A density-independent electrochemical DNA sensor using a steric hindrance and a redox inhibition mechanism

Abstract

An electrochemical steric hindrance hybridization assay system is provided that includes a plurality of capture DNA molecules, a substrate associated with the plurality of capture DNA molecules, and a plurality of signal transduction DNA molecules. The signal transduction DNA molecules have a core nucleic acid sequence that is complementary to a region of the capture DNA molecules and a portion for binding an analyte in proximity to a reporter moiety, and when that portion binds to the analyte, it is configured to inhibit hybridization and reporter activity of the plurality of signal transduction DNA molecules on a surface associated with the plurality of capture DNA molecules. When the analyte binds to the signal transduction DNA molecule in proximity to the reporter moiety, steric hindrance occurs between analytes, between the analyte and the capture DNA molecule, and between the analyte and the substrate, and inhibition of redox activity also occurs due to the interaction with the target substance.

Description

Technical Field

[0001] (Cross - reference to related applications)

[0001] This application claims priority based on U.S. Provisional Application No. 63 / 366,476, filed on June 16, 2022, the content of which is incorporated herein by reference in its entirety.

[0002]

[0002] Provided is an electrochemical steric hindrance hybridization assay for detecting an analyte in a sample.

Background Art

[0003]

[0003] Over the past few decades, electrochemical, DNA - based sensors (eDNA sensors) have been attracting increasing attention because they are highly sensitive, specific, and low - cost, and can detect a wide range of biomarkers. The remarkable versatility of eDNA sensors is due to the fact that any type of recognition element and signal transduction mechanism can be employed. For example, eDNA sensors are designed to utilize nucleic acids, small molecules, peptides, proteins, and even enzymes as recognition elements. eDNA sensors are also designed to utilize many signal transduction mechanisms such as structural transformation, diffusion collision, enzyme catalysis, and steric hindrance. These sensing mechanisms have certain advantages depending on the type of biorecognition element used, but most of them have significant limitations. These performances vary greatly depending on factors that affect surface density, such as manufacturing variations and sensor aging, and also, when used in complex samples such as blood, due to the typical drift of the electrochemical baseline.

[0004]

[0004] Research focusing on optimizing the reproducibility and long-term stability of sensor manufacturing has hardly been conducted. These issues hardly attract academic interest, but on the other hand, even 20 years after the initial development, they still significantly limit the commercialization process of eDNA sensors. To reduce the impact of these limitations, various strategies have been proposed. These methods have improved the accuracy and precision of eDNA sensors, but due to all factors affecting sensor reproducibility, such as variations in sensor density related to manufacturing and aging degradation, these methods remain complex and have limited applications.

[0005]

[0005] Therefore, the development of more stable and improved eDNA sensors is still needed.

Summary of the Invention

[0006]

[0006] According to one embodiment, a system for detecting a target analyte in a sample is provided. This system includes a plurality of capture DNA molecules having a first end and a second free end, a first substrate having a surface associated with each of the first ends of the plurality of capture DNA molecules at a plurality of positions, and a plurality of signal transduction DNA molecules. Each of the signal transduction DNA molecules is substantially complementary to a region of each capture DNA molecule, and has a first end associated with a core nucleic acid sequence that is hybridizable with the capture DNA molecule and a portion for binding an analyte substance in proximity to a reporter portion, and a second end. And when the portion binds to the analyte, it is configured to inhibit the hybridization and reporter activity of the plurality of signal transduction DNA molecules on the surface associated with the plurality of capture DNA molecules. When the analyte binds to the signal transduction DNA molecule in proximity to the reporter portion, steric hindrance occurs between the analytes, between the analyte and the capture DNA molecule, and between the analyte and the substrate.

[0007]

[0007] In one embodiment, the portion for binding the analyte is at a distance of up to 16 nucleotides, up to 13 nucleotides, up to 10 nucleotides, up to 7 nucleotides, up to 4 nucleotides, or preferably up to 1 nucleotide from the reporter portion.

[0008]

[0008] In a further embodiment, this portion is at a distance of up to 16 nucleotides, up to 13 nucleotides, up to 10 nucleotides, up to 7 nucleotides, up to 4 nucleotides, or preferably up to 1 nucleotide from the first end of the signaling DNA molecule.

[0009]

[0009] In another embodiment, the length of the capture DNA molecule is up to 16 nucleotides, up to 13 nucleotides, preferably up to 10 nucleotides, or up to 8 nucleotides.

[0010]

[0010] In one embodiment, the target analyte is a macromolecule or an antibody.

[0011]

[0011] In a further embodiment, this system is for detecting a small molecule or a polypeptide, and the small molecule or polypeptide competes with the analyte for binding to the signaling DNA molecule.

[0012]

[0012] In one embodiment, the small molecule is a drug, such as cocaine.

[0013]

[0013] In a further embodiment, the portion for binding the analyte is an antigen.

[0014]

[0014] In a supplementary embodiment, the reporter moiety is a redox reporter or a fluorophore.

[0015]

[0015] In one embodiment, the redox reporter is methylene blue.

[0016]

[0016] In another embodiment, the substrate is a metal electrode, a 96-well plate, or a tube.

[0017]

[0017] In one embodiment, the metal electrode is a gold electrode or a carbon electrode.

[0018]

[0018] In a further embodiment, the substrate is a glass tube Ag / AgCl reference electrode, a platinum wire counter electrode, or a CTI electrode.

[0019]

[0019] In one embodiment, the substrate is an electrode comprising a gold working electrode (WE), a platinum reference electrode (RE), and a platinum counter electrode (CE).

[0020]

[0020] In another embodiment, the sample is a biological sample from a subject.

[0021]

[0021] In another embodiment, the sample is water, an environmental element, or food.

[0022]

[0022] In a supplementary embodiment, the biological sample is whole blood, saliva, or urine.

[0023]

[0023] In a particular embodiment, the subject is a human or an animal.

[0024]

[0024] A kit comprising the present system in a container is also provided.

[0025]

[0025] In one embodiment, the container is an Eppendorf with a lid. In another embodiment, the lid contains capture DNA molecules.

[0026]

[0026] In a further embodiment, the kit also comprises a potentiostat.

[0027]

[0027] A method for detecting a target analyte in a sample is further provided. The method includes preparing a sample suspected of containing the target analyte, preparing the system described herein, providing or determining a control amount of a plurality of capture DNA molecules hybridized to a plurality of signaling DNA molecules in the system in the absence of the target analyte, contacting the sample with the system, determining a test amount of a plurality of capture DNA molecules hybridized to a plurality of signaling DNA molecules in the system in the presence of the sample, characterizing the sample as containing the target analyte if the test amount is determined to be less than the control amount, and characterizing the sample as not containing the target analyte if the test amount is determined to be greater than or equal to the control amount.

[0028]

[0028] In one embodiment, the method described herein further includes quantifying the concentration of the target analyte in the sample based on a comparison of the control amount and the test amount.

[0029]

[0029] In another embodiment, the method described herein includes determining the control amount by contacting a control sample known to be free of the target analyte with the system described herein, and determining the amount of a plurality of capture DNA molecules hybridized to a plurality of signaling DNA molecules.

[0030]

[0030] In one embodiment, the method described herein further includes electrochemically determining the test amount and / or the control amount.

Brief Description of the Drawings

[0031]

[0031] Reference is now made to the accompanying drawings.

[0032]

Figure 1

[0032] ] Figure 1a shows a conventional electrochemical steric hindrance hybridization assay (eSHHA). The hybridization efficiency of the redox-labeled signal transduction DNA is reduced by the steric hindrance between the large analytes, proteins. Figure 1b shows an electrochemical steric hindrance redox inhibition assay (eSHRI). Since the position of the recognition element is strategically placed closer to the gold surface and the redox molecule, it becomes a new sensing mechanism that can occur between the analyte and the capture DNA layer or the gold surface.

Figure 2

[0033] Figure 2a shows that when the analyte binds near the electrode surface and the redox molecule, the hybridization efficiency between the signal transduction DNA and the complementary capture DNA bound to the electrode surface is significantly reduced. Figure 2b shows the hybridization efficiency of signal transduction DNA labeled with methylene blue (●, 5' end) and biotin (□) at different positions on the strand. The signal transduction DNA is named after the distance between biotin and methylene blue. For example, signal transduction DNA-16 indicates that there are 16 nucleotides between biotin and methylene blue. Square wave voltammograms (SWV) in the absence and presence of streptavidin 5 minutes after the start of hybridization are shown. In the absence of streptavidin, all signal transduction DNAs show similar currents, but in the presence of 100 nM streptavidin, the current decreases significantly as biotin approaches the gold surface and methylene blue.

Figure 3

[0034] It is shown that the hybridization efficiency and kinetics of the signal transduction DNA to the complementary capture DNA located on the sensor surface decrease linearly as the distance between the binding site of the analyte and the electrode surface or redox molecule shortens. Figure 3(a) shows the kinetic profiles of surface hybridization of six signal transduction DNAs and capture DNAs in the absence of streptavidin and in the presence of 100 nM streptavidin. Figure 3(b) shows the raw current after 5 minutes. Figure 3(c) shows the signal gain after 5 minutes. The signal gain is calculated from the current reduction ratios with and without streptavidin (Gain: Cstrep - C0 / C0). Figure 3(d) shows the rate constants with and without streptavidin. Figure 3(e) shows the signal gain of the sensor over time.

Figure 4

[0035] Figures 4(a) - (f) show the raw currents at different surface densities at 30 minutes. Electrodes with different surface densities were fabricated using capture DNAs at concentrations of 300 nM, 100 nM, 50 nM, and 25 nM. The length of the capture DNA used in this assay was 16 nucleotides. The error bars indicate the standard deviation of the current or signal gain obtained from three experiments.

Figure 5

[0036] Reducing the distance between the analyte binding site on the signal transduction DNA and the electrode surface shows that the assay becomes less affected by the capture DNA density due to the redox molecule. Electrodes with different surface densities were fabricated using capture DNAs at concentrations of 300 nM, 100 nM, 50 nM, and 25 nM. The signal gain is calculated from the current reduction ratios with and without streptavidin at 30 minutes. The length of the capture DNA used in this assay was 16 nucleotides (nt). The error bars indicate the standard deviation of the signal gain obtained from three experiments.

Figure 6

[0037] Figures 6(a)-(f) show the bioelectric currents of capture DNAs of different lengths in 30 minutes. Capture DNAs of lengths 16, 13, and 10 nucleotides (nt) were used to vary the hybridization affinity on the surface. Error bars indicate the standard deviation of the current or signal gain obtained from three experiments.

Figure 7

[0038] Reducing the distance between the analyte binding site on the signal transduction DNA and the electrode surface indicates that the assay becomes less susceptible to changes in the hybridization affinity between DNA strands by redox molecules. Capture DNAs of lengths 16, 13, and 10 nucleotides (nt) were used to vary the hybridization affinity on the surface. The signal gain is calculated from the rate of current decrease with and without streptavidin in 30 minutes. Error bars indicate the standard deviation of the signal gain obtained from three experiments.

Figure 8

[0039] The protein-induced fluorescence enhancement of methylene blue indicates support for the contact redox inhibition mechanism. The fluorescence of signal transduction DNA labeled with methylene blue decreases slightly (by about 32% on average) (green curve) when binding to the complementary strand. The fluorescence of signal transduction DNA labeled with methylene blue is significantly enhanced (<4 nm, red curve) when streptavidin binds within 3 nucleotides of methylene blue, also causing significant contact redox inhibitory activity. The concentrations of signal transduction DNA and streptavidin are 100 nM each.

Figure 9

[0040] Highly sensitive eSHRI indicates that it is not affected by variations in sensor manufacturing and factors (such as temperature and matrix buffer) that affect DNA hybridization. In Figure 9(a), the optimal conditions were achieved at a high surface density (300 nM capture DNA) using 100 nM signaling DNA-16 (KD = 19 nM) and signaling DNA-01 (KD = 15 nM) for each of the conventional eSHHA and eSHRI. Figure 9(b) shows the case of a low surface density using 25 nM capture DNA. Figure 9(c) shows the case of weak hybridization affinity using 10 nt capture DNA at a high density. Error bars indicate the standard deviation of the signal gain obtained from three experiments.

Figure 10

[0041] Figure 10(a) shows the synthesis procedure of THC azide. Figure 10(b) shows the binding of THC azide to double-labeled DNA (NH2 and DBCO) of methylene blue. Figure 10(c) shows the synthesis procedure of cocaine azide. Figure 10(d) shows the binding of peptide azide to double-labeled DNA (MB and DBCO).

Figure 11

[0042] Figure 11(a) shows the kinetic profile of the THC antibody assay in buffer using a CTI (Conductive Technologies) electrode, and Figure 11(b) shows the signal gain of the sensor versus time.

Figure 12

[0043] eSHRI is also adapted to the competitive format, indicating that antigens or small molecules can be detected.

Figure 13

[0044] Figure 13(a) shows THC detection in buffer using a competitive eSHRI assay. Figure 13(b) shows the signal gain of the THC sensor versus time (in buffer). Figure 13(c) shows THC detection in saliva using a competitive eSHRI assay. Figure 13(d) shows the signal gain of the THC sensor versus the THC concentration in human saliva. In this assay, free THC molecules were pre-incubated with the THC antibody for 5 minutes, and then the signaling DNA was added to buffer or saliva. Figure 13(e) compares THC detection (100 nM) (squares) in buffer using the THC sensor with cocaine detection (circles) in buffer using the cocaine sensor, both using a competitive eSHRI assay.

Figure 14

[0045] Shows the adaptation of eSHRI in a point-of-care format for rapid detection with a drop of blood. Figure 14(a) shows the conventional three-electrode system used herein. This system utilizes a rod-shaped gold electrode (diameter = 2 mm), a glass tube Ag / AgCl reference electrode, and a platinum wire counter electrode. Figure 14(b) shows the miniaturized integrated electrode from Micrux Technologies. This integrated electrode comprises a miniaturized gold working electrode (WE: 1 mm), a platinum reference electrode (RE), and a platinum counter electrode (CE). Figure 14(c) shows the kinetic profile of a streptavidin assay in buffer using the Micrux electrode. Figure 14(d) shows the kinetic profile of a streptavidin assay performed directly in 5 μL of blood using the Micrux electrode. In this assay, 100 nM signaling DNA-01 and 100 nM streptavidin were used. Error bars indicate the standard deviation of the current or signal gain obtained from three experiments.

Figure 15

[0046] Figure 15a shows a control strategy using an additional "control" electrode in combination with a hybridization assay containing a different antigen / antibody combination that does not interact with molecules present in the clinical sample. Different colors of DNA represent different DNA sequences.

[0047] Figure 15b shows a control strategy in which an additional "control" electrode is used with a hybridization assay that contains antigen-labeled signal transduction DNA but no antibody. This antigen does not interact with molecules in the biological sample.

[0048] Figure 15c shows a control strategy in which an additional electrode is used with a hybridization assay that has no recognition element on the signal transduction strand.

[0049] Figure 15d shows a control strategy in which an additional signal transduction DNA strand and a capture DNA strand that can hybridize on the same electrode are used with another redox element.

[0050] Figure 15e shows a variation of the control electrode proposed in the strategy as shown in Figure 15c.

Figure 16

[0051] Shows a workflow enabling a THC assay. THC contained in the cotton swab used to rub the gums is released into a solution containing a THC antibody. After a short incubation, it is sealed with the lid of an Eppendorf containing dried signal transduction DNA (THC-DNA and control DNA, here cocaine-DNA), and the solution is mixed for 10 seconds to solubilize the signal transduction DNA. Next, this solution is added to the electrodes (THC electrode and cocaine electrode) for analysis (see Figure 15b for the control strategy utilized here). This analysis is performed with a portable potentiostat controlled by an app via Bluetooth. In the absence of THC, both electrodes detect an equal amount of signal transduction DNA. In the presence of THC, the electrochemical signal on the THC electrode increases because the THC antibody that can bind to THC-DNA decreases and the THC-DNA that can bind to the THC electrode increases.

Figure 17

[0052] Shows the hybridization kinetics obtained in saliva samples with different THC concentrations using 67% operon buffer + 33% saliva.

Figure 18

[0053] Shows three analytical methods that achieve good accuracy and are included herein. The dotted line represents the acceptable THC limit (5 ng / ml).

Figure 19

[0054] The coupling curves using the current in 1 minute and the coupling curves using the current gain in 1 minute are shown.

Figure 20

[0056] Direct antibody detection in bovine blood (here, anti-NT-proBNP antibody) using the eSHRI assay and the specific epitope binding to this antibody (ETSGLQEQRNHL) are shown.

Mode for Carrying Out the Invention

[0033]

[0057] According to the present specification, an electrochemical steric hindrance hybridization assay for detecting an analyte in a target sample is provided.

[0034]

[0058] In order to remotely detect an analyte in blood or saliva, an inexpensive and easy-to-operate sensor for analyzing molecules in blood is required. Electrochemical DNA-based sensors (eDNA sensors) are highly sensitive, low-cost, and relatively unaffected by the matrix effect of biological samples, and thus have great potential for rapidly detecting multiple types of blood markers. However, due to several problems, the commercialization of eDNA sensors has been delayed. Among them, the performance of the eDNA sensing mechanism is greatly affected by the variation in the DNA probe density on the electrode surface.

[0035]

[0059] Among the different signal transduction mechanisms developed for eDNA sensors, the recently developed steric hindrance hybridization assay has many advantages. When the sensor is first immersed in a biological sample, since the redox molecule is not located on the surface of the sensor, DNA hybridization can occur in the biological sample without being disturbed. This specific eDNA sensing structure functions efficiently in whole blood without causing signal drift. Although this sensing mechanism is relatively widely recognized, a high sensor density on the gold surface is required to achieve optimal performance. To address this limitation, a novel electrochemical DNA hybridization sensing mechanism is provided. This mechanism utilizes multiple steric hindrance effects and a redox suppression mechanism to enhance the gain and reduce the dependence on sensor density. For example, by strategically arranging the recognition elements on the signal transduction DNA, a new steric hindrance mechanism between the analyte and the gold electrode can be created. In principle, this mechanism will not be affected by the sensor density. Alternatively, by placing the protein, which is the analyte, next to the redox molecule, a contact-induced redox suppression mechanism can also be created. This mechanism will limit the electron transfer rate and the influence of the probe density on the sensor performance.

[0036]

[0060] Thus, in one embodiment, a novel electrochemical hybridization-based assay for detecting proteins is provided. This assay integrates four different signal transduction mechanisms to create a highly sensitive, density-independent sensing mechanism. This mechanism functions reproducibly even in the presence of manufacturing variations and aging degradation. This signal transduction mechanism utilizes the binding of the protein analyte to a recognition element that binds to a redox-active signal transduction DNA, inhibiting hybridization and redox activity on an electrode with a complementary strand. When the protein analyte binds in proximity to the redox molecule, the contact inhibition mechanism may further limit electroactivity. As shown herein, this novel sensing mechanism enables the detection of low-nanomolar concentrations of protein analytes in a drop of blood in less than 3 minutes with nearly optimal signal gain (up to 93%).

[0037]

[0061] As included herein, the sample can be a biological sample such as whole blood, saliva, or urine from a human or animal subject, but is not limited thereto. The sample may also be an environmental element such as water, soil, or food.

[0038]

[0062] In the conventional electrochemical steric hindrance hybridization assay (eSHHA, Figure 1a), signaling DNA having a redox molecule and a recognition element at each end diffuses into the sample and hybridizes with complementary capture DNA located on the electrode surface. When the protein analyte binds to the signaling DNA, the amount of signaling DNA reaching the electrode surface decreases due to the steric hindrance that occurs between large protein analytes (Figure 1a). Unfortunately, since the steric hindrance depends on the distance between the capture DNAs on the surface, in eSHHA, the signal gain is high (signal decrease of -60% or less) only on electrodes with a high surface density. To construct a density-independent sensing mechanism, a new electrochemical steric hindrance redox inhibition (eSHRI) hybridization assay is presented in Figure 1b. The labeling position of the recognition element to which the analyte binds causes a greater steric hindrance. When the analyte binds in proximity to the redox molecule, another steric hindrance occurs between the protein analyte and the capture DNA layer, and also between the analyte and the gold surface. Alternatively, when the protein analyte binds in the vicinity of the redox molecule, a contact-induced redox inhibition mechanism also occurs, significantly reducing the electron transfer rate and improving the signal transduction mechanism of the eDNA sensor.

[0039]

[0063] The hybridization efficiency between the signal - transmitting DNA and the capture DNA varies depending on the position of the recognition element on the signal - transmitting DNA strand (Figure 2a). To study these novel steric - hindrance and redox - suppression mechanisms, six signal - transmitting DNAs were designed with the position of the recognition element (here biotin) varied from the position farthest from the redox molecule (signal - transmitting DNA - 16, Figure 2b) to the position closest to it (signal - transmitting DNA - 01, Figure 2b). These 16 - nucleotide - long signal - transmitting DNAs have the same sequence, all with their 5′ ends labeled with methylene blue, and when hybridized with the complementary DNA that binds to the gold surface, an electrochemical read - out signal is generated. These DNAs were synthesized in - house using a DNA / RNA synthesizer (K&A Laborgeraete, Germany), methylene blue phosphoramidite for labeling the 5′ end, and biotin - modified thymine. In the absence of the protein analysis target, all signal - transmitting DNAs can hybridize with comparable efficiency at the electrode surface. All showed extremely similar electrochemical currents at - 0.3 V after 5 minutes (Figure 2b). This indicates that the position of the small biotin does not affect the hybridization efficiency. On the other hand, when the protein analysis target (here streptavidin) binds to the signal - transmitting DNA, the hybridization efficiency was mainly affected by the position of the recognition element. For example, biotin at the farthest position (signal - transmitting DNA - 16) only reduced the hybridization efficiency by 50%, while when it was at the closest position, the hybridization efficiency was reduced by up to 93% after only 5 minutes.

[0040]

[0064] As used in the context of the present disclosure, the "target analyte" can be any molecule suspected of being present in a sample to be analyzed and capable of binding (in one embodiment, specifically) to a signaling DNA molecule. In the context of the present disclosure, the expressions "specific binding" or "binds specifically" refer to the interaction between two elements and are determinative of the presence of these elements in the presence or absence of a heterogeneous population of molecules that can include nucleic acids, proteins, and other biomolecules. For example, under specified conditions, a certain target binds to a particular macromolecule and does not bind in a significant way to other molecules in the sample. In some embodiments (when the target is considered to be a macromolecule), when the target "binds specifically" to a signaling DNA molecule, steric hindrance occurs on the substrate surface, ultimately resulting in a detectable signal or a detectable signal change.

[0041]

[0065] In one embodiment, the average molecular weight of the target analyte can be at least 10 kDa, at least 20 kDa, at least 30 kDa, at least 40 kDa, at least 50 kDa, at least 60 kDa, at least 70 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, or at least >1000 kDa. In one embodiment, the target can be an antibody (IgA, IgE, IgG, IgM, IgD), a single-chain antibody, or an antibody fragment (e.g., Fab’ fragment). The target can be a cell (e.g., eukaryotic cell (such as immune cells) or prokaryotic cell (such as bacteria)) or a cell fragment (such as red blood cells or platelets). The target can be an infectious agent, e.g., a prokaryotic cell (such as bacteria), a fungal cell (such as yeast or mold), a virus, or a prion. The target can be a macromolecule (e.g., a polynucleotide molecule (such as DNA-based (e.g., cDNA or DNA fragment) or RNA-based (e.g., mRNA, miRNA, tRNA, siRNA)), a polypeptide molecule, or a carbohydrate (such as lectin)), or a monomeric molecule (such as a lipid). When the macromolecule is a polynucleotide molecule, it contains at least 20, 30, 40, 50, or at least 100 nucleobases. Examples of polypeptides / proteins include, but are not limited to, antibodies, cell receptors, secreted polypeptides, immunomodulatory polypeptides (such as interleukins, interferons), hormones (such as growth factors), coagulation factors, DNA-binding polypeptides (such as transcription factors), etc. The target can be a single molecule, a combination of two or more molecules (such as a glycosylated antibody), or an aggregate of two or more molecules. The target can be a naturally occurring molecule or a synthetic (e.g., artificial) molecule.

[0042]

[0066] The influence of the protein-binding position of the signal transduction DNA on the hybridization kinetics was further characterized. In the absence of streptavidin, the kinetics (Figure 3a), the current after 5 minutes (Figure 3b), and the hybridization rate (Figure 3d) of the six signal transduction DNAs were all very similar. On the other hand, when streptavidin binds, all six signal transduction DNAs showed that the hybridization efficiency decreased in inverse proportion to the distance between biotin and methylene blue (Figure 3a). For example, when the position of biotin was moved from the farthest position (signal transduction DNA-16) to the closest position to the gold surface (signal transduction DNA-01), the hybridization after 5 minutes decreased by 86% (Figure 3b), and the hybridization rate decreased to one-fourth (Figure 3d). Interestingly, when the position of biotin was brought closer to the gold surface or methylene blue, the signal gain of these streptavidin sensors increased linearly from -49% (signal transduction DNA-16) to -93% (signal transduction DNA-01) (Figure 3c). In addition to significantly improving the signal gain of the sensor, this assay is also extremely rapid, and the maximum signal gain can be obtained as early as 2 minutes later (Figure 3e).

[0043]

[0067] The main advantage of eSHRI (electrochemical steric hindrance redox inhibition hybridization assay) is that its performance is, in principle, hardly affected by the variation in the capture DNA density on the electrode surface. The conventional eSHHA mechanism is significantly affected by the variation in the capture DNA density. When the average distance between two capture DNAs becomes wider than the size of the protein analyte, the steric hindrance disappears or becomes slight. Therefore, for eSHHA to function optimally, a high surface density is required (and thus it is sensitive to sensor degradation). In the eSHRI mechanism, the analyte is also thought to cause steric hindrance with the DNA layer (which also depends on density) and the electrode surface. When the protein analyte approaches methylene blue, methylene blue may bind to the surface of the analyte and cause redox signal inhibition, which can be a new signal transduction mechanism within the eDNA sensor. The steric hindrance and contact-induced redox suppression mechanism between the analyte and the electrode are likely to be relatively unaffected by the variation in the capture DNA density.

[0044]

[0068] To verify this hypothesis, streptavidin assays were performed at different capture DNA densities. As expected, decreasing the capture DNA density significantly reduced the electrochemical current (Figure 4). Next, the efficiency of the steric hindrance assay was evaluated by testing these different sensors in the presence and absence of streptavidin. As expected, decreasing the capture DNA density decreased the signal gain of the conventional eSHHA assay (signal transduction DNA-16) from -53% to -7% (Figure 5a). Moving the biotin position 3 nucleotides within the DNA layer (signal transduction DNA-13) increased the steric hindrance at high surface densities (-65% signal gain, Figure 5b). Since this effect mainly depends on the density of the capture DNA, the enhanced steric hindrance is likely to occur between streptavidin and the DNA layer. Deeper positioning of the biotin within the DNA layer (e.g., signal transduction DNA-10 and signal transduction DNA-07) further enhanced the steric hindrance at high surface densities (-80% and -78% signal gain, Figures 5c and 5d), but decreasing the density of the DNA layer also resulted in a decrease in steric hindrance ( -8% and -21% at low surface coverage, Figures 5c and 5d). On the other hand, bringing the binding site of the analyte closer to the electrode surface, such as in signal transduction DNA-04 and signal transduction DNA-01, resulted in large signal gains even at low densities (-76% and -83%, Figures 5e and 5f). These results suggest that the observed increase in the steric hindrance effect is no longer solely due to (1) steric hindrance between analytes or (2) steric hindrance between the analyte and the DNA layer. The increase in signal gain could be due to (3) a steric hindrance mechanism between the analyte and the electrode or (4) a redox inhibition mechanism between methylene blue and streptavidin. Since the DNA density typically changes due to the manufacturing method or sensor aging, and thus the sensor performance changes, this new sensing mechanism is independent of the capture DNA density and has significant advantages in terms of reproducibility.

[0045]

[0069] The captured DNA molecule consists of any combination of known natural nucleobases or synthetic nucleobases, and its backbone may be modified from the naturally occurring backbone. Naturally occurring oligonucleotides contain phosphodiester bonds, and synthetic oligonucleotides containing nucleic acid analogs may have another backbone, including, for example, phosphoramide bonds, phosphorothioate bonds, phosphorodithioate bonds, O-methyl phosphoramidite bonds, and peptide nucleic acid backbones and linkages. Other nucleic acid analogs include those having positive backbones, non-ionic backbones, and non-ribose backbones. Nucleobases containing one or more carbocyclic sugars are also included in the definition of the envisioned nucleobases.

[0046]

[0070] To further study the characteristics of the eSHRI mechanism, the length of the captured DNA was varied to confirm its involvement in the formation of steric hindrance between the "analyte and DNA layer". Shortening the length of the captured DNA will have the following two effects. a) The affinity of the captured DNA decreases, reducing the signal transduction DNA that can hybridize on the surface, thus reducing the steric hindrance between the "analyte and analyte". b) The average distance between the analyte and the DNA layer increases, reducing the steric hindrance between the "analyte and DNA layer".

[0047]

[0071] To investigate these effects, tests were conducted with the length of the capture DNA shortened to 16, 13, and 10 nucleotides (nt). As expected, it was observed that the affinity of the 10-nt capture DNA for the signaling DNA decreased significantly (Figure 6). Since there is less signaling DNA that can hybridize to the surface modified with 10-nt capture DNA, the steric hindrance between the analytes is extremely reduced. Even when streptavidin is added to Signaling DNA-16 or Signaling DNA-13, the signal gain hardly increases (Figures 7a and 7b). Notably, when biotin penetrates deeper into the DNA layer, as in the case of Signaling DNA-10 or Signaling DNA-07, the assay is no longer affected by the number of signaling DNAs on the surface. Even with the 10-nt capture DNA with low affinity, the addition of streptavidin results in a -50% signal gain (Signaling DNA-10, Figure 7c) and a -75% signal gain (Signaling DNA-07, Figure 7d). These results indicate that when the analyte binds to the center of the capture DNA layer, the dominant steric hindrance effect is no longer (1) the steric hindrance between the analytes, but rather (2) the steric hindrance between the analyte and the DNA layer or (3) the steric hindrance between the analyte and the electrode. Finally, when biotin is brought closer to the electrode, as in the case of Signaling DNA-04 or Signaling DNA-01, the signal gain further increases and becomes constant for all capture DNA lengths (Figures 7e and 7f). These results suggest that when methylene blue binds to the surface of the protein analyte, another steric hindrance and potential redox inhibition mechanism with the gold electrode are brought about.

[0048]

[0072] An electrochemical hybridization-based signal transduction mechanism for detecting an analyte that is a protein is provided. This mechanism utilizes two new steric hindrance mechanisms, steric hindrance between the analyte and the DNA layer and steric hindrance between the analyte and the electrode. This mechanism is not affected by the variation in sensor density and gives a nearly optimal signal gain (>-90%), so it has many advantages over the signal transduction mechanisms (e.g., diffusion collision and structural conversion) that have already been developed. However, a third effect (or mechanism) may also contribute to the overall performance of this novel sensor. That is, the redox inhibition mechanism between methylene blue and the analyte protein may also limit the electron transfer rate. To demonstrate the potential binding-induced redox inhibition mechanism observed when streptavidin binds adjacent to methylene blue, the fluorescence of methylene blue was measured after streptavidin was bound at various distances (Figure 8). When binding to the complementary strand, the fluorescence of the signal transduction DNA labeled with methylene blue decreased slightly (by an average of about 32%) (Figure 8). This is a characteristic effect observed when the tumbling rate decreases as the size of the fluorescently labeled molecule increases. Next, when streptavidin was added to various double-stranded signal transduction DNAs, it was revealed that the fluorescence changed significantly depending on the distance between streptavidin and methylene blue (Figure 8). When streptavidin was bound at a position distant from methylene blue (i.e., 16, 13, 10, 7, and 4 nucleotides away), a 30% decrease in fluorescence intensity was observed. On the other hand, when streptavidin binds near methylene blue (at an interval of less than 3 nucleotides), a large protein-induced fluorescence enhancement is observed, which also correlates with significant contact redox inhibitory activity. Therefore, from these results, the presence of physical contact between methylene blue and streptavidin is clear, and thus a novel electrochemical signal transduction mechanism based on contact-induced redox inhibition is suggested.

[0049]

[0073] In eSHRI, the signal gain is higher than that of conventional eSHHA, and the influence on sensor manufacturing parameters (such as surface density and length of capture DNA) and sensor deterioration is extremely low. To confirm whether eSHRI can reduce the detection limit and improve the sensitivity, the dose-response curves obtained using signal transduction DNA-16 (conventional eSHHA) and signal transduction DNA-01 (eSHRI) were compared. Under optimal conditions (i.e., high surface density and long capture DNA), both assays showed sigmoid curves centered around K D values of around 19 nM and 15 nM, respectively (Figure 9a). In conventional eSHHA, the signal gain changes by 22% when the streptavidin concentration is between 10 nM and 30 nM, while in eSHRI, the signal gain decreases by 66%, indicating that the sensitivity is significantly improved by three times (Figure 9a). As described above, the performance of eSHRI is not affected by various sensor manufacturing parameters. For example, when the surface density decreases due to manufacturing variations or sensor aging, the performance of conventional eSHHA decreases extremely, while that of eSHRI does not change (Figure 9b). Similarly, when the hybridization efficiency is affected by factors that reduce the affinity between signal transduction DNA and capture DNA (such as high temperature or unstable matrix / buffer), the performance of eSHRI is not affected, while conventional eSHHA is extremely affected (Figure 9c). Overall, these results clearly show that eSHRI not only improves the sensitivity but is also not affected by various external factors (manufacturing batch, temperature, and matrix) that affect sensor deterioration and sensor performance.

[0050]

[0074] To confirm the versatility and potential universality of eSHRI, this assay was adapted for the detection of another more clinically relevant protein, such as an antibody. As a proof of concept, the specific antibody was detected using tetrahydrocannabinol (THC), a model antigen (or small recognition element). First, a dual-labeled signal transduction DNA in which the redox molecule methylene blue was bound to the antigen THC was designed (Figure 10b). For this purpose, THC was first modified to THC azide and then subjected to a coupling reaction (Figure 10a). Using DNA labeled with NH2 and DBCO obtained from IDT (Integrated DNA Technologies, Inc., Iowa, USA), methylene blue was bound by an ester-amino reaction, and then THC azide was bound by an azide-DBCO coupling reaction. In the absence of the THC antibody, this dual-labeled signal transduction DNA efficiently hybridized, and the current exceeded 300 nA after 10 minutes (Figure 11). On the other hand, in the presence of 100 nM THC antibody, the hybridization efficiency decreased by more than 60% even a few seconds later (Figure 11b). The fact that the signal decrease reaches its maximum even at the initial stage of the hybridization reaction further indicates that the binding rate of THC to the antibody is much faster than the hybridization rate of the signal transduction DNA to the complementary DNA bound to the electrode. Although excellent, it was also clear that this 80% signal gain decrease was relatively small compared to the 93% signal decrease observed in the streptavidin-biotin system (-93%). This may be due to the fact that while one antibody is expected to bind to two signal transduction DNAs, streptavidin can in principle bind to four antibodies. Alternatively, the anti-THC antibody may be close to methylene blue when it binds to THC but may not be able to bind to methylene blue and may not even be able to inhibit the redox activity (see, for example, Figure 5f). The versatility of this signal transduction structure was further demonstrated by replacing the THC bound on the signal transduction DNA with cocaine (Figure 10c) or a peptide epitope (Figure 10d).

[0051]

[0075] To expand the range of analytes detected by eSHRI, the utility of this assay was also demonstrated in a competitive format for the detection of antigens or small molecules. As shown in Figure 12, in the absence of an antigen (or small molecule), a reagent antibody (or specific protein) can be used to bind to the signal transduction DNA having a copy of the antigen. As a result, due to steric hindrance and redox inhibition, the hybridization efficiency becomes extremely low, and thus the electrochemical signal also becomes low. In the presence of an antigen, the antibody preferentially binds to this antigen rather than the antigen bound to the signal transduction DNA. Therefore, the signal transduction DNA remains unbound and efficiently hybridizes to the capture DNA on the electrode surface, resulting in a high electrochemical signal (Figure 13a). As included herein, after mixing the sample with the antibody for 0 to 1 minute, the signal transduction DNA is added, and this sample is added onto the electrode. Thereby, free antigen can be bound to the antibody before the antibody binds to the signal transduction DNA. Therefore, it is possible to increase the gain depending on the rate of the antigen-Ab interaction or the sample viscosity.

[0052]

[0076] The utility of this competitive assay was further confirmed by detecting free THC molecules using a proof-of-concept THC antibody (Figs. 13a-d). In this experiment, 100 nM signaling DNA and 100 nM THC antibody were used. In the absence of free THC molecules, the THC antibody bound to the signaling DNA labeled with THC, and the hybridization efficiency and current decreased (Fig. 13a). In the presence of 100 nM free THC molecules, the THC antibody first bound to this THC (as in the control experiment, the antibody was first reacted with the sample for 5 minutes and then the signaling DNA was added), and as a result, the signaling DNA did not bind and reached the electrode surface with high efficiency (Fig. 13a). As included herein, the incubation time may be as short as 30 seconds, depending on the assay. In this competitive eSHRI assay, almost optimal gain is obtained 1 second after the start of hybridization (Fig. 13b). The ability to perform this competitive eSHRI in complex biological media was also tested by directly detecting free THC molecules in human saliva (Fig. 13c). As shown in Fig. 13c, it was revealed that the performance of this assay did not significantly decrease even when performed directly in biological samples. The decrease in the total bioelectric current in saliva may be due to some matrix effects of saliva that reduce electron transfer rate or hybridization efficiency. Finally, this competitive assay is quantitative, and the detection limit in saliva is shown to be approximately 10 nM (3.14 ng / mL). This is within the range of the detection limit in saliva (5 ng / mL) required for commercially available lateral flow THC detection kits. Similar assays for detecting other small amounts of drugs, antigens, or molecules are also feasible simply by replacing THC with another molecule and replacing the THC antibody with an antibody that binds to this specific molecule. See, for example, the detection of cocaine using cocaine-labeled DNA (Fig. 10c) and a cocaine antibody (Fig. 13e).

[0053]

[0077] The ability of eSHRI to directly detect the protein analyte in complex biological samples such as whole blood, using only 1 drop (5 μL) of blood, is further elucidated. For this purpose, streptavidin detection was directly tested in 1 drop of blood using a miniaturized integrated electrode. All the results shown so far were obtained using a conventional three - electrode system comprising a rod - shaped gold working electrode, a glass tube Ag / AgCl reference electrode, and a platinum wire counter electrode, which usually requires a large sample volume of 1000 μL (Figure 14a). Instead, the CTI electrode was used to detect THC in saliva, which required a 70 μL sample. To test the sensor for point - of - care applications with 1 drop of blood, this assay was adapted to a small disposable electrode manufactured by Micrux Technologies using photolithography. This Micrux electrode has a gold working electrode (WE), a platinum reference electrode (RE), and a platinum counter electrode (CE) on a small glass chip (6×10 mm) (Figure 14b). Since the surface area of the electrode is small (0.785 mm 2 ), only 5 μL of sample (a small drop of blood) is required to perform the assay. Streptavidin was first detected in buffer to compare the performance of Micrux and the rod - shaped electrode. Both electrodes showed similar high signal gains (Micrux = - 96% (5 min), inset in Figure 14c; rod - shaped electrode = - 93% (5 min), Figure 3c) and kinetics (Micrux = 0.035 min -1 , Figure 14c; rod - shaped electrode = 0.04 min -1 , Figure 3d). The performance of these small streptavidin sensors was further directly tested with 1 drop of blood (Figure 14d). The results in blood showed a similarly high signal gain (- 95% in 5 min) as in buffer, but slightly faster kinetics (0.1 min -1 ). It should be noted that the bio - current in blood is about 25% lower than that in buffer, which may be due to the decreased electron transfer rate in blood. From these results, it is clear that the high specificity and selectivity of the signal transduction mechanism of eSHRI are not affected by the non - specific absorption of proteins on the sensor surface.

[0054]

[0078] As included herein, the substrate provides a surface for binding at least two capture DNA molecules such that the capture DNA molecules do not freely diffuse in solution / suspension. In yet another embodiment, the substrate is a metal electrode (such as gold, silver, platinum) or a non-metal electrode (such as carbon or silicon). The conductive and semiconductive materials may be metals or non-metals, and the reporter moiety is a redox reporter (such as organic redox moieties such as viologen, anthraquinone, ethidium bromide, daunomycin, methylene blue, and their derivatives, organometallic redox moieties such as ferrocene, ruthenium, bispyridine, tripyridine, bisimidazole, and their derivatives, and biological redox moieties such as cytochrome c, plastocyanin, and cytochrome c’).

[0055]

[0079] In certain embodiments, the detection system described herein is capable of identifying targets at nanomolar or picomolar concentrations in a sample. In some embodiments, the system has a dynamic range of at least 10, 20, 30, 40, 50, 60, 70, or 80. For example, in an embodiment where the signaling oligonucleotide is a single molecule and is used at a concentration of 100 nM, the system is capable of detecting targets at concentrations of 1 nM to 1 μM, such as 1 nM to 750 nM, 1 nM to 500 nM, or 1 nM to 250 nM, such as 1 nM to 100 nM or 2 nM to 160 nM.

[0056]

[0080] Therefore, a density-independent electrochemical eDNA sensing mechanism is provided that utilizes a novel electrochemical steric hindrance redox inhibition mechanism (eSHRI). This new strategy aims to overcome many of the limitations of current eDNA sensors, such as the limits of signal gain and the high dependence on sensor manufacturing and aging degradation. By shortening the distance between the analyte binding site and the electrode, a novel steric hindrance redox inhibition mechanism was constructed. These mechanisms enhanced the signal gain up to -93% (close to -100% of the highest theoretical value) and achieved a signal transduction mechanism that is independent of the sensor density on the electrode surface. Importantly, this new method enables the rapid (<3 minutes) one-step detection of protein analytes in the low nanomolar range directly in a single drop of blood.

[0057]

[0081] Compared to conventional eSHHA, which utilizes only one steric hindrance mechanism, eSHRI utilizes three different steric hindrance mechanisms: (1) between analytes, (2) between the analyte and the DNA layer, and (3) between the analyte and the electrode surface. Furthermore, for the first time, a (4) contact-induced redox inhibition mechanism that occurs when the protein analyte contacts and binds to the redox molecule (methylene blue) is described. eSHRI is a potentially universal signal transduction mechanism and will likely be applicable to the detection of other protein analytes of clinical significance, such as clinically relevant antibodies (Covid-19 and HIV antibodies), simply by replacing biotin with a specific antigen or epitope molecule (e.g., peptide). Alternatively, this assay could be utilized as a competitive assay to detect any antigen. For example, a method is provided for easily adapting this assay to directly detect THC in saliva.

[0058]

[0082] Accordingly, a means for enabling rapid quantification of an analyte is provided. Current measurements are typically reported as gain, which is the percentage increase (or decrease) in current measured when a specific analyte (e.g., THC) is added to a sample. Using a calibration curve of gain vs. [analyte], the concentration of the analyte (e.g., [THC]) can be determined from the gain. To simplify the quantification process of the analyte (e.g., THC), five different strategies are proposed. This allows the gain to be determined without performing additional calibration steps in a sample that does not contain the analyte (Figure 15).

[0059]

[0083] Strategy 1: For applications where the detection of the analyte needs to be performed in a biological sample, develop a "sensor for detecting rare non-biological analytes" that can detect molecules not present in clinical samples (Figure 15a). This sensor will provide the necessary background current that will inform that the analyte cannot be present in the clinical sample. For example, using a fluorescein sensor (FAM) similar to the THC sensor that utilizes FAM-labeled signaling DNA and FAM antibody, the stability of the signaling DNA, the stability of the antibody, and the hybridization rate in this specific sample at specific conditions (e.g., different temperatures) can be recorded.

[0060]

[0084] Strategy 2: As another simple control, an additional electrode can be used to perform another hybridization assay (different DNA sequences) with another small molecule on the signaling strand (Figure 15b). The concentration of this second signaling strand that hybridizes to the second electrode may be adjusted such that its current represents the current obtained at the THC electrode in the absence of free THC.

[0061]

[0085] Strategy 3: As another simple control, additional electrodes can be used to perform another hybridization assay (with a different DNA sequence) without THC on the signal transduction strand (Figure 15c). The concentration of this second signal transduction strand that hybridizes to the second electrode may be adjusted such that its current represents the "acceptable" legitimate concentration of THC (5 ng / ml) or another analyte.

[0062]

[0086] Strategy 4: As another simple control, additional signal transduction DNA strands and capture DNA strands with different DNA sequences and redox elements (e.g., ferrocene, which exchanges electrons at a different voltage than methylene blue) are used. These can hybridize on the same electrode (which is functionalized with two different capture DNAs) (Figure 15d). The hybridization of this second signal transduction strand can be adjusted as needed (e.g., by changing the ratio of capture DNA) to increase or decrease the signal, which is relatively less affected. The current generated by this control signal transduction DNA can be adjusted to represent the background current or the current normally obtained with [THC] (5 ng / ml).

[0063]

[0087] Strategy 5: Illustrates a variation of the proposed previous control electrode. This utilizes an additional signal transduction DNA strand that has no recognition element but has a different redox element (e.g., ferrocene, which exchanges electrons at a different voltage than methylene blue) and hybridizes to the same capture strand as the THC signal transduction DNA strand (Figure 15e). The hybridization of this second signal transduction strand competes with the THC signal transduction strand. In the absence of THC, this signal transduction DNA is likely to be dominant over the THC signal transduction bound to the antibody. In the presence of THC, this signal transduction DNA will hybridize at a comparable rate to the THC signal transduction strand. This system will enable a ratiometric output and a higher gain.

[0064]

[0088] The present disclosure also provides kits and commercial packages comprising the detection systems, multiplex systems or control systems described herein. The kit may include any one of a reaction vessel (such as an electrode or an Eppendorf microtube) for each substrate provided, a control sample known to be free of the target to be detected, a control sample containing a known amount of the target to be detected, a control value or a series of control values associated with the absence or presence of a known amount of the target, a solution or suspension containing a substrate or a signal-transducing DNA molecule, and instructions for a method of determining the presence / amount of the target based on the provided system. The instructions may be present in the kit in various forms, and one or more of them may be present in the kit. One form in which these instructions may exist is information printed on a suitable medium or substrate, such as one or more sheets of paper on which the information is printed, the packaging of the kit, an accompanying document, etc. Another means may be a computer-readable medium on which information is recorded or stored, such as a diskette, CD, DVD, Blu-ray (registered trademark), computer-readable memory, etc. Yet another means that may exist is a website address used to access information remotely via the Internet. In one embodiment, the kit is an Eppendorf, and the signal-transducing DNA molecule is separately added inside the cap of the tube or microtube. (FIG. 16). Thereafter, the Eppendorf is sealed and shaken.

[0065]

[0089] As shown in FIG. 16, THC contained in a cotton swab used to rub the gums is released into a solution containing a THC antibody. After a short incubation, the solution is sealed with an Eppendorf lid containing dry signaling DNA (THC-DNA and control DNA, here cocaine-DNA), and the solution is mixed for 10 seconds to solubilize the signaling DNA. Next, this solution is added to electrodes (THC electrode and cocaine electrode) for analysis. As included herein, this analysis can be performed on a portable potentiostat controlled by an app via Bluetooth. In the absence of THC, both electrodes detect an equal amount of signaling DNA. In the presence of THC, the THC antibody that can bind to THC-DNA decreases, and the THC-DNA that can bind to the THC electrode increases, so the electrochemical signal on the THC electrode rises.

[0066]

[0090] The methods described herein are used in a variety of different applications where determination and / or quantification of the presence or absence of one or more targets in a sample is desired.

[0067]

[0091] For example, the presence, survival, or significant change over time in the concentration of a target in a sample can be utilized to perform disease risk diagnosis / evaluation, disease diagnosis / evaluation in an individual, or adjustment of disease treatment in an individual. For example, the presence of a specific target or target panel can affect the choice of drug treatment and dosing schedule for an individual. In the evaluation of potential drug therapies, the presence or concentration of a target may be used as an alternative to natural endpoints such as survival or irreversible pathological conditions. When a target related directly to the improvement of health changes due to treatment, the target can serve as an alternative endpoint for evaluating the clinical benefit of a specific treatment or dosing schedule. Therefore, individualized diagnosis and treatment based on specific targets or target panels detected in an individual are facilitated by the present system and method. Furthermore, since the present system and method are highly sensitive as described above, early detection of targets related to diseases is facilitated. Having multiplexity capable of detecting multiple targets in a single assay, along with selectivity, sensitivity, and ease of use, the system and method of the present disclosure are used in quantitative, point-of-care assays or bedside biomolecular assays.

[0068]

[0092] In a further embodiment, the presence of an infectious agent (such as bacteria that may be resistant to one or more antibiotics in some embodiments) can be detected in a biological sample, food, or water.

[0069]

[0093] In yet another embodiment, the presence, survival, or significant change over time in the concentration of a target in a sample can be utilized to examine the contamination risk and presence of contamination in food.

[0070]

[0094] The present system and method are used for diagnostic tests. Examples thereof include, but are not limited to, detection and / or quantification of a target as described above, screening tests for periodically testing samples for asymptomatic subjects, prognostic tests for predicting the possible disease course using the presence and / or amount of a target, stratification tests for predicting the response of a subject to different drug treatments, efficacy tests for monitoring the efficacy of drug treatments, and the like.

[0071]

[0095] The present system and method are also used for validation tests. For example, a validation test may be utilized to verify or confirm that a potential disease biomarker is an indicator that reliably indicates the presence or absence of a disease across diverse individuals. Since the present system and method have a short test time, the throughput can be easily increased, and multiple samples can be screened in the shortest time.

[0072]

[0096] In certain embodiments, the present system and method are utilized for the detection of antibodies in a sample. In some cases, the present system and method may be used to detect the presence or absence of a specific antibody, or an increase or decrease in the concentration of a specific antibody in a sample.

[0073]

[0097] In another embodiment, the present system can be used to screen for reagents that can modulate the binding between two biological substances. For example, the present system can be used to screen a drug library to identify antagonists or agonists that can enhance or weaken the binding between two biological substances.

[0074] Example I

[0098] Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and 6-mercaptohexanol (MCH) were purchased from Sigma-Aldrich. Streptavidin was obtained from New England BioLabs. Whole blood (newborn calf) was purchased from Innovative Research. The column and reagents for DNA synthesis were purchased from Biosearch Technologies and ChemGenes Corporation, respectively. The buffer used for streptavidin was 50 mM NaH2PO4, 150 mM NaCl, pH 7.0. The buffer used for the THC antibody or THC assay was 69.5 mM boric acid, 254 mM NaCl, 18.8 mM Na2HPO4, 9.7 mM Chaps, 6.7 mM Proclin-150.

[0075]

[0099] DNA was synthesized using a DNA / RNA synthesizer (K&A Laborgeraete, Germany). Unlabeled DNA was purified by a reverse-phase cartridge (RPC), and labeled DNA (methylene blue-labeled or biotin-labeled) was purified using high-performance liquid chromatography (HPLC) equipped with an XBridge Oligonucleotide BEH C18 column (130 Å, 2.5 μm, 4.6 mm × 50 mm, 1 / pkg). The DNA sequences are shown in Table 1.

[0076]

Table 1

[0077]

[0100] A gold working electrode (rod-shaped) (diameter 0.2 cm, surface area 0.0314 cm 2 , West Lafayette, Indiana) was cleaned based on the literature (Xiao et al., 2007, Nat Protoc, 2:2875). The capture DNA was immobilized on the clean gold electrode by the following procedure. First, 1 μL of 100 μM capture DNA and 2 μL of 10 mM TCEP were mixed at room temperature for 1 hour to reduce the disulfide bond. Next, the reduced capture DNA was diluted to a final concentration of 300 nM with a buffer (50 mM NaH2PO4, 150 mM NaCl, pH 7.0), and then the gold electrode was incubated with the diluted capture DNA solution (300 nM) at room temperature for 2 hours. Next, the gold electrode was washed with deionized water to remove the capture DNA not immobilized on the surface, and further incubated with a 2 mM MCH solution at room temperature for 2 hours to remove the physically adsorbed capture DNA and passivate the gold electrode. Finally, the functionalized gold electrode was washed with deionized water and used for the next measurement or stored at 4 °C in buffer until use.

[0078]

[0101] Immediately after introducing the gold electrode functionalized with capture DNA into a sample solution containing 100 nM signaling DNA and the protein analysis target, electrochemical measurements were initiated. Electrochemical data were recorded in the range of -0.1 to -0.5 V using square wave voltammetry (SWV). Peak currents were collected using the manual fitting mode of PSTrace5.4 (2018) software. Current vs. time, signal gain vs. time, and the kinetic profiles of the binding curves were fitted using Kaleidagraph, version 4.1 (2009). Electrochemical measurements were performed at room temperature using an EmStatMUX potentiostat multiplexer (Palmsens Instruments, Netherlands) equipped with a standard three-electrode cell including a working electrode (rod-shaped gold electrode), a counter electrode (platinum, Sigma-Aldrich), and a reference electrode (Ag / AgCl (1 M KCl), CH Instruments).

[0079]

[0102] Micrux electrodes (ED-SE1-AuPt, MicruX Technologies, Asturias, Spain) were assembled in the following procedure. First, the Micrux electrodes were cleaned using cyclic voltammetry (-1.5 to +1.5 V, scan rate 0.1 V / s, number of scans 10) in 0.05 M H2SO4. Next, the Micrux electrodes were functionalized with capture DNA in the same procedure as the rod-shaped gold working electrode. Electrochemical measurements on the Micrux electrodes were performed by adding 5 μL of blood containing 100 nM signaling DNA and the protein analysis target onto the Micrux electrode surface. Experimental data were recorded in the range of -0.2 to -0.65 V using square wave voltammetry (SWV). Peak currents were collected using the manual fitting mode of PSTrace5.4 (2018) software.

[0080]

[0103] CTI electrodes (Conductive Technologies, York, Pennsylvania, USA) were assembled following the procedures below. First, the CTI electrodes were washed with isopropanol for 2 minutes. Next, the CTI electrodes were functionalized with capture DNA following the same procedure as for the rod-shaped gold working electrodes. Electrochemical measurements on the CTI electrodes were performed by adding 70 μL of buffer or saliva containing 100 nM signaling DNA, 100 nM THC antibody, and the THC analyte to the CTI electrode surface. Experimental data were recorded in the range of -0.1 to -0.55 V using square wave voltammetry (SWV). Peak currents were collected using the manual fitting mode of PSTrace5.4 (2018) software.

[0081]

[0104] The workflow shown in Figure 16 was performed using two electrodes, and THC in saliva was detected as follows. 1. Two different capture DNAs, namely 16nt capture DNA and 16nt capture DNA-2, were functionalized on two CTI electrodes. 2. Prepare an Eppendorf tube containing 28.5 nM anti-THC antibody. 3. Prepare an Eppendorf cap containing two types of dried signaling DNA. Specifically, 2.5 μL of 10 μM THC-labeled signaling DNA and 1.35 μl of 5 μM cocaine-labeled signaling DNA were added simultaneously. The cap was dried in the dark at room temperature for 2 - 3 hours. 4. Use a cotton swab to collect THC from the gums for 30 seconds. Next, place the cotton swab into the Eppendorf tube and rotate it for 10 seconds. Then, remove the cotton swab and incubate the sample for 50 seconds. 5. Finally, cover the Eppendorf tube with the prepared cap containing dried signaling DNA. Invert it up and down for 10 - 20 seconds to mix. Then, add 3 drops (or 2 drops if there is a lid on the electrode surface) of the sample to the two CTI electrodes for detection.

[0082]

[0105] The sequences of the capture DNA and signaling DNA used are as follows. 16 nt capture DNA: 5’-ACG AGA ATG AGC AGGA-SH-3’ (SEQ ID NO: 7) 16 nt capture DNA-2: 5’-TGG ACA AAG AGG AGCA-SH-3’ (SEQ ID NO: 10) THC-labeled signal transduction DNA: 5’-MB-(THC)TCCT GCT CAT TCT CGT-3’ (SEQ ID NO: 11) Cocaine-labeled signal transduction DNA: 5’-MB-(cocaine)TGCT CCT CTT TGT CCA-3’ (SEQ ID NO: 12) (Cocaine DNA was used as control DNA according to the control procedure described in Fig. 15b).

[0083]

[0106] Electrochemical measurements for antibody detection in whole blood were also performed. Immediately after mixing the signal transduction DNA with the sample solution (bovine whole blood with or without antibody) and applying it to the gold electrode functionalized with the capture DNA, the electrochemical measurement was started. Electrochemical data were recorded in the range of -0.10 to -0.55 V using square wave voltammetry (SWV). The peak current was collected using the manual fitting mode of PSTrace5.9 (2022) software. The current vs. time, signal gain vs. time, and kinetic profiles of the binding curves were fitted using Kaleidagraph, version 4.1 (2009). Electrochemical measurements were performed at room temperature using a MultiEmStat3 (Palmsens Instruments, Netherlands) equipped with 4 channels.

[0084]

[0107] CTI electrode (Conductive Technologies, York, Pennsylvania, USA) was used as described above (

[0085] ) It was prepared as follows. The antibody was diluted to 10 uM with buffer. The 100 uM stock solution of signaling DNA was diluted to 10 uM with water. Electrochemical measurements on the CTI electrode were performed by adding 70 μL of bovine whole blood containing 100 nM of signaling DNA and 60 nM of antibody to the surface of the CTI electrode. Experimental data were recorded in the range of -0.10 to -0.55 V using square wave voltammetry (SWV). Peak currents were collected using the manual fitting mode of PSTrace 5.9 (2022) software.

[0086] Synthesis of THC-C9-azide linker

[0108] To a stirred solution of (±)-11-nor-9-carboxy-Δ9-THC (2 mg) in DMF (500 mL), N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (2.3 mg) and 1-hydroxybenzotriazole hydrate (HOBt) (0.8 mg) were added, and the mixture was stirred at room temperature for 10 minutes. After 10 minutes, 3-azido-1-propanamine (0.7 mg) and DIPEA were added, and the mixture was stirred at room temperature overnight. After completion of the reaction, the crude product was purified by HPLC using an acetonitrile / water system (see Figure 10a).

[0087] Synthesis of benzoylecgonine-azide linker

[0109] To a stirred solution of benzoylecgonine (2 mg) in DMF (200 μL), N,N,N’,N’-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (2.3 mg) and 1-hydroxybenzotriazole hydrate (HOBt) (0.8 mg) were added, and the mixture was stirred at room temperature for 10 minutes. After 10 minutes, 3-azido-1-propanamine (0.7 mg) and DIPEA were added, and the mixture was stirred at room temperature overnight. After completion of the reaction, the crude product was purified by HPLC using an acetonitrile / water system (see Figure 10c).

[0088] Synthesis of DNA conjugated with methylene blue

[0110] DBCO-conjugated amino-DNA (IDT-DNA) was dissolved in water. Methylene blue-NHS (20 mM) was added to IDT-DNA (2.6 mM) at the 5'-terminal NH2 of iAmMC6T in sodium bicarbonate buffer (pH 8.5) and stirred overnight at room temperature. After completion of the reaction, the reaction mixture was purified by HPLC using acetonitrile and triethylamine (TEA) / triethylammonium acetate (TEAA) buffer. DNA conjugated with methylene blue (IDT-MB) was obtained in good yield (91%). See Figure 10b.

[0089] Synthesis of IDT-MB conjugated with THC-C9

[0111] THC-C9-azide (2 mM) was added to IDT-MB (1.2 mM) in water and stirred overnight at room temperature. After completion of the reaction, the reaction mixture was purified by HPLC using acetonitrile and triethylamine (TEA) / triethylammonium acetate (TEAA) buffer. IDT-MB DNA conjugated with THC-C9 was obtained in good yield (90%). See Figure 10b.

[0090] Synthesis of IDT-MB conjugated with benzoylecgonine

[0112] Benzoylecgonine-azide (2 mM) was added to IDT-MB (1.2 mM) in water and stirred overnight at room temperature. After completion of the reaction, the reaction mixture was purified by HPLC using acetonitrile and triethylamine (TEA) / triethylammonium acetate (TEAA) buffer. IDT-MB DNA conjugated with benzoylecgonine was obtained in a considerable yield (26%).

[0091] Synthesis of IDT-MB conjugated with peptide

[0113] Peptide-azide (10 mg / mL) in 1 mM PBS buffer (pH 7.4) was added to IDT-MB (0.2 mM) in water and stirred overnight at room temperature. After completion of the reaction, the reaction mixture was purified by HPLC using acetonitrile and triethylamine (TEA) / triethylammonium acetate (TEAA) buffer. IDT-MB DNA conjugated with peptide was obtained in good yield (90%).

[0092]

Table 2

[0093] Example II Assay optimization example for THC detection Optimization of the dynamic range of the assay

[0114] The dynamic range of the sensor can be adjusted by changing the antibody concentration. Since the antibody binds to two signal transduction DNA molecules, it is necessary to ensure that there are always more antibody binding sites than signal transduction DNA molecules. Usually, the signal transduction concentration is made less than twice the Ab concentration to reduce the background.

[0094]

[0115] The sensitivity of THC near the detection limit of THC (5 ng / ml or 15 nM) was optimized using 20 nM antibody and 35 nM signal transduction DNA (see Figure 16). Using higher concentrations of antibody and signal transduction DNA will only shift the dynamic range of the sensor to the higher THC concentration side.

[0095] Various methods are available for quantifying the THC concentration via current or gain

[0116] One method is to utilize the current obtained after a specific time (Figure 16). It was found that the current obtained before 1 minute has low reproducibility. Figure 17 shows the binding curve obtained using the current from 1 minute to 3 minutes after applying the sample to the electrode. In the assay using 20 nM antibody and 35 nM, the observed dynamic range is usually in the low nM (3 - 60 nM or 1 - 20 ng / ml) range. The sensor current (C C ) can be easily converted to signal gain using the control electrode current (C S ). Gain (%) = 100×(C S - C C ) / C C

[0096]

[0117] Another method is to utilize the hybridization kinetics (the slope of the hybridization curve shown in FIG. 17). The slope during a period of usually 1 to 3 minutes is utilized. It has been observed that increasing the waiting time further improves the accuracy.

[0097]

[0118] Examples of binding curves obtained from saliva samples containing different concentrations of THC are shown (see FIG. 19). In these data, the saliva samples were mixed 1:3 with a “THC release buffer” that aids in solubilizing THC.

[0098]

[0119] Although the present specification has been described in relation to specific embodiments, it will be understood that further modifications are possible. This application is intended to cover any variations, uses, or adaptations that depart from the present disclosure but are within the scope of common knowledge or practice in the art and that can apply to the essential features described hereinbefore and are within the scope of the appended claims.

Claims

1. A system for detecting a target analyte in a sample, comprising: a plurality of capture DNA molecules having a first end and a second free end; a first substrate having a surface associated with each of the first ends of the plurality of capture DNA molecules at a plurality of positions; a plurality of signal transduction DNA molecules, each of the signal transduction DNA molecules having: a core nucleic acid sequence substantially complementary to and hybridizable with a region of each of the capture DNA molecules; a first end associated with a portion for binding an analyte in proximity to a reporter portion; a second end; and configured such that when the portion binds to the analyte, hybridization and reporter activity of the plurality of signal transduction DNA molecules are inhibited on the surface associated with the plurality of capture DNA molecules; wherein the analyte binds to the signal transduction DNA molecule in proximity to the reporter portion, resulting in steric hindrance between: (i) the analytes, (ii) between the analyte and the capture DNA molecule, and (iii) between the analyte and the substrate.

2. The system according to claim 1, wherein the portion for binding the analyte is at a distance of up to 16 nucleotides from the reporter portion.

3. The system according to claim 1 or 2, wherein the portion for binding the analyte is at a distance of up to 13 nucleotides, up to 10 nucleotides, up to 7 nucleotides, up to 4 nucleotides, or up to 1 nucleotide from the reporter portion.

4. The system according to any one of claims 1 to 3, wherein the portion is at a distance of up to 16 nucleotides, up to 13 nucleotides, up to 10 nucleotides, up to 7 nucleotides, up to 4 nucleotides, or up to 1 nucleotide from the first end of the signal transduction DNA molecule.

5. The system according to any one of claims 1 to 4, wherein the length of the capture DNA molecule is up to 16 nucleotides, up to 13 nucleotides, up to 10 nucleotides, or up to 8 nucleotides.

6. The system according to any one of claims 1 to 5, wherein the target analyte is a macromolecule or an antibody.

7. For detecting a small molecule or a polypeptide, wherein the small molecule or the polypeptide competes with the binding of the signal transduction DNA molecule of the analyte, the system according to any one of claims 1 to 5.

8. The system according to claim 7, wherein the small molecule is a drug.

9. The system according to claim 8, wherein the drug is cocaine.

10. The system according to any one of claims 1 to 9, wherein the moiety for binding the analyte is an antigen.

11. The system according to any one of claims 1 to 10, wherein the reporter moiety is a redox reporter or a fluorophore.

12. The system according to claim 11, wherein the redox reporter is methylene blue.

13. The system according to any one of claims 1 to 12, wherein the substrate is a metal electrode, a 96-well plate, or a tube.

14. The system according to claim 13, wherein the metal electrode is a gold electrode or a carbon electrode.

15. The system according to any one of claims 1 to 14, wherein the substrate is a glass tube Ag / AgCl reference electrode, a platinum wire counter electrode, or a CTI electrode.

16. The system according to any one of claims 1 to 14, wherein the substrate is an electrode comprising a gold working electrode (WE), a platinum reference electrode (RE), and a platinum counter electrode (CE).

17. The system according to any one of claims 1 to 16, wherein the sample is a biological sample from a subject.

18. The system according to any one of claims 1 to 17, wherein the sample is water, an environmental element, or food.

19. The system according to claim 17, wherein the biological sample is whole blood, saliva, or urine.

20. The system according to claim 17, wherein the subject is a human or an animal.

21. A kit comprising the system according to any one of claims 1 to 20 in a container.

22. The kit according to claim 21, wherein the container is an Eppendorf with a lid.

23. The kit according to claim 22, wherein the lid contains the signal transduction DNA molecule.

24. The kit according to any one of claims 21 to 23, further comprising a potentiostat.

25. A method for detecting a target analyte in a sample, comprising: preparing the sample suspected of containing the target analyte; Preparing the system according to any one of claims 1 to 20; Providing or determining a control amount of the plurality of capture DNA molecules hybridized with the plurality of signal transduction DNA molecules in the system in the absence of the target analyte; Contacting the sample with the system; Determining a test amount of the plurality of capture DNA molecules hybridized with the plurality of signal transduction DNA molecules in the system in the presence of the sample; Characterizing that the sample contains the target analyte when it is determined that the test amount is less than the control amount, and characterizing that the sample does not contain the target analyte when it is determined that the test amount is equal to or greater than the control amount. A method comprising:

26. The method according to claim 25, further comprising quantifying the concentration of the target analyte in the sample based on a comparison between the control amount and the test amount.

27. Determining the control amount by contacting a control sample known to be free of the target analyte with the system according to any one of claims 1 to 20, and determining the amount of the plurality of capture DNA molecules hybridized with the plurality of signal transduction DNA molecules. The method according to claim 25 or 26, further comprising:

28. The method according to any one of claims 25 to 27, further comprising electrochemically determining the test amount and / or the control amount.

29. The method according to any one of claims 25 to 27, wherein the sample is a biological sample from a subject.

30. The method according to any one of claims 25 to 27, wherein the sample is water, an environmental element, or food.

31. The method according to claim 29, wherein the biological sample is whole blood, saliva, or urine.

32. The method according to claim 29, wherein the subject is a human or an animal.

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