Impedance-based liquid biopsy system and method for cancer detection and screening

Abstract

The present disclosure provides impedance-based detection of nucleic acids for cancer screening. The disclosure is based on the use of impedance spectroscopy to detect nucleic acids in solution in the clinical concentration range without the use of any labels, amplification steps, bioreceptors, surface modifications, or complex electrode configurations. More specifically, the disclosure exploits structural variations in nucleic acids from healthy individuals and cancer patients to classify samples as cancer-positive or cancer-negative based solely on the electrophysiochemical properties of the samples. The disclosure also provides a simple, rapid, label-free, miniaturized biosensor for cancer detection and screening.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Indian Patent Application No. 202311005850 entitled "IMPEDANCE-BASED LIQUID BIOPSY SYSTEM AND METHOD FOR DETECTING AND SCREENING CANCER," filed on January 30, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing, filed in extensible markup language (xml) entitled "P12615PC00_Sequence_Listing_ST.26.xml," having a size in bytes of 17 kb, created on January 18, 2024. The information contained in this electronic file is incorporated herein by reference in its entirety.

[0003] Field The present disclosure relates generally to the field of cancer screening. More specifically, the present disclosure provides a novel approach for DNA detection using impedance spectroscopy for cancer screening. [Background technology]

[0004] Liquid biopsy is a minimally invasive diagnostic approach that has emerged as a promising tool for cancer screening and management through the evaluation of blood-based biomarkers. Circulating tumor cells (CTCs), genomic DNA (gDNA), circulating tumor DNA (ctDNA), and cell-free DNA (cfDNA) are some of the most widely studied biomarkers under this approach. ctDNA is tumor-derived fragmented DNA released into the blood by apoptotic or necrotic cancer cells. Therefore, their concentration levels increase in the blood during cancer and can be used to track disease progression. The most common platforms for DNA detection are next-generation sequencing (NGS), digital PCR, real-time PCR, and mass spectrometry. All of these methods require highly complex and expensive infrastructure, which limits their adoption in clinical settings. They are also very time-consuming because they lack the sensitivity required to produce results without signal or target amplification. Currently, no single technology exists that can detect ctDNA directly in the blood at low concentrations without an additional amplification step in a simple and rapid manner.

[0005] In addition to sequencing, aberrant DNA methylation has also been widely studied as an indicator of cancer development and progression. Some common techniques for cancer detection based on methylation patterns in plasma DNA include methylated DNA immunoprecipitation sequencing (MeDIP-seq), bisulfite sequencing, and hydroxymethylcytosine sequencing. MeDIP-seq utilizes a 5mC (methylcytosine)-specific antibody to capture nonspecifically fragmented methylated DNA strands. The unenriched DNA is then labeled with different fluorescent dyes (e.g., Cy5 and Cy3) and co-hybridized on a microarray platform. The ratio of the fluorescence intensities of the two dyes reflects the methylation status of the region of interest. The main limitations of this approach are the need for single-stranded DNA for analysis and the fact that the quality of the anti-5mC antibody can vary greatly. Bisulfite sequencing uses bisulfite treatment of DNA to deaminate cytosines to uracils, which are then read as thymines. The challenge is that bisulfite conversion can lead to DNA fragmentation, resulting in the generation of chimeric products. Because circulating DNA is already short, further fragmentation during bisulfite treatment can be a concern, as further fragmentation could potentially reduce the sensitivity of the assay.

[0006] Recent relatively fast methods for DNA detection include colorimetry, fluorescence, and impedance spectroscopy. In particular, DNA-electrochemical biosensors have attracted significant attention due to their robustness, ease of miniaturization, low detection limits, and compatibility with biological fluids. Traditionally, DNA detection using integrated sensors requires immobilization of molecules on an electrode surface using bioaffinity receptors, followed by signal amplification using a set of probes or chemically active compounds. This not only adds extra steps and reagent costs to the process, but also increases the chance of false positives due to nonspecific adsorption of molecules on the surface. Therefore, electrode design / materials must undergo extensive optimization for various binding steps, while chips generally cannot be reused, making the process less environmentally friendly and economically inefficient. Some recent studies have attempted to address these issues by measuring the electrical properties of DNA directly in solution, but their signal sensitivity is low and significantly affected by the type of solvent used. In deionized (DI) water, the lower limit of detection (LOD) of DNA was found to be >10 mg / ml (Ma et al., Scientific reports, 2013, 3, 2730).

[0007] Thus, available methods suffer from one or more limitations, and there is a need for the development of improved systems and methods for label-free detection of DNA for cancer diagnosis and screening without the use of surface modifications, tedious amplification steps, or complex electrode structures. Summary of the Invention [Problem to be solved by the invention]

[0008] This disclosure provides a novel impedance approach for DNA detection and cancer stratification by exploiting the effects of structural abnormalities in DNA on its electrophysiochemical properties. This approach involves the use of impedance spectroscopy to ensure direct, rapid, sensitive, and label-free detection of DNA for cancer screening. [Means for solving the problem]

[0009] An aspect of the present disclosure is to provide a biosensor for analyzing a test sample that may contain cfDNA.The biosensor also includes a microchamber for holding the test sample suspended in a zwitterionic buffer, the microchamber enclosing a plurality of electrodes, and an impedance analyzer operatively connected to the microchamber and a programmable controller with a digital processing unit for receiving information from the plurality of electrodes, and an impedance signal is measured by placing the test sample on the plurality of electrodes.

[0010] In the biosensor, the microchamber may comprise a material selected from polydimethylsiloxane, polylactic acid, polylactic-co-glycolic acid, polyetheretherketone, silicone, nitrile, polyurethane, soft vinyl chloride resin, polypropylene, polyamide, polyethylene, polycarbonate, acrylonitrile butadiene styrene (ABS) resin, polystyrene, and poly(methyl methacrylate).

[0011] The biosensor may be for use in rapid, label-free, and amplification-free screening for cancer.

[0012] Another aspect of the present disclosure is to provide a liquid biopsy method.This method also includes: collecting a biological sample, extracting cfDNA fraction from this biological sample, suspending this cfDNA fraction in a zwitterionic buffer to prepare a test sample, loading this test sample into a plurality of electrodes enclosed in a microchamber, using an impedance analyzer to measure the electrical conductivity of the test sample, and analyzing the difference between the electrical conductivity of the test sample and that of a control sample.

[0013] In the method, the biological sample may include a biological fluid selected from whole blood, plasma, platelets, saliva, white blood cells, serum, and urine. In an example where the biological sample includes plasma, the method may include separating the plasma from the whole blood by double centrifugation, which may include a first cycle at 1500 g to 2500 g, preferably 2000 g, for 7 to 12 minutes, preferably 10 minutes, at 4°C, and a second cycle at 15000 g to 17000 g, preferably 16000 g, for 7 to 12 minutes, preferably 10 minutes.

[0014] The zwitterionic buffer may be selected from (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), piperazine-1,4-bis(2-hydroxypropanesulfonic acid), and 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid.

[0015] This method can be used for quantitative and qualitative detection of cfDNA.

[0016] The method may have a limit of detection of 0.4 ng / ml or 5.14 pm for cfDNA in HEPES buffer and a sensitivity and specificity of about 95%, respectively.

[0017] The method may have a detection time of less than 5 minutes, preferably less than 1 minute.

[0018] One general embodiment involves the use of impedance spectroscopy in the detection of cancer based on the electrical conductivity of a test sample, which may contain cfDNA in solution.

[0019] One general embodiment includes a label-free method for detecting cfDNA using impedance spectroscopy. This label-free method also includes: a. collecting a biological sample and extracting a cfDNA fraction from the biological sample; b. suspending the cfDNA fraction in a liquid to prepare a test sample; c. loading the test sample onto a plurality of electrodes enclosed in a microchamber; d. using an impedance analyzer to measure the electrical conductivity of the test sample; and e. analyzing the difference between the electrical conductivity of the test sample and that of a control sample.

[0020] Implementations may include one or more of the following features: In the method, the liquid is selected from a zwitterionic buffer, deionized water, and purified water; The biological sample may include a biological fluid selected from whole blood, plasma, platelets, saliva, white blood cells, serum, and urine; The biological sample may include plasma, and the method may include separating the plasma from the whole blood by double centrifugation, which may include a first cycle at 1500 g to 2500 g, preferably 2000 g, for 7 to 12 minutes, preferably 10 minutes, and a second cycle at 15000 g to 17000 g, preferably 16000 g, for 7 to 12 minutes, preferably 10 minutes, at 4°C. The buffer may comprise a zwitterionic buffer selected from (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), piperazine-1,4-bis(2-hydroxypropanesulfonic acid), and 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid. The method can be used for quantitative and qualitative detection of cfDNA. The method has a detection time of less than 5 minutes, preferably less than 1 minute.

[0021] One general embodiment involves a method for detecting DNA using impedance spectroscopy, which also includes the steps of: a. collecting a biological sample and extracting a nucleic acid fraction from the biological sample; b. preparing a test sample by suspending the nucleic acid fraction in a zwitterionic buffer; c. loading the test sample onto a plurality of electrodes enclosed in a microchamber; d. measuring the electrical conductivity of the test sample using an impedance analyzer; and e. analyzing the difference between the electrical conductivity of the test sample and that of a control sample.

[0022] The nucleic acid fraction may comprise at least one of genomic DNA (gDNA), cell-free DNA (cfDNA), mitochondrial DNA (mtDNA), and complementary DNA (cDNA).

[0023] The biological sample may comprise a biological fluid selected from whole blood, plasma, platelets, saliva, white blood cells, serum, and urine.

[0024] In embodiments where the biological sample comprises whole blood, the method may include separating a buffy layer from the whole blood. The zwitterionic buffer is selected from (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), piperazine-1,4-bis(2-hydroxypropanesulfonic acid), and 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid. The method may be used for quantitative and qualitative detection of DNA.

[0025] The method may have a detection time of less than 5 minutes, preferably less than 1 minute.

[0026] Additional objects, advantages, and novel features of the present disclosure will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the biosensors and methods described herein. [Brief explanation of the drawings]

[0027] The accompanying drawings, together with the description, illustrate some of the embodiments of the present disclosure, and are provided by way of example and not by way of limitation.

[0028] [Figure 1A] FIG. 1A is a schematic diagram depicting a biosensor for impedance-based detection of DNA for cancer screening.

[0029] [Figure 1B] FIG. 1B is a schematic diagram depicting a method for impedance-based detection of DNA for cancer screening.

[0030] [Figure 2] Figure 2 shows frequency optimization for distinguishing healthy versus cancer cfDNA. The largest change in signal was observed between 10 kHz and 1 MHz. Results are shown for three healthy and three cancer samples with the following concentrations: 40.92 ng / ml, 54.18 ng / ml, 58.31 ng / ml, 79.92 ng / ml, 89.54 ng / ml, and 99.90 ng / ml.

[0031] [Figure 3] Figure 3 shows the effect of different media on the impedance response of cancer and healthy plasma samples (cfDNA concentration 53.50 ng / ml). Data are reported as the mean ± 2 SD of triplicates. Samples prepared in Milli-Q™ water and DI water are shown on the left-Y axis, and the remaining samples prepared in buffer solution are shown on the right-Y axis.

[0032] [Figure 4A] Figure 4A shows a box plot of cfDNA concentrations in healthy and cancer clinical plasma samples. The upper boundary of the box indicates the upper quartile (75th percentile), the lower boundary indicates the lower quartile (25th percentile), and the horizontal line within the box is the median.

[0033] [Figure 4B]FIG. 4B is a graph showing a comparative analysis of impedance signals in an elution buffer containing 15 mM HEPES pH 7.4.

[0034] [Figure 4C] FIG. 4C is a graph showing the number of samples for the cancer types examined.

[0035] [Figure 4D] FIG. 4D is a graph showing the ROC analysis of clinical samples.

[0036] [Figure 5] Figure 5 shows the effect of added cfDNA concentration on the impedance signal. Data are reported as the mean ± 1 SD of two replicates.

[0037] [Figure 6] Figure 6 shows representative TEM micrographs of healthy and cancer cfDNA suspended in 15 mM HEPES buffer, pH 7.4. The images show clear morphological differences between the two cfDNA types at various concentrations.

[0038] [Figure 7A] FIG. 7A is a graph showing the effect of methylation on the impedance signal of single-stranded (ss) and double-stranded (ds) synthetic oligonucleotides.

[0039] [Figure 7B] FIG. 7B is a series of TEM images of 25% and 100% methylated ds-oligonucleotides taken at concentrations of 70 ng / ml and 140 ng / ml.

[0040] [Figure 8A] FIG. 8A is a graph of impedance as a function of oligo concentration for various sequences.

[0041] [Figure 8B] FIG. 8B is a chart of the sequences tested in FIG. 8A. DETAILED DESCRIPTION OF THE INVENTION

[0042] First, it should be understood that the following description merely illustrates specific embodiments of the present invention. However, such specific embodiments are merely exemplary embodiments and do not imply any limitations on the scope of the present disclosure. Therefore, the descriptions and examples should be understood as exemplary embodiments for teaching the present disclosure and are not intended to be limiting.

[0043] The details of one or more embodiments of the present disclosure are set forth, by way of example, in the accompanying description below, including specific details of the best mode contemplated by the inventors for carrying out the invention. It will be apparent to those skilled in the art that the present disclosure may be practiced without limitation to these specific details. Abbreviations used [Table 1]

[0044] definition The use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It is to be understood that both the foregoing general description and this detailed description are exemplary and explanatory only and are not restrictive.

[0045] As used herein, biotechnology terms have their conventional meanings as exemplified by the following exemplary definitions.

[0046] As used herein, the term "impedance" refers to the effective resistance of a current or electrical component to alternating current resulting from the combined effects of ohmic resistance and reactance.

[0047] As used herein, the term "biomarker" refers to a biological marker that is a measurable indicator of some biological state or condition.

[0048] "Zwitterionic" is used herein to describe a molecule that has both a positive and a negative charge. The terms "zwitterion," "inner salt," or "zwitterion" may be used interchangeably herein to describe a zwitterionic molecule.

[0049] Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0050] The foregoing has outlined broadly the features and technical advantages of the present disclosure in order that the detailed description of the disclosure that follows may be better understood. It will be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying the disclosed methods or for carrying out the same purposes of the present disclosure.

[0051] The present disclosure provides a novel approach for detecting nucleic acids in solution based on the effect of structural abnormalities in nucleic acids on the electrophysiochemical properties of nucleic acids to differentiate cancer and healthy samples using impedance spectroscopy. In some embodiments, the present disclosure combines impedance spectroscopy with a zwitterionic buffer solution with inherently low conductivity at physiological pH. The buffer's background structure and low ionic conductivity allow for the measurement of even minimal differences in electrical conductivity between nucleic acid molecules in healthy individuals and cancer patients. These differences are likely due to differences in the solvation properties of cancer and healthy nucleic acids due to structural variations in their methylation patterns. These structural differences can change significantly in genomic DNA, and therefore in shorter DNA molecules such as cfDNA, resulting in differences in their dielectric properties.

[0052] Aberrant methylation is a hallmark of tumorigenesis, and differences in methylation patterns between tumors and benign tissues have been reported in many cancer types. A distinctive feature of epigenetic remodeling in cancer gDNA is differential hypomethylation in coding and intergenic regions and differential hypermethylation in CpG-rich regulatory regions, resulting in a significantly hypomethylated entire genomic landscape. gDNA-derived cfDNA fragments are more selectively enriched in coding and intergenic regions compared to gene promoter regions in both malignant and benign samples. This suggests that the structural variations in cancer and healthy cfDNA described in the Examples herein arise from differences in their methylation patterns, with healthy cfDNA containing a much greater number of methyl groups. It is the presence of these nonpolar functional methyl groups that leads to the hydrophobic self-assembly of healthy cfDNA in aqueous solution. Because the loss of methyl groups from cytosines is gradual, the morphology changes from microaggregates to dispersed nanoaggregates as cancer progresses. More importantly, because the global loss of methylation is a common signature shared by multiple cancer types, the electrophysicochemical properties and impedance signals also behave similarly across different cancer types.

[0053] The present disclosure provides highly sensitive impedance measurements (readouts) with high signal-to-noise ratios (S / N), and therefore, the present impedance spectroscopy-based approach can be used for direct, rapid, and label-free detection of DNA for cancer screening and diagnosis.

[0054] In one embodiment, the present disclosure provides a biosensor (100) for analyzing a test sample, the biosensor (100) comprising: a microchamber (101) configured to hold the test sample suspended in a zwitterionic buffer, the microchamber (101) enclosing a plurality of electrodes (102); and an impedance analyzer (105) operably connected to a programmable controller (104) with a digital processing unit for receiving information from the microchamber and the interdigitated microelectrodes, wherein impedance signals are measured by placing the test sample on the plurality of electrodes enclosed within the microchamber. Figure 1A provides a schematic diagram of a biosensor for impedance-based detection of cfDNA using a zwitterionic buffer.

[0055] The electrodes (102) may include any suitable type of electrode, including, but not limited to, interdigitated microelectrodes (IDEs), microdisk electrodes, microband electrodes, and three-dimensional microelectrode arrays.

[0056] The microchamber (101) may include an opening for receiving a test sample. The opening of the microchamber (101) may be covered with a seal (103). Any suitable material may comprise the seal (103), including, but not limited to, glass, film, pressure-sensitive adhesive tape, polymers, etc. In certain non-limiting examples, the seal (103) comprises a glass cover slip, Parafilm™, Scotch™ Tape, or a transparency slide. In some examples, the seal (103) is removably attachable to the microchamber (101). In further examples, the seal (103) is placed on the microchamber (101). The seal (103) may reduce evaporation and contamination of the test sample.

[0057] The microchambers in the biosensor may comprise a material selected from, but not limited to, polydimethylsiloxane (PDMS), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), polyetheretherketone, silicone, nitrile, polyurethane, soft vinyl chloride resin, polypropylene, polyamide, polyethylene, polycarbonate, acrylonitrile butadiene styrene (ABS) resin, polystyrene, and poly(methyl methacrylate) (PMMA).

[0058] 1B is a schematic diagram illustrating an impedance spectroscopy method (110). In the example described herein, the method (110) is performed by a biosensor (100). The method includes: a. collecting a biological sample and extracting a nucleic acid fraction from the sample (112); b. suspending the nucleic acid fraction in a liquid to prepare a test sample (114); c. loading (116) the test sample onto a plurality of electrodes enclosed within the microchamber of claim 1; d. measuring the electrical conductivity of the test sample using an impedance analyzer (118); e. analyzing (120) the difference in electrical conductivity of the test sample by comparison with a standard; Includes:

[0059] Block 112 involves collecting a biological sample and extracting a nucleic acid fraction from the sample. The biological sample may include a biological fluid selected from, but not limited to, whole blood, plasma, platelets, saliva, white blood cells, serum, urine, saliva, cerebrospinal fluid, amniotic fluid, bone marrow, and synovial fluid.

[0060] In embodiments where the biological sample comprises plasma, the plasma may be obtained by separating a blood sample. In a particular example, the plasma is separated from the blood by double centrifugation at about 4°C at 1500g-2500g, preferably 2000g, for about 7-12 minutes, preferably 10 minutes, followed by 15000g-17000g, preferably 16000g, for about 7-12 minutes, preferably 10 minutes.

[0061] The nucleic acid fraction may comprise any suitable type of DNA, including, but not limited to, genomic DNA (gDNA), cell-free DNA (cfDNA), mitochondrial DNA (mtDNA), complementary DNA (cDNA), guide RNA (gRNA), and combinations thereof.

[0062] Any suitable means known in the art may be used to extract nucleic acid fractions from biological samples, including, but not limited to, phenol-chloroform, cetyltrimethylammonium bromide, silica columns, anion exchange columns, Chelex™ resin, salting out, solid-phase reversible immobilization (SPRI), and combinations thereof. The method of nucleic acid extraction may be selected to extract a subset of the total nucleic acids in the biological sample.

[0063] In specific examples where the nucleic acid fraction comprises cfDNA, the cfDNA may be extracted using the MagMax™ Cell-Free DNA Isolation Kit (Thermo Fisher Scientific, Waltham, Massachusetts).

[0064] In a specific example where the nucleic acid fraction includes guide RNA or genomic DNA, extraction may involve centrifuging whole blood at 2000 g for 10 minutes at 4°C to obtain three layers: plasma (top), buffy coat (middle), and red blood cells (bottom). In a specific example where the nucleic acid fraction includes gRNA, the buffy coat layer may be collected and mixed with TRIzol® Reagent (Invitrogen, catalog number 15596026). The RNA pellet may be dissolved in RNAase-free water and quantified by UV-visible spectroscopy within two hours of isolation. The extract may be used directly for analysis or stored at -80°C. In a specific example where the nucleic acid includes gDNA, gDNA may be extracted from the buffy coat layer using the KingFisher™ Blood DNA Kit (Thermo Fisher Scientific, catalog number 97010196) or any other standard method.

[0065] In a specific example where the nucleic acid fraction contains mtDNA, mtDNA may be extracted by isolating PBMCs (peripheral blood mononuclear cells) from whole blood using Ficoll-Hypaque density gradient centrifugation. Briefly, blood is layered on Ficoll-Hypaque and spun at 1800 rpm for 30 minutes. This results in four layers: plasma, buffy coat (containing PBMCs), Ficoll, and red blood cells (RBCs). The white buffy coat layer is collected and mixed with PBS to a total volume of 45 ml. This mixture is centrifuged at 1300 rpm for 7 minutes to pellet the cells, which are then washed with PBS. mtDNA is isolated from PBMCs using the Mitochondrial DNA Isolation Kit (Abcam, ab65321).

[0066] In some examples, block 112 further includes amplifying the nucleic acid. Any suitable amplification method may be used, including, but not limited to, polymerase chain reaction (PCR), isothermal amplification, multiple displacement amplification, and ligase chain reaction.

[0067] Block 114 involves preparing a test sample by suspending the nucleic acid fraction in a liquid. The volume of the liquid may be selected so that the concentration of nucleic acid in the test sample is equal to or approximately equal to the concentration of the nucleic acid fraction in the biological sample. In another example, the volume is selected so that the concentration of nucleic acid in the test sample is equal to or approximately equal to a predetermined standard. As part of the block 114 process, the concentration of nucleic acid in the nucleic acid fraction may be quantified. The method for quantifying nucleic acid is not particularly limited. In a specific example, nucleic acid may be quantified by UV-visible spectroscopy (Nanodrop™ Lite, Thermo Fisher Scientific).

[0068] The liquid in which the nucleic acid fraction is suspended may include, but is not limited to, water, zwitterionic buffers, and combinations thereof. In certain instances where the liquid is water, the liquid may include ultrapure water, Milli-Q™ water (MilliporeSigma, Burlington, Massachusetts), deionized water, etc.

[0069] In examples where the liquid includes a zwitterionic buffer, the zwitterionic buffer may include Good's buffer. Suitable examples of Good's buffer include, but are not limited to, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), piperazine-1,4-bis(2-hydroxypropanesulfonic acid) (POPSO), and 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid (EPPS).

[0070] The concentration of the zwitterionic buffer may be about 1 mM to about 100 mM. In a specific example, the concentration of the zwitterionic buffer is about 5 mM. In a specific example, the concentration of the zwitterionic buffer is about 10 mM. In a further example, the concentration of the zwitterionic buffer is about 15 mM. In yet a further example, the concentration of the zwitterionic buffer is about 20 mM.

[0071] Block 114 may further include adjusting the pH of the liquid. In a specific, non-limiting example, the pH of the liquid is adjusted to about 7.4.

[0072] Block 116 includes loading the test sample onto the plurality of electrodes 102 in the biosensor 100. Block 116 may include adding the test sample to the microchamber such that the test sample covers at least two of the plurality of electrodes 102. After loading the test sample into the microchamber, the microchamber 101 may be encapsulated with a sealant 103 to prevent evaporation of the test sample.

[0073] Block 118 includes measuring the electrical conductivity of the test sample using an impedance analyzer. As part of block 118, the impedance analyzer (105) applies a test signal to one of the electrodes (102). The frequency of the test signal may be in the range of about 1 Hz to about 1 MHz. In a particular example, the frequency of the test signal may be about 0.1 MHz. Generally, the frequency of the test signal is selected to maximize the difference between measurements of the cancerous sample and the healthy sample. As part of block 118, the impedance analyzer (105) measures a response signal at one of the electrodes. To calculate the electrical conductivity of the test sample, the impedance analyzer (105) compares the test signal to the response signal. In some examples, the test signal is applied to a first one of the electrodes and the response signal is measured at a second one of the electrodes. In other examples, the test signal is applied to the same electrode at which the response signal is measured.

[0074] Block 120 involves analyzing the difference in electrical conductivity of the test sample by comparison with a standard. In the biosensor 100, block 120 may be performed by the impedance analyzer 105 or a processor connected to the impedance analyzer. The standard may be retrieved from memory in the impedance analyzer or processor. In some examples, the standard is obtained by measuring the electrical conductivity of a liquid in which nucleic acids are not suspended. In other examples, the standard is obtained by measuring the electrical conductivity of a known concentration of synthetic oligonucleotides. In some examples, the standard is obtained by measuring the electrical conductivity of nucleic acids from subjects known to be non-cancerous or healthy. In yet further examples, the standard is obtained by measuring the electrical conductivity of nucleic acids from subjects known to be cancerous. Generally, the standard should be measured using the same method and the same or identical biosensor used to measure the electrical conductivity of the test sample. Microchamber dimensions and other variables can affect the electrical conductivity determined through method 110.

[0075] The standard may include a single value or a range of values. In some examples, block 120 includes comparing the electrical conductivity of the test sample to multiple standards.

[0076] Based on the comparison with the standard, block 120 may further include determining whether the biological sample is cancerous. The determination may be output on a display connected to the impedance analyzer (105) or the processor.

[0077] In some instances, the standard includes a predetermined threshold value. If the electrical conductivity of the test sample is above the predetermined threshold value, the test sample is determined to be cancerous. If the electrical conductivity of the test sample is below the predetermined threshold value, the test sample is determined to be non-cancerous (healthy).

[0078] In some examples, block 120 includes determining whether the electrical conductivity of the test sample is close to that of a non-cancerous (healthy) standard or a cancerous standard. If the electrical conductivity value is close to the non-cancerous standard, the biological sample is determined to be non-cancerous. If the electrical conductivity value is close to the cancerous standard, the biological sample is determined to be cancerous.

[0079] In examples where the standard includes a range of values, the determination in block 120 may include determining whether the electrical conductivity of the test sample is within that range. If the electrical conductivity of the test sample is within the cancerous standard range, the biological sample is determined to be cancerous. If the electrical conductivity of the test sample is within the non-cancerous standard range, the biological sample is determined to be healthy.

[0080] While block 120 is described with respect to cancerous and non-cancerous standards, it should be understood that block 120 can similarly determine qualitative traits of biological samples, such as cancer type or progression. In some examples, the standards represent nucleic acid samples obtained from one or more subjects with a particular type of cancer, such as breast cancer, prostate cancer, lung cancer, colorectal cancer, etc. In other examples, the standards represent nucleic acid samples obtained from one or more subjects at various stages of cancer progression, such as pre-cancerous, stage I, stage II, stage III, stage IV, or remission.

[0081] In another embodiment, the processes of the present disclosure can be used for quantitative and qualitative detection of cfDNA.

[0082] In a further embodiment, the process of the present disclosure has an LOD of 0.4 ng / ml or 5.14 pM for cfDNA in HEPES buffer, and a sensitivity and specificity of about 95%, respectively.

[0083] In another embodiment, the conductivity measurement and analysis can be performed in less than 5 minutes. In a particular example, the conductivity measurement and analysis can be performed in less than 3 minutes. In a further example, the conductivity measurement and analysis can be performed in less than 1 minute.

[0084] In one embodiment, the biosensor and process of the present disclosure can be used for rapid, label-free screening of cancer.

[0085] The present disclosure further provides a novel use of impedance spectroscopy in the detection of cancer based on the electrical conductivity of a test sample containing cfDNA in solution.

[0086] It will now be apparent to those skilled in the art that the present disclosure offers several advantages over the prior art. The biosensors and methods described herein enable the direct, rapid, and label-free detection of methylated nucleic acids, which are important markers in cancer diagnosis. This approach is sensitive enough to detect methylated nucleic acids within the clinical concentration range and eliminates the need for labeling, amplification, surface modification, or complex electrode structures. A key advantage is the reusability and cost-effectiveness of the electrodes, which can be reused indefinitely with proper maintenance and cease functionality only upon physical wear. Furthermore, these methods and biosensors are notable for their excellent sensitivity and specificity, approximately 95%, respectively, ensuring high accuracy and reliability in detecting cancerous changes.

[0087] Building on these advantages, the described impedance spectroscopy method offers a universal screening tool for a wide variety of cancer types, thanks to the high incidence of aberrant methylation across all cancers. This approach contrasts with existing multi-cancer screening methods, which primarily rely on assays or DNA sequencing, and offers a faster, more cost-effective alternative. Patients would benefit from faster, more affordable diagnostic results, while healthcare providers would be permitted to screen more frequently. Early detection is critical to successful cancer treatment, and the increased screening frequency facilitated by this method increases the chances of catching cancer early. Furthermore, the label-free nature of these methods contributes to reduced waste generation in clinical laboratories, consistent with sustainable practices in medical testing.

[0088] The biosensors and methods are further described with respect to the examples provided herein. [Example]

[0089] Example 1: Sample collection and plasma separation A total of 131 cancer samples and 40 healthy controls were collected from Maulana Azad Medical College and Rajiv Gandhi Cancer Institute and Research Centre, Delhi. The cancer samples collected were of the following types: breast (26), thyroid (19), colorectal (4), head and neck (17), kidney (31), gallbladder (5), lymph node (4), brain (4), penis (1), prostate (7), ovary (5), pancreas (2), cervix (1), and unknown origin (5). Two milliliters of whole blood was collected from each subject via routine venipuncture into K3EDTA tubes, and plasma was isolated from the blood by double centrifugation at 4°C (2,000 g for 10 minutes, followed by 16,000 g for 10 minutes). The plasma layer was then transferred to cryovials and immediately frozen.

[0090] Example 2: Extraction of cf / ctDNA from plasma For cfDNA extraction, the plasma samples were first thawed at room temperature, and then cfDNA was extracted using the MagMax™ cfDNA isolation kit according to the manufacturer's protocol. Finally, cfDNA was eluted using elution buffer, and its amount was quantified by UV-visible spectroscopy (Nanodrop™ Lite, Thermo Fisher Scientific). To prepare the cfDNA sample, 2 ml of eluted cfDNA in the MagMax™ elution buffer was spiked into 98 ml of 15 mM HEPES buffer at pH 7.4. A sample containing only 15 mM HEPES with 2% v / v elution buffer (i.e., no cfDNA) was used as a reference.

[0091] Example 3: Frequency optimization and solvent selection for impedance measurements First, we optimized the operating frequency and the type of solvent used to obtain the maximum reliable signal change between cancer and healthy samples. Because the maximum signal difference was obtained in the range of 10 kHz to 1 MHz (Figure 2), 0.1 MHz was fixed for all subsequent measurements. The phase angle at this frequency was much lower than 90 degrees (data not shown), which meant that the impedance response was essentially mostly resistive. Therefore, throughout this specification, the term conductance is used interchangeably with impedance. Furthermore, the impedance change (ΔZ) of cfDNA samples was also measured in various liquids, including deionized (DI) water, Milli-Q™ water, 10 mM PBS, and 10 mM tris-EDTA, as well as different zwitterionic buffers such as HEPES, EPPS, POPSO, and PIPES, all at 15 mM. The results showed that the S / N ratio of ΔZ was highest in HEPES, followed by POPSO and EPPS (Figure 3 and Table 1). [Table 2]

[0092] Table 1 above shows the comparative performance based on S / N values ​​of the different media shown in Figure 3. HEPES had the highest S / N and therefore the best performance. The S / N was lowest for PBS and intermediate for water, as expected based on the conductivity values. Therefore, HEPES was fixed as the buffer of choice for all remaining experiments. All buffers were at pH 7.4, approximately the pH of human blood, to stabilize DNA.

[0093] Example 4: Detection of cf / ctDNA using an impedance analyzer Impedance readings were performed by placing 20 μl of test sample onto a clean pair of IDEs (100 μm × 100 μm platinum on glass) enclosed in a PDMS microchamber (4 mm diameter, 1.6 mm thick), then covering the PDMS microchamber with a glass coverslip, which was sealed using vacuum grease. Care was taken to keep the diameter and height of the microchamber constant to maintain a constant geometric cell constant of the system. Impedance readings were recorded at 100 mV over a wide frequency range from 20 Hz to 10 MHz using an impedance analyzer (E4990A, Keysight™). Readings were recorded 3 minutes after the sample was placed on the electrode. The results were expressed as ΔZ = (Z cfDNA -Z ref ) and reported as the mean ± 1SD of two experiments.

[0094] The results of the clinical samples showed that the absolute value of the ΔZ signal of the cancer samples was significantly higher than that of the healthy samples (Figure 4B). After ROC analysis, the threshold for classifying samples as cancer positive was set at -34 ohms, and samples with readings above this value were identified as cancer positive, while samples with readings below this value were identified as cancer negative (or healthy). Based on this classification, the sensitivity and specificity of the system were found to be 95%, respectively.

[0095] Example 5: Examining the reasons behind signal differences The present inventors have found that the electrical conductivity measured in the currently proposed system is dominated by the ionic conductivity of the system. As evidenced by the results presented in Figure 5, the ΔZ response is highly non-monotonic with respect to DNA concentration, and the solvent plays an essential role in the signal quality. According to Marzano et al., Molecules 2019, 24, 654, G4 oligonucleotides have higher conductivity and therefore lower impedance than normal DNA due to their enhanced base stacking ability and the presence of trapped ions. To check whether the underlying nucleotide sequence influences the conductivity response in any way, synthetic oligonucleotide sequences with increasing GC content were tested (Figures 8A and 8B). Figure 8A shows the lack of impedance variation as a function of oligonucleotide concentration. The oligonucleotides (SEQ ID NOS: 1-5) had different degrees of GC content, as shown in the sequences in Figure 8B (bold in Figure 8B indicates methylated cytosines). The results of this test showed no discernible differences between the various sequences, ruling out the contribution of electronic conductivity. Figure 5 shows that the electrical conductivities of cancer and healthy tissue are significantly different even at the same DNA concentration, pointing to the underlying role of DNA morphology during impedance measurements.

[0096] As measured in Figure 4A, we designed an experiment in which the methylation content was systematically varied along an oligonucleotide sequence corresponding to the hTERT gene promoter region. 77-mer sequences with corresponding 25% and 100% methylation contents were received from Sigma-Aldrich (India). In actual samples, cfDNA from healthy individuals is expected to have a higher methylation rate compared to cfDNA from cancer patients. Because cfDNA is known to exist in both double-stranded (ds) and single-stranded (ss) forms, this experiment was performed using both forms of DNA. To cover the typical clinical range of cfDNA found in non-healthy tissues, the experiment was also performed at two DNA concentrations (70 ng / ml and 140 ng / ml). The results, shown in Figure 7A, indicate that dsDNA is more conductive than ssDNA, which is consistent with the literature. Furthermore, impedance varied non-monotonically with methylation, with 25% methylated oligos having lower signals compared to 100% methylated oligos, regardless of the cfDNA concentration used or the ds / ss nature of the DNA strands.

[0097] As shown by TEM images of differentially methylated oligos (Figure 7) and clinical samples (Figure 6), different degrees of methylation can significantly alter DNA morphology. Figure 7 shows that 25% methylated DNA is more dispersed at both 70 ng / ml and 140 ng / ml concentrations. 100% methylated oligos exhibited a higher impedance change compared to the 25% methylated sample. As methylation increases up to 100%, even larger aggregates are formed, extending well beyond the size of tens of micrometers. The same morphological behavior is observed in real samples, revealing that healthy cfDNA with higher methylation content forms large aggregates, whereas cancer cfDNA with lower methylation content is relatively well dispersed (Figure 6).

[0098] Example 6: Effect of methylation on the impedance signal of single-stranded (ss) and double-stranded (ds) synthetic oligonucleotides Morphological differences may lead to the nonmonotonic behavior observed in Figure 7A. The reason the 25% methylated oligos are more dispersed than the unmethylated ones is the presence of a methyl group at the 5-position of cytosine, which tends to stabilize the DNA molecule. However, the presence of such a nonconductive functional group may interfere with the charge mobility of counterions along the DNA backbone, resulting in a partial dip in the ΔZ response. In the case of 100% methylated oligos, the large number of closely spaced hydrophobic methyl groups can collapse the cfDNA into micro-sized domains surrounded by unmethylated hydrophilic regions. This reduces the total surface area per unit volume of the aggregate. In other words, the relative contribution of counterion charges to the overall conductivity is reduced compared to the nonconductive core mass of the aggregate. This results in a difference in the magnitude of the signals achieved from the 100% and 25% methylated oligos. This aggregation behavior persisted across different cfDNA concentrations within the clinical range (Figure 6), implying that the currently proposed method is applicable regardless of the cfDNA concentration obtained from plasma. Moreover, the effect of methylation on impedance followed the matching trends in Figures 4B and 7A, confirming that the method provides true information of epigenomic signatures.

[0099] Example 7: Comparative analysis with other available methods This approach was compared and analyzed with various known techniques for various parameters including LOD, detection time, receptor type, biomarker type, etc. The results are shown in Table 2 below. [Table 3(1)] [Table 3(2)] [Table 4]

[0100] In Table 3, underlining indicates 5-methylcytosine.

[0101] The many features and advantages of the present invention are apparent from the detailed specification, and it is, therefore, intended by the appended claims to cover all such features and advantages of the present invention that fall within the true spirit and scope of the invention. Further, because numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation as shown and described, and therefore, all suitable modifications and equivalents are to be embraced within the scope of the present invention.

Claims

1. A biosensor (100) for analyzing a test sample containing DNA, comprising: a microchamber (101) configured to hold the test sample suspended in a zwitterionic buffer, the microchamber enclosing a plurality of electrodes (102); an impedance analyzer (105) operatively connected to a programmable controller (104) with a digital processing unit for receiving information from the microchamber and the plurality of electrodes; wherein an impedance signal is measured by placing the test sample on the plurality of electrodes.

2. 10. The biosensor of claim 1, wherein the microchamber comprises a material selected from polydimethylsiloxane, polylactic acid, polylactic-co-glycolic acid, polyetheretherketone, silicone, nitrile, polyurethane, soft vinyl chloride resin, polypropylene, polyamide, polyethylene, polycarbonate, acrylonitrile butadiene styrene (ABS) resin, polystyrene, and poly(methyl methacrylate).

3. 10. The biosensor of claim 1 for use in rapid, label-free, and amplification-free screening of cancer.

4. 1. A method of liquid biopsy using a zwitterionic buffer and impedance spectroscopy, comprising: a. collecting a biological sample and extracting a cfDNA fraction from said biological sample; b. suspending the cfDNA fraction in the zwitterionic buffer to prepare a test sample; c. loading the test sample onto a plurality of electrodes enclosed within a microchamber; d. measuring the electrical conductivity of the test sample using an impedance analyzer; e. Analyzing the difference between the electrical conductivity of the test sample and the electrical conductivity of a control sample; A method comprising:

5. 5. The method of claim 4, wherein the biological sample comprises a biological fluid selected from whole blood, plasma, platelets, saliva, white blood cells, serum, and urine.

6. The biological sample comprises plasma, and the method comprises: Separating the plasma from the whole blood by double centrifugation, the double centrifugation comprising a first cycle at 1500g-2500g, preferably 2000g, for 7-12 minutes, preferably 10 minutes, and a second cycle at 15000g-17000g, preferably 16000g, for 7-12 minutes, preferably 10 minutes, at 4°C. The method of claim 4 further comprising:

7. 5. The method of claim 4, wherein the zwitterionic buffer is selected from 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, piperazine-1,4-bis(2-hydroxypropanesulfonic acid), and 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid.

8. 5. The method of claim 4, wherein the method can be used for quantitative and qualitative detection of cfDNA.

9. 8. The method of claim 7, wherein the zwitterionic buffer comprises 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and the method has a limit of detection of 0.4 ng / ml or 5.14 pM and a sensitivity and specificity of about 95%, respectively.

10. 5. The method of claim 4, wherein the electrical conductivity measurement and analysis is performed in less than 5 minutes, preferably less than 1 minute.

11. Use of impedance spectroscopy in the detection of cancer based on the electrical conductivity of a test sample containing cfDNA in solution.

12. 1. A label-free method for detecting cfDNA using impedance spectroscopy, comprising: a. collecting a biological sample and extracting a cfDNA fraction from said biological sample; b. suspending the cfDNA fraction in a liquid to prepare a test sample; c. loading the test sample onto a plurality of electrodes enclosed within a microchamber; d. measuring the electrical conductivity of the test sample using an impedance analyzer; e. Analyzing the difference between the electrical conductivity of the test sample and the electrical conductivity of a control sample; A method comprising:

13. 13. The method of claim 12, wherein the liquid is selected from a zwitterionic buffer, deionized water, and purified water.

14. 13. The method of claim 12, wherein the biological sample comprises a biological fluid selected from whole blood, plasma, platelets, saliva, white blood cells, serum, and urine.

15. The biological sample comprises plasma, and the method comprises: Separating the plasma from the blood by double centrifugation, said double centrifugation comprising a first cycle at 1500g-2500g, preferably 2000g, for 7-12 minutes, preferably 10 minutes, and a second cycle at 15000g-17000g, preferably 16000g, for 7-12 minutes, preferably 10 minutes, at 4°C. The method of claim 12 further comprising:

16. 14. The method of claim 13, wherein the liquid comprises a zwitterionic buffer selected from (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), piperazine-1,4-bis(2-hydroxypropanesulfonic acid), and 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid.

17. 13. The method of claim 12, wherein the method can be used for quantitative and qualitative detection of cfDNA.

18. 13. The method of claim 12, wherein the electrical conductivity measurement and analysis is performed in less than 5 minutes, preferably less than 1 minute.

19. 1. A method for detecting DNA using impedance spectroscopy, comprising: a. collecting a biological sample and extracting DNA from said biological sample; b. preparing a test sample by suspending the DNA fraction in a zwitterionic buffer; c. loading the test sample onto a plurality of electrodes enclosed within a microchamber; d. measuring the electrical conductivity of the test sample using an impedance analyzer; e. Analyzing the difference between the electrical conductivity of the test sample and the electrical conductivity of a control sample; A method comprising:

20. 20. The method of claim 19, wherein the DNA fraction comprises at least one of genomic DNA (gDNA), cell-free DNA (cfDNA), mitochondrial DNA (mtDNA), and complementary DNA (cDNA).

21. 20. The method of claim 19, wherein the biological sample comprises a biological fluid selected from whole blood, plasma, platelets, saliva, white blood cells, serum, and urine.

22. 20. The method of claim 19, wherein the biological sample comprises whole blood, and the method further comprises separating a buffy coat layer from the whole blood.

23. 20. The method of claim 19, wherein the zwitterionic buffer is selected from 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, piperazine-1,4-bis(2-hydroxypropanesulfonic acid), and 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid.

24. 20. The method of claim 19, wherein the method can be used for quantitative and qualitative detection of DNA.

25. 20. The method of claim 19, wherein the electrical conductivity measurement and analysis is performed in less than 5 minutes, preferably less than 1 minute.

Images