or immunolysis due to insufficient blood supply. This could result in smaller fragments and potentially single-stranded DNA fragments.

[0108] In fact, there is evidence that ctDNA containing usable mutations in early-stage cancer is smaller in size than both wild-type DNA and ctDNA present in advanced cancer (Liu et al., 2019, EBioMedicine, 41: 345-56). This report demonstrates that ctDNA from early-stage pancreatic cancer patients has fragment sizes significantly smaller than 100 bp. This study utilized a single-strand DNA sequencing strategy, and therefore, the extremely short ctDNA fragments observed may not only be smaller than 100 base pairs but also single-stranded. Other studies using single-strand sequencing have also demonstrated that ctDNA is smaller in size than circulating DNA of other origins (Burnham et al., 2016, Sci Rep, 2016, 6: 27859; Underhill et al., 2016, PLOS Genet. 12: e1006162). Preliminary studies in our laboratory have shown that mutant ctDNA sequences detected in NSCLC patients are indeed 35–44 bp long. Other modalities, such as ddPCR, cannot detect these small fragments. A unique characteristic of the eLB platform is that its preferred template is extremely small single-stranded DNA, which allows eLB to preferentially detect ctDNA while ignoring circulating DNA from other sources.

[0109] Another possibility is that eLB measures exosomal DNA. Fernando and colleagues (Fernando et al., 2018, Clin Chim Acta, 483: 39-47) reported that the majority of exosomal DNA is approximately 76 bp in length. Further research is needed to explain in detail the origin and nature of the fragments analyzed by eLB.

[0110] Another advantage of eLB over other techniques is the ability to use non-standard sample types. In this report, we were able to verify that other sample types, such as urine, pleural fluid, and cerebrospinal fluid, are also likely to be suitable for eLB analysis, using both qualitative and quantitative eLB assays on saliva.

[0111] The EFIRM methodology is plate-based, allowing the entire assay to be easily performed within 4 hours, with very little manual handling time. Therefore, the turnaround time is far superior to that of NGS-based methods, which often take several days to complete and 10-14 days to report, and is also faster than ddPCR assays.

[0112] The ultimate advantage of eLB is cost. eLB can be performed for a few hundred dollars and can therefore be used for continuous monitoring for treatment response, detection of early recurrence, or assessment of minimal residual disease. If the sensitivity and specificity are sufficient, eLB can also be used to screen smokers or individuals with indeterminate lung nodules on CT scans. The sensitivity of eLB as a screening test is currently limited by the fact that three variants are present among NSCLC patients. While the incidence is as high as 40% in China (Zhang et al., 2016, Oncotarget, 7: 78985-93), the fraction of NSCLC tumors with one of these three mutations is low in the United States. According to Forbes SA et al. (Curr. Protoc. Hum. Genet., Hoboken, NJ, USA, John Wiley & Sons, Inc., 2008, p. hg1011s57), the incidence of EGFR mutations is 2.7%, with a confidence that the true incidence does not exceed 3.6%. For adenocarcinoma, the overall incidence is 23%. This is 19% for men and 28% for women. The overall incidence is 47% for smokers and 14% for non-smokers (Midha et al., 2015, Am J Cancer Res, 2015, 5: 2892-911).

[0113] To enhance detection sensitivity, additional variants are added to the eLB, increasing the detection rate in NSCLC patients to 50%. In summary, this report presents the technical validation of four eLB assays: qualitative and quantitative assays for plasma and saliva. The platform is stable and suitable for clinical laboratories. Detection of minor allele fractions by this assay is 0.1% for p.T790M and 1% for exon 19del and p.L858R. These specificities are significantly better than those reported by the only FDA-approved in vitro diagnostic test for these EGFR variants.

[0114] The materials and methods used in the experiment are described here.

[0115] eLB assay The eLB assay is an open-platform signal amplification technique based on a 96-well gold electrode microtiter plate and an EFIRM (Electric Field Induced Release and Measurement) electrochemical reader device (EZLife Bio). Three TKI-sensitive EGFR variants, exon 19del, p.L858R, and p.T790M, were validated using the eLB assay. Briefly, this assay uses a variant-specific capture probe immobilized on a conductive polymer gel at the bottom of a 96-well gold electrode plate. The sample (either saliva or plasma) is added directly to the hybridization buffer without prior DNA isolation or other procedures. The sample-hybridization mixture is then added directly to the wells at room temperature, and alternating current is applied to promote specific hybridization. The plate is then thoroughly washed to remove any unbound fragments. Subsequently, the biotinylation detection probe in the hybridization buffer is added to the wells and subjected to electrically promoted hybridization. Table 1 lists three synthesized (Integrated DNA Technologies) TKI-sensitive mutation-specific probe pairs (capture and detector).

[0116] The following lists the capture and detection probes for three EGFR variants. * indicates biotinylation of the 3' nucleotide.

[0117] L858R detection probe (SEQ ID NO: 1)

[0118] [ka]

[0119] L858R detection probe (for 15-base pair capture probe) (SEQ ID NO: 2)

[0120] [ka]

[0121] Control / Calibration material: Oligo L858R wild type (SEQ ID NO: 3)

[0122] [ka]

[0123] Control / Calibration material oligo L858R mutant (SEQ ID NO: 4)

[0124] [ka]

[0125] Exon-19 del capture probe (SEQ ID NO: 5)

[0126] [ka]

[0127] Exon 19 del detection probe (SEQ ID NO: 6)

[0128] [ka]

[0129] Control / Calibration material: Oligoexon 19 mutation Del (SEQ ID NO: 7)

[0130] [ka]

[0131] Control / Calibration material: Oligoexon 19 del wild type (SEQ ID NO: 8)

[0132] [ka]

[0133] T790M detection probe (SEQ ID NO: 9)

[0134] [ka]

[0135] T790M detection probe (SEQ ID NO: 10)

[0136] [ka]

[0137] Control / Calibration material: Oligo T790M (SEQ ID NO: 11)

[0138] [ka]

[0139] Control / Calibration material oligo T790M wild-type sequence (SEQ ID NO: 12)

[0140] [ka]

[0141] The capture probe (100 nM) was first copolymerized with pyrrole (W336805, Sigma-Aldrich) and a bare gold electrode by applying a periodic square wave electric field of 350 mV for 1 second and then 1100 mV for 1 second. In total, the polymerization was carried out for 4 cycles of 2 seconds each.

[0142] Synthetic 50 bp oligonucleotides (Integrated DNA Technologies) corresponding to the mutant alleles were added to 20 μl to 40 μl volumes of control plasma and saliva (patient samples from individuals without the respective EGFR mutations). The synthetic oligonucleotides and plasma or saliva were diluted in a 1:2 ratio in Ultrahyb-Oligo Hybridization Buffer (ThermoFisher). The resulting diluted buffer was added to two wells.

[0143] Hybridization was performed for 150 cycles, with each cycle lasting 2 seconds, at 300mV for 1 second and 500mV for 1 second, followed by a 30-minute incubation period at room temperature in an eLB instrument. The plate was then removed and washed in an automated 96-well plate washer (Biotek, Model 405LSR, Winooski, VT). This involved first aspirating the existing liquid from each well of the microtiter plate, and then distributing 500 μL of washing buffer (2×SSC with 0.5% SDS) into each well. The washing buffer was aspirated, and this process was repeated for a total of two cycles.

[0144] After washing, the detection probe was mixed with casein-phosphate buffered saline (Invitrogen) at a dilution of 1:100 and transferred to the electrode. Hybridization was performed at room temperature for 150 cycles at 300 mV for 1 second and 500 mV for 1 second. Subsequently, streptavidin polyHRP (ThermoFisher) was mixed with casein-phosphate buffered saline (Invitrogen) at a ratio of 1:1000 and incubated on the electrode at room temperature for 30 minutes.

[0145] After adding HRP, an anti-HRP antibody (ab195239, Abcam) at a concentration of 1 / 60 μg / mL was added with casein-phosphate buffered saline (Invitrogen), followed by incubation at room temperature for 30 minutes and rinsing with PBS-T buffer. Subsequently, streptavidin polyHRP80 conjugate (Fitzgerald Industries) mixed with casein-phosphate buffered saline (Invitrogen) in a 25:975 ratio was added and incubated for 30 minutes to increase the amount of available HRP molecules. After this procedure, the amplified signal was measured in nanoamperes by current generated through a gold electrode. The EFIRM® reader (EZLife Bio) used for the electrochemical measurement is capable of detecting current in the nanoampere range, and therefore this falls well within the technical capabilities of the instrument.

[0146] [Table 1]

[0147] RNase processing method RNase treatment was performed by adding an RNase cocktail (0.025 U / μl RNase A and 1 U / μl T1 RNase) to casein / PBS and incubating at room temperature for 30 minutes. Washing was performed using 2×SSC and 0.5% SDS wash buffer. This treatment was performed after the sample capture step of the eLB protocol.

[0148] DNase processing method DNase treatment was performed to evaluate the single-stranded / double-stranded state of DNA strands. First, proteinase K was added to the plasma sample to a concentration of 2 μg / μl. The sample was then incubated at 50°C for 30 minutes. After this digestion, the proteinase K was inactivated by heating it to 65°C for 10 minutes in a thermocycler, and the solution was then cooled to 4°C for 1 hour (using a cooling rate of 0.1°C / s). Following this initial proteinase K digestion to remove interfering proteins, exonuclease VII was introduced at a concentration of 0.33 units / μl and incubated at 37°C for 30 minutes. The exonuclease was then inactivated by heat treatment at 95°C for 10 minutes, and then cooled to 4°C for 1 hour (gradient rate of 0.1°C / s) to re-anneal the double-stranded DNA present in the sample. The final solution was then assayed using the EFIRM protocol described above.

[0149] cell line To generate genomic DNA standards for point mutations p.L858R and p.T790M, genomic DNA was isolated from the NCI-H1975 cell line (ATCC) using QuickgDNA miniprep (Zymo Research). For exon 19del testing, genomic DNA was obtained from four different cell lines containing variants of the top four exon 19del mutations (COSM6623 / 6225 / 12370 / 12382) (Applied Stem Cells).

[0150] software The multi-channel EFIRM electrochemical reader device hardware was controlled via a USB 2.0 connection, and parameters were configured using a custom in-house software suite from EZLife Bio (Los Angeles, CA). Data from each EFIRM experiment was exported to a comma-separated values ​​file format and analyzed using R-Language for statistical analysis.

[0151] Biological samples Saliva was collected from healthy individual volunteers at American Dental Association meetings between 2006 and 2011. The participants were mixed-gender, mostly non-smokers, aged 18-80 years, and of mixed ethnicities. Informed consent was obtained from all participants before collection. Each participant sputumed approximately 5 mL of total saliva into a 50 cc conical tube placed on ice. Total saliva was processed within 1 / 2 hour of collection. Samples were centrifuged in a refrigerated centrifuge at 2600 g for 15 minutes at 4°C. The supernatant (cell-free saliva) was then pipetted into two 2 mL cryotubes, and the following reagents were added to preserve RNA and DNA: Superase-In 1.1 μL / 1 mL supernatant (Ambion, Austin TX). After adding additional reagents, the tubes were inverted and mixed. The samples were then frozen with dry ice and stored later in a -80°C freezer.

[0152] The results of the experiment are described here.

[0153] DNA or RNA? EFIRM technology can detect either DNA or RNA molecules. We investigated whether EGFR variants detected in clinical samples from NSCLC patients originated from circulating DNA or RNA. To investigate this, we developed an RNase treatment method as described above. An eLB assay for the microRNA species mir415a was used as a control for digestion completeness. In the control experiment, synthetic mir415a was added to normal plasma and subjected to eLB analysis both before and after RNase treatment. The results are shown in Figure 1A. The left panel shows the case without mi415a spike-in, and the right panel shows the case with mir415a addition. In both panels of Figure 1A, red represents the eLB signal without RNase treatment, and blue represents the signal after RNase treatment. As is clearly visible in the right panel of Figure 1A, RNase treatment completely eliminated the signal from the eLB reaction for microRNA, indicating that RNase digestion was complete.

[0154] The left panel of Figure 1B shows normal plasma samples before and after treatment, analyzed for the EGFR exon 19del variant. The right panel shows plasma from NSCLC patients from a previously published study using advanced NSCLC disease, where the tumor was exon 19del positive and the eLB results were also positive (Pu et al., 2016, Thorac Cancer, 7: 428-436). The signal reduction after RNase treatment was only 10%, indicating that the circulating nucleic acid measured in the eLB assay was primarily DNA and not RNA.

[0155] Single-stranded DNA vs. Double-stranded DNA Next, we investigated whether the molecules measured by EFIRM were single-stranded or double-stranded DNA. For these experiments, we used enzymatic treatment with the single-strand specific nuclease exonuclease VII. Figure 2 includes data from three control experiments and analysis of clinical samples. Figure 2A shows plasma from a healthy control, where only a background signal is demonstrated in both the untreated sample and the exonuclease VII-digested sample. Figure 2B demonstrates that when dsDNA containing the mutant spikes in the plasma, no decrease in signal is observed after exonuclease VII digestion. As predicted, when synthetic ssDNA was added, exonuclease VII digestion resulted in a significant decrease in the eLB signal (Figure 2C). Figure 2D represents a plasma sample from a patient with advanced NSCLC in which the p.T790M EGFR mutant was demonstrated from previous tissue and eLB results. Exonuclease VII treatment resulted in a dramatic decrease in eLB signaling, similar to the decrease observed in the ssDNA spike-in experiment shown in Figure 2C. Taken together, these data demonstrate that eLB primarily measures single-stranded DNA targets.

[0156] Technical validation of qualitative eLB assays for three EGFR variants. One use of eLB is for screening for malignancies in asymptomatic individuals with a history of smoking, or in individuals in whom indeterminate nodules have been detected by screening radiography. For this purpose, a "positive" or "negative" result is sufficient. For this purpose, technical validation of qualitative eLB was performed in both plasma and saliva.

[0157] Exon 19del is not a single mutant, but rather a family of closely related mutants with slight variations in the length of exon 19. Due to the nature of eLB, it was possible to design capture probe-signal probe combinations that could detect the majority, though not all, of the exon 19del mutants. The data presented below demonstrate that a single capture probe-detection probe pair can detect the four most common exon 19del mutants. Further discussion of this issue can be found in the description of the quantitative assay.

[0158] Qualitative assay of plasma To validate the qualitative assay, plasma was purchased from 100 healthy individuals as described elsewhere in this specification. eLB technology was performed on these samples in 2-row sets for three EGFR variants: exon 19del, p.L858R, and p.T790M. The results are shown in Figure 3. Nanoampere (nA) current is plotted on the Y-axis. The samples are healthy plasma samples with nA normalized by subtracting the mean nA of two untemplated controls (NTCs) in each plate. The standard 2SD reference range is plotted as a dotted line. The solid line represents the control oligonucleotide added to each plate to confirm the assay performance. The assay performance is adequate for the qualitative assay. The confirmed reference range varies slightly for each variant. For the qualitative assay of plasma, cutoff values ​​for exon 19del, p.L858R, and p.T790M are shown in Table 2, respectively. The reference range was set to less than 3SD of the unaffected control. As can be seen in Figure 3, the internal standard is well above the cutoff value for each mutant.

[0159] Both inter-assay and intra-assay variability experiments were performed for the qualitative assay using the derived reference ranges described above. For intra-assay variability, negative and positive samples were run multiple times on a single plate. Each sample was assayed at least eight times (eight replicates for mutation-negative controls and twelve replicates for mutation-positive controls, each testing for one of the three mutations). There were no inconsistencies; all negative samples were below the cutoff, and all positive samples were above the cutoff.

[0160] Regarding inter-assay variability, the assay was performed on three different plates with mutation-negative and mutation-positive controls (spiked with 12 pM oligonucleotides in biological fluid) by two different operators (each plate had 8 replicates for mutation-negative normality and 12 replicates for mutation-positive normality). Again, no conflicting results were found, resulting in zero intra-assay and inter-assay variability.

[0161] [Table 2]

[0162] Validation of a qualitative eLB assay for saliva Figure 4 shows the same experiment using saliva samples obtained from healthy dentists who gave their consent at the American Dental Association's annual meeting. The plate design and validation design were identical to those for the plasma validation. The performance of the assay was the same as that of the plasma assay. Intra-assay and inter-assay variability were similarly zero, and all replicated experiments were consistent for positive or negative results. As with the plasma assay, the reference range varied slightly for each variant. Table 2 summarizes the reference range determinations for six assays, namely plasma and saliva, three EGFR variants, exon 19del, p.L858R, and p.T790M. There was small variation in the reference range for each assay, but positive clinical samples well above these cutoff values. Internal positive controls well above the 3SD cutoff for all variants.

[0163] As with all potential screening tests, the reference range may need to be adjusted according to clinical performance. One advantage of eLB over other techniques is that both saliva and plasma can be analyzed from the same patient. Both tests can be performed simultaneously for each patient, and patients with positive results for both plasma and saliva are considered screening positive. For individuals who test positive on one and negative on the other, repeat samples are obtained and analyzed to resolve the discrepancy.

[0164] Clinical sensitivity and specificity of eLB qualitative assay The usefulness of any assay is determined by its sensitivity / specificity and positive and negative predictive values. Regarding eLB platforms, data on these parameters for detecting EGFR variants in NSCLC patients is limited. Two published trials involving patients with end-stage (III and IV) NSCLC, data (Wei et al., 2014, Am J Respir Crit Care Med, 190: 1117-1126; Pu et al., 2016, Thorac Cancer, 7: 428-436), show that qualitative eLB assays detected tumors in all 23 patients with the p.L858R variant and all 15 patients with the exon 19del variant. There were no false positives in the 28 NSCLC patients whose tumors did not contain any of these variants. Therefore, qualitative eLB is used with high sensitivity and specificity in selecting treatment options for these end-stage NSCLC patients.

[0165] Performance testing of plasma qualitative eLB in early (I and II) NSCLC patients revealed a sensitivity of 92% for p.L858R and 77% for exon 19del. Specificity was 95% for both variants in the control group, which consisted of patients with benign lung nodules. Notably, there was 100% agreement between histological variant analysis and eLB nodule biopsy. No contradictory results were observed in these studies.

[0166] eLB quantitative assay Quantitative evaluation of eLB was performed in two parts. First, detection limits, linearity, and linear range were confirmed using genomic DNA controls. However, biological material is not ideal for use as an internal calibrator in assays. Therefore, confirmation of detection limits, linearity, and linear range using genomic DNA was performed, followed by the development of a clinical assay using synthetic oligonucleotides as internal standards and calibrators.

[0167] EGFR exon-19del Exon 19del variants of EGFR are actually a closely related family of variants. Table 2 lists the five most prevalent variants within this family. Because the interruptions of these deletions are close together, it is theoretically possible to design a capture / detection probe set that can detect most, though not all, variants of the exon 19del family (Table 3). To demonstrate that the eLB assay can detect four of the most common exon 19del variants, DNA was obtained from four cell lines, each containing one of the common variants. Genomic DNA was processed and subjected to the eLB reaction at various concentrations.

[0168] Figure 5 shows the results obtained using serial dilutions of genomic DNA containing the four most common EGFR 19del variants. As can be seen, the EGFR assay can detect all four of these variants with similar performance.

[0169] The assay was linear for all four variants, and R 2 The values ​​were 0.99, 0.99, 0.95, and 0.94 for mutants c.2235_2249 del18, c2236_2250 del18, c2240_2257 del18, and c.2239_2248 del11, respectively. The linear range was from 20,000 copies / 25 μL to 500 copies / 25 μL. It is important to note that sheared genomic DNA fragments are not the optimal template for the eLB reaction. Therefore, the detection limit may be significantly underestimated. Thus, these data should be considered as the minimum sensitivity of the eLB reaction.

[0170] [Table 3]

[0171] Linearity and detection limits of eLB for T790M Figure 6 shows data from various dilutions of genomic DNA containing the mutant p.T790M. The assay is linear and ranges from 300 to a single-digit copy number per 25 μL. 2 The value was 0.99.

[0172] Linearity in the detection limit of eLB for p.L858R Figure 7 demonstrates the linearity and range for p.L858R. The linearity is excellent, with R exhibiting a range of 300 copies to a single-digit copy number per 25 μL. 2 The value was 0.998.

[0173] Analytical validation of eLB for three types of EGFR mutants After determining linearity and LOD separately for each mutant, the performance of eLB for the combined mutants in a single assay was investigated. One measure of the sensitivity of a liquid biopsy platform is the assay's ability to detect mutant sequences in a background of non-mutant sequences. For this purpose, reference materials containing an artificial mixture of wild-type and mutant sequences are available. Reference materials were purchased from Horizon Diagnostics, and these samples were subjected to eLB analysis (see Figure 8). From the data, the eLB results for three EGFR mutants were demonstrated, and it was found that the T790M eLB could detect the mutant at the 0.1% level. Although slightly less sensitive for exon 19del and p.L858R, the mutant sequences could be clearly detected at the 1% level. Reference materials are not available between 1% and 0.1%, and therefore it is not possible to determine how low a minor allele fraction below 1% can be detected by EFRIM for exon 19del and p.L858R. These detection levels are better than those of the in vitro diagnostic kits available from Roche for EGFR detection in liquid biopsy.

[0174] For the three mutant assays with quantitative eLB, synthetic oligonucleotide standards were used as calibration and internal standards. Titration measurements were performed in quadruplicate using various dilutions of oligonucleotides containing each of the three mutants at concentrations ranging from 0.02 pM to 1600 pM. The linearity was excellent, and the R 2 values were 0.99, 0.98, and 0.99 for p.T790M, p.L858R, and exon19del, respectively.

[0175] Verification results (linearity, inter-assay and intra-assay CV, reference range) Quantitative verification of eLB was achieved by conducting a series of experiments using plasma and saliva matrices to evaluate the linearity of the assays, the CV of the assays, and the reference range for p.L858R, exon19 deletion, and p.T790M. For linearity, two-fold dilutions ranging from 1000 pM to 24 femtomolar were performed by spiking synthetic oligonucleotide targets into plasma or saliva matrices. Each dilution was performed 4 times. From the results, for all of p.L858R (R 2 = 0.99), exon19 deletion (R 2 = 0.99), and p.T790M (R 2 = 0.99), R 2 ≧0.99 was demonstrated when within the ranges of 0 - 10 pM, 0 - 50 pM, and 0 - 12.5 pM in saliva, respectively. Similarly, in plasma, for p.L858R (R 2 = 0.996), exon19 deletion (R 2 = 0.999), and p.T790m (R 2 = 0.996) within the ranges of 0 - 6.25 pM, 0 - 12.5 pM, and 0 - 12.5 pM, respectively, R 2A value of ≥0.99 was achieved. The detection limit was calculated using copy number versus oligonucleotide targeted equivalence. The detection limits for saliva were measured at 27.51 fM (3 copies), 144.86 fM (31 copies), and 60.38 fM (9 copies) for p.L858R, exon 19 deletion, and p.T790M mutations, respectively. For plasma, the detection limits were measured at 41.63 fM (119 copies), 28.26 fM (211 copies), and 27.72 fM (142 copies), respectively.

[0176] For intraassay mutations, four checkpoint dose curves were used for each mutation in plates of 88 replicate experiments of biofluid spiked with a 3.25 pM oligonucleotide target (concentrations of 3.25 pM, 1.56 pM, 0.78 pM, and 0 pM). Intraassay CVs for p.L858R, exon 19 deletion, and p.T790M were 8.72%, 18.6%, and 12.5% ​​in plasma, and 29.3%, 25.2%, and 23.3% in saliva, respectively.

[0177] For inter-assay CV, four runs were performed, with each run assay involving 24 replicates of each mutation and three checkpoint dose curves at 3.25 pM in biological fluid. The inter-assay CVs for p.L858R, exon 19 deletion, and p.T790M were 42.2%, 45.9%, and 30.7% for plasma, and 40.6%, 35.9%, and 29.0% for saliva, respectively.

[0178] To assess the variability of signal levels within a healthy population, for each biofluid, four checkpoint level curves were used, running over four or five plates for each of the three mutations in healthy plasma and saliva from 40 healthy subjects. Based on three standard deviations from the mean, the cutoff values ​​for p.L858R, exon 19 deletion, and p.T790M were confirmed to be values ​​4.04 nA, 4.34 nA, and 6.40 nA above normalized background current measurements for saliva, and 7.10 nA, 9.18 nA, and 12.57 nA above normalized background current measurements for plasma, respectively.

[0179] The entire disclosure of each patent, patent application, and publication cited herein is thus incorporated herein by reference.

[0180] Although the present invention has been disclosed with reference to specific embodiments, it will be apparent to those skilled in the art that other embodiments and variations of the present invention can be devised without departing from the true spirit and scope of the invention. The appended claims shall be construed to encompass all such embodiments and equivalent variations.

Claims

1. A system for detecting target nucleic acid molecules in a sample, a) A multiwell plate comprising an array of sensors, wherein each well comprises an electrode tip comprising a working electrode, a counter electrode, and a reference electrode, and at least one unit of the working electrode is coated with a conductive polymer; b) A probe set comprising at least one pair of capture probes and detection probes, wherein the capture probe comprises a poly-A region comprising at least 65 nucleotides, at least 80% of which are adenine, the poly-A sequence is interrupted by non-adenine nucleotides and is embedded or functionalized in a conductive polymer, and the detection probe is biotin-labeled at its 3' terminal nucleotide; c) Multiwell plate washer; and d) A multi-channel electrochemical reader that controls the electric field applied to the array sensor and simultaneously reports the measured current. A system that includes this.

2. The system according to claim 1, wherein at least one of the capture probe and the detection probe hybridizes with a nucleic acid molecule containing a lung cancer marker.

3. The system according to claim 2, wherein the lung cancer marker is a variant of the epidermal growth factor receptor (EGFR) selected from the group consisting of exon 19 deletion, T790M, L858R, c. 2235_2249 del18, c. 2236_2250 del18, c. 2240_2257 del18, c. 2239_2248TTAAGAGAAG>C, c. 2239_2247 delTTAAGAGAA and c. 2239_2248 del11.

4. The system according to claim 3, wherein the lung cancer marker is selected from the group consisting of exon 19 deletion, T790M, and L858R.

5. A pair of capture probes and detection probes, a) A probe pair for detecting exon 19 deletion, wherein the capture probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 13, and the detection probe comprises SEQ ID NO: 6; b) A probe pair for detecting L858R, wherein the capture probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 14, and the detection probe comprises SEQ ID NO: 2; and c) A probe pair for detecting T790M, wherein the capture probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 15, and the detection probe comprises SEQ ID NO:

10. The system according to claim 4, selected from the group consisting of the following.

6. A method to assist in the detection of lung cancer in a subject, a) A step of mixing a first portion of at least one sample derived from the subject with a solution containing a biotin-labeled detection probe; b) For use in the system according to claim 1, a step of adding the mixture to a single well of a multiwell plate, wherein each well of the multiwell plate includes an electrode tip comprising a working electrode, a counter electrode, and a reference electrode, the working electrode being coated with a conductive polymer in which a capture probe is embedded, the capture probe comprising a poly-A region comprising at least 65 nucleotides, wherein at least 80% of the nucleotides are adenine, and the sequence of the poly-A is interrupted by non-adenine nucleotides; c) Applying a periodic square wave electric field to the electrode tip; d) Adding horseradish peroxidase (HRP) conjugated with streptavidin from the first round to the wells; e) Adding biotin-labeled anti-HRP antibody to the wells; f) The step of adding HRP conjugated with streptavidin in the second round to the well, g) A step of measuring the current in an electrode tip, wherein the change in current correlates with the presence of a lung cancer-related marker in the sample. Methods that include...

7. The method according to claim 6, further comprising at least one washing step, wherein the multiwell plate is washed using an automated plate washer.

8. The method according to claim 6, wherein at least one reagent is maintained at 4°C before use.

9. The method according to claim 6, wherein at least one of the capture probe and the detection probe hybridizes with a nucleic acid molecule containing a lung cancer marker.

10. The method according to claim 9, wherein the nucleic acid molecule is a circulating tumor DNA (ctDNA) molecule.

11. The method according to claim 6, wherein the lung cancer marker is a variant of EGFR selected from the group consisting of exon 19 deletion, T790M, L858R, c. 2235_2249 del18, c. 2236_2250 del18, c. 2240_2257 del18, c. 2239_2248TTAAGAGAAG>C, c. 2239_2247delTTAAGAGAA and c. 2239_2248 del11.

12. The method according to claim 11, wherein the lung cancer marker is selected from the group consisting of exon 19 deletion, T790M, and L858R.

13. A pair of capture probes and detection probes, a) A probe pair for detecting exon 19 deletion, wherein the capture probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO: 13, and the detection probe comprises SEQ ID NO: 6; b) A probe pair for detecting L858R, wherein the capture probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 14, and the detection probe comprises SEQ ID NO: 2; and c) A probe pair for detecting T790M, wherein the capture probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 15, and the detection probe comprises SEQ ID NO:

10. The method according to claim 12, selected from the group consisting of the following.

14. The method according to claim 6, wherein at least one sample is selected from the group consisting of a saliva sample, a blood sample, a plasma sample, and a serum sample.

15. The method according to claim 6, wherein a saliva sample from the subject is added to the first well of a multiwell plate, and a plasma sample from the same subject is added to the second well of a multiwell plate.

16. The method according to claim 15, wherein if a lung cancer marker is detected in both a saliva sample and a plasma sample from the same subject, the subject is determined to have lung cancer or be at risk of lung cancer.

17. The method according to claim 6, wherein if a lung cancer marker is detected, the need for treatment is indicated.

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