Detection of target analytes using multimodal signal probes

Abstract

A dual-labeled signal probe comprising an electron transfer portion (ETM) and an optical signaling portion (OSM) is disclosed. When a target analyte binds to the signal probe, the composition is transported to the electrode surface. In some embodiments, the ETM and OSM are then excited by applying a voltage to the electrode, respectively. In some embodiments, the ETM and OSM signals are subsequently detected using a single detector (or two or more detectors) to allow for the determination of the presence or absence of the target analyte. In some embodiments, the ETM and OSM signals are correlated before determining the presence or absence of the target analyte. In some embodiments, the dual-labeled signal probe enables real-time detection using an endpoint electrochemical detection system. In some embodiments, the dual-labeled signal probe may be used to evaluate the location and density of the capture probe on the electrode.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnostic methods, and particularly to the detection of one or more analytes containing biological molecules in a obtained sample.

Background Art

[0002] Bloodstream infection (BSI) is associated with significant morbidity, mortality, and prolonged length of hospital stay (LOS). Delayed administration of effective antibiotics increases the risk of death, and thus it is most important to correctly select an antibiotic regimen early in the treatment process. Delays in the identification of the causative microorganism and antibiotic resistance or susceptibility often cause delays in optimal antibiotic treatment. Rapid diagnostic tests (RDTs), including tests such as polymerase chain reaction (PCR), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and peptide nucleic acid fluorescence in situ hybridization (PNA-FISH), have improved conventional microbiological methods, shortened the time to microorganism identification, optimized antimicrobial therapy, and as a result, improved clinical outcomes including mortality. Commercially available molecular RDTs (mRDTs) are available for the direct testing of positive blood culture bottles (BCBs) and provide timely results compared to conventional subculture and phenotypic susceptibility testing. Examples of commercially available mRDT devices include the applicant's ePLEX® diagnostic system.

[0003] Electrochemical detection systems are useful tools that are highly sensitive and can detect small amounts of targets. Since the electrical and electrochemical monitoring of nucleic acid amplification does not require optical assistance, the system can be simplified, miniaturized, and integrated into a small chip by using a complementary metal oxide semiconductor (CMOS)-compatible manufacturing process, leading to the production of a scalable high-throughput analysis system for point-of-care applications.

[0004] In these electrochemical systems, the target amplicon is typically mixed with a ferrocene-labeled signal probe (or osmium-labeled signal probe) complementary to a specific target on the panel. The target sequence hybridizes to the complementary signal probe and capture probe coupled to a gold-plated electrode, as shown in Figure 1a. The presence of each target is determined by voltammetry, which generates a specific electrical signal from the ferrocene-labeled signal probe (or osmium-labeled signal probe). The use of microfluidic systems in the electrochemical detection of target analytes is described in detail in U.S. Patents 10,005,080, 9,557,295, 8,501,921, 6,600,026 and 6,740,518, the disclosure of which is incorporated herein by reference in its entirety.

[0005] A potential drawback of electrochemical detection techniques is their reliance on endpoint analysis, which is disadvantageous because (1) the system requires additional time for amplification post-processing before detection results are achieved, and (2) quantification is difficult due to a narrower dynamic range compared to fluorescence-based real-time PCR. Therefore, further improvements to electrochemical detection systems are still needed.

[0006] Fluorescence-based bioassays are considered to be unsensitive and require expensive optical equipment. Furthermore, the biorecognition events in these assays are inherently slow (minutes to hours). The sensitivity of fluorescence-based assays can be improved without the use of high-end optical equipment by incorporating plasmon resonance particles (PSPs). This improved sensitivity is thought to be enabled by an increase in the fluorescence signature and a decrease in the lifetime of fluorophores positioned in close proximity to the PSPs, a phenomenon explained by a phenomenon called metal-enhanced fluorescence (MEF). In MEF-based bioassays, PSPs (generally silver nanoparticles) are deposited on a planar surface, and the bioassay is built on the PSPs. Since the size of most biomolecules is smaller than the PSPs (20–100 nm), the fluorophores are positioned within a distance where their emission is increased due to their interaction with surface plasmons of the PSPs. Digital fluorescence detection is disclosed in U.S. Patent No. 9,810,637, which is incorporated in its entirety herein by reference. In these MEF bioassays, the fluorophores are enhanced by the wavelength of light, rather than by electricity. Furthermore, these MEF bioassays do not use signal probes that have both electron transport moieties (ETMs) and optical signaling moieties (OSMs). Moreover, these MEF bioassays do not utilize capture probes to hold the OSMs in close proximity to the surface. Furthermore, a goal in MEF bioassays is to eliminate the need for photodetectors, such as photomultiplier tubes or charge-coupled device (CCD) cameras, to convert the photon flux into a digital signature, which are still required under the disclosed methodologies. [Overview of the project]

[0007] A first aspect of this disclosure is a signal probe comprising a first signaling portion and a second signaling portion, the first and second signaling portions having different detection modalities (referred to herein as “dual-labeled signal probe”). In some embodiments, the first signaling portion is an electron transfer portion (ETM), and the second signaling portion is an optical signaling portion (OSM). In some embodiments, the dual-labeled signal probe is bound to a target analyte (e.g., nucleic acid) to form a target / dual-labeled signal probe complex. In some embodiments, the target / dual-labeled signal probe complex is bound to a capture probe to form a capture probe-dual-labeled signal probe-nucleic acid system complex. In some embodiments, the capture probe-dual-labeled signal probe-nucleic acid system complex is bound to an electrode ("capture probe-signal probe-nucleic acid system complex").

[0008] A second aspect of the present disclosure is a composition comprising (i) a first dual-labeled signal probe including a first signaling portion having a first detection modality and a second signaling portion having a second detection modality, and (ii) a second dual-labeled signal probe including a third signaling portion having a first detection modality and a fourth signaling portion having a second detection modality, wherein at least the signals generated by the second signaling portion and the fourth signaling portion are distinguishable.

[0009] A third aspect of the present disclosure is one or more capture probe / stained DNA hybridization complexes, each of which comprises a fluorescently stained nucleic acid on an electrode bound to a capture probe. In some embodiments, a voltage can be applied to one or more capture probe / stained DNA hybridization complexes. In some embodiments, the fluorescently stained nucleic acid produces a detectable signal in response to the applied voltage.

[0010] In each of the above compositions, the detectable signal may be detected by an optical detector, an electrochemical detector, or both an optical detector and an electrochemical detector. In this way, the fluorescently stained nucleic acid (or OSM on a signal probe) can be measured by applying a voltage. In some embodiments, the detectable signal is a dipole moment, a plasmon current, and / or fluorescence.

[0011] A fourth aspect of this disclosure is a method for detecting a target analyte (e.g., nucleic acid) in a sample. In some embodiments, the method includes introducing a signal probe into a obtained sample containing a target analyte such that the target analyte binds to a signal probe (e.g., a dual-labeled signal probe) and a capture probe to form a capture probe-signal probe-target analyte system complex (e.g., a capture probe-dual-labeled signal probe-target analyte system complex). In some embodiments, the signal probe is a dual-labeled signal probe comprising a first signaling portion and a second signaling portion, the first and second signaling portions having different detection modalities. In some embodiments, the first signaling portion is an ETM and the second signaling portion is an OSM. In some embodiments, after excitation, signals from the first signaling portion and signals from the second signaling portion are detected (using either a single detector or multiple detectors of different modalities). After detection, the signals from the first signaling portion and signals from the second signaling portion are correlated as disclosed herein.

[0012] In some embodiments, the optical signal is detected by an optical detector after a voltage is applied to a double-labeled signal probe. In some embodiments, the optical signal is detected by an electrochemical detector after a voltage is applied to a double-labeled signal probe. In some embodiments, the optical signal is detected by both an electrochemical detector and an optical detector after a voltage is applied to a double-labeled signal probe. Thus, the disclosure provides detection of optical signals such as colorimetric signals, fluorescence signals, emission signals, chemiluminescence signals, and / or phosphorescence signals without requiring additional activation means (such as a light source) to excite the label (and, in some embodiments, without adding an optical detector).

[0013] A fifth aspect of the present disclosure is a method for detecting either a redox reaction or an oxidation reaction, comprising: (a) introducing a signal probe into a obtained sample; (b) irradiating the obtained sample with light or laser light; and (c) detecting either a redox reaction or an oxidation reaction from one or more signaling moieties (e.g., ETM or OSM) coupled to the signal probe. For example, in some embodiments, a dual-labeled signal probe including a first signaling moiety and a second signaling moiety is introduced into a obtained sample, where the first signaling moiety is an ETM and the second signaling moiety is an OSM. Subsequently, the obtained sample (including the introduced signal probe) is irradiated with an electromagnetic radiation source (e.g., light or a laser beam) to detect a redox reaction or an oxidation reaction. In some embodiments, the redox reaction or oxidation reaction is detected by an optical detector after irradiation of the obtained sample with light or a laser beam. In some embodiments, the redox reaction or oxidation reaction is detected by an electrochemical detector after irradiation of the obtained sample with light or a laser beam. In some embodiments, the redox or oxidation reaction is detected by both an electrochemical detector and an optical detector after irradiating the resulting sample with light or a laser beam. Therefore, this disclosure provides detection of an ETM signal without the need for an ETM activating means (such as an electrode) to excite the ETM (and, in some embodiments, without the addition of an electrochemical detector). In some embodiments, instead of exciting the ETM with light or a laser beam, the ETM is excited by the application of a voltage and detected by an electrochemical detector, an optical detector, or both.

[0014] In some embodiments, detection includes applying a voltage (without optical stimulation) and detecting a first signaling portion and a second signaling portion of a dual-labeled signal probe, wherein the first and second signaling portions are of different modalities (e.g., ETM and OSM). In some embodiments, detection includes applying an optical stimulation such as a laser beam or light (without voltage) and detecting a first signaling portion and a second signaling portion, wherein the first and second signaling portions are of different modalities (e.g., ETM and OSM).

[0015] A sixth aspect of this disclosure is a method (not optical stimulation) for applying a voltage to a sample including a signal probe, wherein the signal probe includes only OSM (without ETM). In other words, a voltage is applied to a sample including a signal probe, and the signal probe consists of OSM.

[0016] A seventh aspect of this disclosure is a method for applying an optical stimulus (such as a laser beam or light) (without voltage) to a sample including a signal probe, wherein the single probe includes only an ETM (without an OSM). In other words, an optical stimulus is applied to a sample including a signal probe consisting of an ETM.

[0017] The eighth part of this disclosure relates to the real-time detection of amplification. In a typical real-time PCR reaction, no post-PCR processing is required. However, as disclosed herein, a PCR product containing OSM must undergo post-PCR processing, i.e., binding to a capture probe, for detection. In some embodiments, the PCR product (without OSM) must be hybridized to a signal probe containing OSM. The signal probe / amplicon complex must then be hybridized to a capture probe. In any case, energy is applied to the OSM-labeled signal probe or OSM-labeled amplicon to excite the optical signaling moiety, which is then detected by an optical detector, an electrochemical detector, or both.

[0018] A ninth aspect of this disclosure relates to multiplex real-time PCR. In some embodiments, dual-labeled signal probes (containing signaling portions having different modalities) can enhance the multiplexing capability of the assay cartridge disclosed herein. In a typical detection system, 20 targets can be detected if each of 20 pads is labeled with a capture probe for a single target. Here, multiple targets can be detected per electrode because different optical labels can be detected at different potentials. Furthermore, different regions of the same target can be detected because different optical labels can be detected at different potentials. Thus, one aspect of this subject relates to multiplex detection. Nucleic acids are extracted from a sample. The sample is amplified. The amplified sample is mixed with at least two signal probes, the first signal probe containing a first ETM and a first optical signaling portion, and the second signal probe containing a first ETM and a second optical signaling portion, which are distinct from each other, unlike the first and second optical signaling portions. In some embodiments, at least two different pathogens are detected. In some embodiments, at least two different amplicons are detected (the amplicons originate from the same or different pathogens).

[0019] A tenth aspect of this disclosure relates to SNP analysis. In particular, a method for identifying a single nucleotide residue of interest at a position within a stretch of consecutive nucleotide residues in a DNA molecule is disclosed herein. In one embodiment, a method is provided for detecting a SNP in a target nucleic acid in a sample, comprising: an amplification step, in which, if any target nucleic acid is present in the sample, the sample is contacted with a primer comprising a first nucleic acid sequence to produce an amplification product; a hybridization step, in which the amplification product is contacted with a SNP-specific signal probe comprising a second nucleic acid sequence complementary to the SNP-containing region of the amplification product, wherein the SNP-specific signal probe comprises a first signaling portion and a second signaling portion (the first and second signaling portions being of different modalities); and detection of the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of a SNP at the target nucleic acid target, and the absence of the amplification product indicates the absence of a SNP at the target nucleic acid target.

[0020] An eleventh aspect of this disclosure relates to measuring the position of a capture probe on an electrode. Instead of using a second signaling portion to detect the analyte, the second signaling portion is used to determine the position of the capture probe. The position of the optical signaling portion during (or after) detection can indicate whether the capture probe is uniformly distributed across the electrode. In some embodiments, the optical signaling portion is attached to the capture probe. Detection of the position / distribution of the capture probe on the electrode may be used to evaluate or improve quality control.

[0021] A twelfth aspect of the present disclosure is a detection system comprising a single signal exciter and a first detector and a second detector, wherein the first and second detectors are of different modalities. In some embodiments, the first signal exciter is a voltage generator. In some embodiments, the first signal exciter is an optical or laser beam generator. In some embodiments, the first detector is an ECM detector and the second detector is an optical detector. In some embodiments, the system does not include an optical / laser beam source (such as a laser, photodiode, or lamp) for activating an optical signaling label on a signal probe. In some embodiments, the system does not include a laser, a high-intensity mercury (Hg) arc lamp, an optical fiber light source, or other high-intensity light source for activating an optical signaling label on an optically labeled signal probe or optically labeled nucleic acid. In some embodiments, the system does not include a voltage generator for activating an ETM on a signal probe. In some embodiments, the system comprises a first signal exciter, a second signal exciter, a first detector, and a second detector, the first signal exciter being capable of generating a voltage, the second signal exciter being capable of generating light of a first wavelength, the first detector detecting a redox / oxidation reaction, and the second detector detecting light of a second wavelength.

[0022] In some embodiments, ETMs (such as ETMs included as part of a signal probe) are detected by an ETM detector, an optical detector, or both. In some embodiments, OSMs (such as OSMs included as part of a signal probe) are detected by an ETM detector, an optical detector, or both. In some embodiments, the detector is an ammeter that measures a plasmon voltage from the OSM. In some embodiments, the detection device is designed to detect the OSM and excite the optical signaling portion without using light or a laser. In some embodiments, the detection device is designed to detect the optical signaling portion without using an optical detector. In some embodiments, the detection device is designed to detect the optical signaling portion without using light or a laser to excite the optical signaling portion and without using an optical detector. In some embodiments, the detection device is designed to detect the ETM without using a voltage generator. In some embodiments, the detection device is designed to detect the ETM without using an ETM detector. In some embodiments, the detection device is designed to detect the ETM without using a voltage generator to excite the ETM and without using an ETM detector. [Brief explanation of the drawing]

[0023] [Figure 1a]A typical sandwich assay used for electrochemical detection is shown. In Figure 1b, the sandwich comprises three main elements: a capture probe (2), a signal probe (3), and a target (4). The target may be synonymous with a PCR amplicon sequence in the embodiment of nucleic acids. The target may have a portion (4b) that specifically binds to or hybridizes to a desired portion (3b) of the signal probe, a portion (4a) that binds to or hybridizes to the corresponding capture probe portion (2b), and optionally one or more adjacent portions, e.g. (4c). The capture probe may include a linker (2a) that connects, links, or binds the capture probe (2) to the electrode surface (1). As illustrated, the signal probe has a detectably labeled portion or label (3a) adjacent to the electrode surface. Here, circles represent ETMs, squares represent optical signaling portions, and triangles represent colorimetric portions. Circles may represent a single or multiple ETMs. Squares may represent a single or multiple optical signaling portions. The triangle can represent a single colorimetric portion or multiple colorimetric signaling portions. In some embodiments, the labeled portion 3a is conjugated to or located within the signal probe binding portion (3b). In some embodiments, a self-assembled monolayer (SAM) is also attached to the electrode surface via one or more linkers of a similar form to the capture probe linker (2a) and is not depicted, which helps prevent or reduce undesirable electron transfer events ("noise") to the electrode surface. [Figure 1b]A typical sandwich assay used for electrochemical detection is shown. In Figure 1b, the sandwich comprises three main elements: a capture probe (2), a signal probe (3), and a target (4). The target may be synonymous with a PCR amplicon sequence in the embodiment of nucleic acids. The target may have a portion (4b) that specifically binds to or hybridizes to a desired portion (3b) of the signal probe, a portion (4a) that binds to or hybridizes to the corresponding capture probe portion (2b), and optionally one or more adjacent portions, e.g. (4c). The capture probe may include a linker (2a) that connects, links, or binds the capture probe (2) to the electrode surface (1). As illustrated, the signal probe has a detectably labeled portion or label (3a) adjacent to the electrode surface. Here, circles represent ETMs, squares represent optical signaling portions, and triangles represent colorimetric portions. Circles may represent a single or multiple ETMs. Squares may represent a single or multiple optical signaling portions. The triangle can represent a single colorimetric portion or multiple colorimetric signaling portions. In some embodiments, the labeled portion 3a is conjugated to or located within the signal probe binding portion (3b). In some embodiments, a self-assembled monolayer (SAM) is also attached to the electrode surface via one or more linkers of a similar form to the capture probe linker (2a) and is not depicted, which helps prevent or reduce undesirable electron transfer events ("noise") to the electrode surface. [Figure 2-1]Embodiments of the signal probe disclosed herein are shown. In some embodiments, a first signaling portion (shown as three circles) is directed toward, near, or located toward the 5' end of the signal probe, and a second signaling portion of a modality different from the first signaling portion (shown as three squares) is conjugated inside the signal probe, as shown in Figure 2a. In some embodiments, as shown in Figure 2b, a first signaling portion (shown as one circle, but there may be multiple signaling portions) is directed toward, near, or located toward the 5' end of the signal probe, and a second signaling portion of a modality different from the first signaling portion (shown as one square, but there may be multiple signaling portions) is also directed toward, near, or located toward the 5' end of the signal probe. In some embodiments, as shown in Figure 2c, a third signaling portion of a modality different from the first or second signaling portion is also located toward the 5' end of the signal probe. In some embodiments, a first signaling portion (shown as a circle) is directed toward, near, or located toward the 5' end of the signal probe, together with a second signaling portion of a different modality (shown as a square), and in some embodiments, a third signaling portion of a different modality (shown as a triangle) is also directed toward, near, or located toward the 5' end of the signal probe, and the first signaling portion (shown as a circle) is conjugated internally to the signal probe, together with the second signaling portion of a different modality (shown as a square), and in some embodiments, the third signaling portion of a different modality (shown as a triangle), as shown in Figure 2d. In this example, the circle may be the ETM, the square may be the optical signaling portion, and the triangle may be the colorimetric portion.In some embodiments, the first signaling portion (shown as three circles) is located on the 3' end of the signal probe, and the second signaling portion of a modality different from the first signaling portion (shown as three squares) is conjugated inside the signal probe, as shown in Figure 2e. In some embodiments, as shown in Figure 2f, the first signaling portion (shown as one circle) is located on the 3' end of the signal probe, and the second signaling portion of a modality different from the first signaling portion (shown as one square) is also located on the 3' end of the signal probe. In some embodiments, as shown in Figure 2g, the third signaling portion of a modality different from the first or second signaling portion is also located on the 3' end of the signal probe. In some embodiments, a first signaling portion (shown as a circle) is located on the 3' end of the signal probe, along with a second signaling portion (shown as a square) of a different modality than the first signaling portion; in some embodiments, a third signaling portion (shown as a triangle) of a different modality than the first and second signaling portions is located on the 3' end of the signal probe; and the first signaling portion (shown as a circle) is conjugated internally to the signal probe as shown in Figure 2h, along with the second signaling portion (shown as a square) of a different modality than the first signaling portion, and in some embodiments, the third signaling portion of a different modality (shown as a triangle). In this example, the circle may be an ETM, the square may be an optical signaling portion, and the triangle may be a colorimetric portion. In some embodiments, the first signaling portion (shown as three circles) is directed toward, near, or located toward the 5' end of the signal probe, and the second signaling portion of a modality different from the first signaling portion is conjugated toward, near, or located toward the 3' end of the signal probe (Figure 2i).In other words, the probe has two signaling regions, which can be introduced toward, near, or at any location in the 5' end of the sequence or in the middle of the sequence. In Figure 2(a-i), ii is the detection region, which has one or more detection regions, e.g., ferrocene or fluorophores, such as fluorescein / FITC, rhodamine, or Alexa Fluor, and i is the annealing region, e.g., the region that binds to the target analyte. The annealing region may or may not have one or more detection regions. Alternatively, the signal probe may contain extra terminal nucleosides at the ends of the nucleic acid (n+1 or n+2), which are used to covalently attach the signaling regions but do not participate in base pair hybridization during detection. Figure 2j shows an extra terminal nucleoside ligating the signaling region at the 5' end of the signal probe, with a second signaling region, which is a different modality from the first signaling region, conjugated inside the signal probe itself. Figure 2k shows an extra terminal nucleoside at the 5' end of the signal probe that connects the first signaling portion, the second signaling portion of a different modality from the first signaling portion, and the third signaling portion of a different modality from the first and second signaling portions, with the first signaling portion, the second signaling portion of a different modality from the first signaling portion, and the third signaling portion of a different modality from the first and second signaling portions being conjugated inside the signal probe itself. Figure 2l shows an extra terminal nucleoside at the 5' end of the signal probe that connects the first signaling portion, the second signaling portion of a different modality from the first signaling portion, and the third signaling portion of a different modality from the first and second signaling portions. In this example, the circle may be an ETM, the square may be an optical signaling portion, and the triangle may be a colorimetric portion. Alternatively, the primer may contain a linker used to covalently attach the electron transfer portion.Figure 2m shows that a linker connecting a signal transduction portion at the 5'-end of the signal probe and a second signal transduction portion of a different modality from the first signal transduction portion is conjugated inside the signal probe itself. Figure 2n shows, at the 5'-end of the signal probe, a linker connecting a first signal transduction portion, a second signal transduction portion of a different modality from the first signal transduction portion, a first signal transduction portion, and a third signal transduction portion of a different modality from the second signal transduction portion of the first signal transduction portion. The first signal transduction portion, the second signal transduction portion of a different modality from the first signal transduction portion, the first signal transduction portion, and the third signal transduction portion of a different modality from the second signal transduction portion are conjugated inside the signal probe itself. Figure 2o shows, at the 5'-end of the signal probe, a linker connecting a first signal transduction portion, a second signal transduction portion that is of a different modality from the first signal transduction portion, and a third signal transduction portion that is of a different modality from the first signal transduction portion and the second signal transduction portion. In this example, the circle can be an ETM, the square can be an optical signal transduction portion, and the triangle can be a colorimetric portion. Figure 2p shows the linker on the 5'-end connecting the first signal transduction portion and the 3'-end connecting the second signal transduction portion, and the second signal transduction portion is of a different modality from the first signal transduction portion. In each of the embodiments of Figures 2a to 2p showing three signal transduction modalities, only two signal transduction modalities, namely ETM and OSM, can be used. In each of the embodiments of Figures 2a to 2p showing two signal transduction modalities, three signal transduction modalities can be used. In each of the embodiments of Figures 2a to 2p showing three signal transduction modalities, more than three signal transduction modalities can be used. In each of the embodiments of Figures 2a to 2p, each circle, square, or triangle can represent a single or multiple signal transduction portions. [Figure 2-2]Embodiments of the signal probe disclosed herein are shown. In some embodiments, a first signaling portion (shown as three circles) is directed toward, near, or located toward the 5' end of the signal probe, and a second signaling portion of a modality different from the first signaling portion (shown as three squares) is conjugated inside the signal probe, as shown in Figure 2a. In some embodiments, as shown in Figure 2b, a first signaling portion (shown as one circle, but there may be multiple signaling portions) is directed toward, near, or located toward the 5' end of the signal probe, and a second signaling portion of a modality different from the first signaling portion (shown as one square, but there may be multiple signaling portions) is also directed toward, near, or located toward the 5' end of the signal probe. In some embodiments, as shown in Figure 2c, a third signaling portion of a modality different from the first or second signaling portion is also located toward the 5' end of the signal probe. In some embodiments, a first signaling portion (shown as a circle) is directed toward, near, or located toward the 5' end of the signal probe, together with a second signaling portion of a different modality (shown as a square), and in some embodiments, a third signaling portion of a different modality (shown as a triangle) is also directed toward, near, or located toward the 5' end of the signal probe, and the first signaling portion (shown as a circle) is conjugated internally to the signal probe, together with the second signaling portion of a different modality (shown as a square), and in some embodiments, the third signaling portion of a different modality (shown as a triangle), as shown in Figure 2d. In this example, the circle may be the ETM, the square may be the optical signaling portion, and the triangle may be the colorimetric portion.In some embodiments, the first signaling portion (shown as three circles) is located on the 3' end of the signal probe, and the second signaling portion of a modality different from the first signaling portion (shown as three squares) is conjugated inside the signal probe, as shown in Figure 2e. In some embodiments, as shown in Figure 2f, the first signaling portion (shown as one circle) is located on the 3' end of the signal probe, and the second signaling portion of a modality different from the first signaling portion (shown as one square) is also located on the 3' end of the signal probe. In some embodiments, as shown in Figure 2g, the third signaling portion of a modality different from the first or second signaling portion is also located on the 3' end of the signal probe. In some embodiments, a first signaling portion (shown as a circle) is located on the 3' end of the signal probe, along with a second signaling portion (shown as a square) of a different modality than the first signaling portion; in some embodiments, a third signaling portion (shown as a triangle) of a different modality than the first and second signaling portions is located on the 3' end of the signal probe; and the first signaling portion (shown as a circle) is conjugated internally to the signal probe as shown in Figure 2h, along with the second signaling portion (shown as a square) of a different modality than the first signaling portion, and in some embodiments, the third signaling portion of a different modality (shown as a triangle). In this example, the circle may be an ETM, the square may be an optical signaling portion, and the triangle may be a colorimetric portion. In some embodiments, the first signaling portion (shown as three circles) is directed toward, near, or located toward the 5' end of the signal probe, and the second signaling portion of a modality different from the first signaling portion is conjugated toward, near, or located toward the 3' end of the signal probe (Figure 2i).In other words, the probe has two signaling regions, which can be introduced toward, near, or at any location in the 5' end of the sequence or in the middle of the sequence. In Figure 2(a-i), ii is the detection region, which has one or more detection regions, e.g., ferrocene or fluorophores, such as fluorescein / FITC, rhodamine, or Alexa Fluor, and i is the annealing region, e.g., the region that binds to the target analyte. The annealing region may or may not have one or more detection regions. Alternatively, the signal probe may contain extra terminal nucleosides at the ends of the nucleic acid (n+1 or n+2), which are used to covalently attach the signaling regions but do not participate in base pair hybridization during detection. Figure 2j shows an extra terminal nucleoside ligating the signaling region at the 5' end of the signal probe, with a second signaling region, which is a different modality from the first signaling region, conjugated inside the signal probe itself. Figure 2k shows an extra terminal nucleoside at the 5' end of the signal probe that connects the first signaling portion, the second signaling portion of a different modality from the first signaling portion, and the third signaling portion of a different modality from the first and second signaling portions, with the first signaling portion, the second signaling portion of a different modality from the first signaling portion, and the third signaling portion of a different modality from the first and second signaling portions being conjugated inside the signal probe itself. Figure 2l shows an extra terminal nucleoside at the 5' end of the signal probe that connects the first signaling portion, the second signaling portion of a different modality from the first signaling portion, and the third signaling portion of a different modality from the first and second signaling portions. In this example, the circle may be an ETM, the square may be an optical signaling portion, and the triangle may be a colorimetric portion. Alternatively, the primer may contain a linker used to covalently attach the electron transfer portion.Figure 2m shows that a linker connecting the signaling portion at the 5' end of the signal probe and the second signaling portion of a different modality from the first signaling portion is conjugated inside the signal probe itself. Figure 2n shows a linker at the 5' end of the signal probe that connects the first signaling portion, the second signaling portion of a different modality from the first signaling portion, and the third signaling portion of a different modality from the first and second signaling portions, with the first signaling portion, the second signaling portion of a different modality from the first signaling portion, and the third signaling portion of a different modality from the first and second signaling portions being conjugated inside the signal probe itself. Figure 2o shows a linker at the 5' end of the signal probe that connects the first signaling portion, the second signaling portion of a different modality from the first signaling portion, and the third signaling portion of a different modality from the first and second signaling portions. In this example, a circle could be an ETM, a square could be an optical signaling portion, and a triangle could be a colorimetric portion. Figure 2p shows linkers on the 5' end linking the first signaling portion and the 3' end linking the second signaling portion, where the second signaling portion is a different modality from the first signaling portion. In each embodiment of Figures 2a to 2p where three signaling modalities are shown, only two signaling modalities, namely ETM and OSM, may be used. In each embodiment of Figures 2a to 2p where two signaling modalities are shown, three signaling modalities may be used. In each embodiment of Figures 2a to 2p where three signaling modalities are shown, more than three signaling modalities may be used. In each embodiment of Figures 2a to 2p, each circle, square, or triangle may represent one or more signaling portions. [Figure 3a]An embodiment of the system disclosed herein is shown. As seen in Figure 3a, capture probes coupled to detectable labels (shown as circles) can be spotted individually or sequentially at the detection site. As seen in Figure 3b, detectable labels (shown as squares) can be located near the electrode surface. [Figure 3b] An embodiment of the system disclosed herein is shown. As seen in Figure 3a, capture probes coupled to detectable labels (shown as circles) can be spotted individually or sequentially at the detection site. As seen in Figure 3b, detectable labels (shown as squares) can be located near the electrode surface. [Figure 4a]An embodiment of the system disclosed herein is shown. In some cases, it may be beneficial to have a longer amplicon and multiple capture probes coupled to different portions of the amplicon. The method can utilize one or more capture probes on the same detection electrode coupled to different portions of the amplicon / target. In some embodiments, portion (a) of the amplicon / target coupled to a first capture probe (e) does not cross-hybridize with a second capture probe (d) (see Figures 4a and 4b). In some embodiments, portion (b) of the amplicon / target coupled to a first capture probe (d) does not cross-hybridize with a second capture probe (e) (see Figures 4a and 4b). In some embodiments, a second portion (b) of the amplicon / target is coupled to a first capture probe (d) and does not cross-hybridize with a second capture probe (e) coupled to a second portion (a) of the amplicon / target. In some embodiments, a first portion (a) of the amplicon / target can bind to a first capture probe (d) and a second capture probe (e), and a second portion (b) of the amplicon / target cross-hybridizes with a second capture probe (e) and a first capture probe (d). In some embodiments, a first portion (a) of the amplicon / target can bind to a first capture probe (d) and a second capture probe (e), but a second portion (b) of the amplicon / target can bind only to a second capture probe (e). As shown herein, both the first and second signaling portions are bound to signal probes that are coupled to a section (c) of the amplicon. Here, circles represent ETMs, while squares represent fluorescent portions (FMs). Circles and squares represent one or more signaling portions. [Figure 4b]An embodiment of the system disclosed herein is shown. In some cases, it may be beneficial to have a longer amplicon and multiple capture probes coupled to different portions of the amplicon. The method can utilize one or more capture probes on the same detection electrode coupled to different portions of the amplicon / target. In some embodiments, portion (a) of the amplicon / target coupled to a first capture probe (e) does not cross-hybridize with a second capture probe (d) (see Figures 4a and 4b). In some embodiments, portion (b) of the amplicon / target coupled to a first capture probe (d) does not cross-hybridize with a second capture probe (e) (see Figures 4a and 4b). In some embodiments, a second portion (b) of the amplicon / target is coupled to a first capture probe (d) and does not cross-hybridize with a second capture probe (e) coupled to a second portion (a) of the amplicon / target. In some embodiments, a first portion (a) of the amplicon / target can bind to a first capture probe (d) and a second capture probe (e), and a second portion (b) of the amplicon / target cross-hybridizes with a second capture probe (e) and a first capture probe (d). In some embodiments, a first portion (a) of the amplicon / target can bind to a first capture probe (d) and a second capture probe (e), but a second portion (b) of the amplicon / target can bind only to a second capture probe (e). As shown herein, both the first and second signaling portions are bound to signal probes that are coupled to a section (c) of the amplicon. Here, circles represent ETMs, while squares represent fluorescent portions (FMs). Circles and squares represent one or more signaling portions. [Figure 5]A typical assay used herein is shown. In Figure 5, each signal from multiple probes has the same ETM but different optical signaling moieties (shown as squares 1 and 2, but may include three or more optical signaling moieties). In some embodiments, the first optical signaling moiety is fluorescein and the second optical signaling moiety is rhodamine. In some embodiments, the first optical signaling moiety is fluorescein and the second optical signaling moiety is Alexa Fluor 405. [Figure 6] N6 is a mark that can be used, and its synthesis is described in U.S. Patent No. 7,393,645, owned by the same applicant, which is incorporated herein by reference in whole. [Figure 7] This indicates QW56. [Figure 8] QW80 is shown. QW56 and QW80 are essentially ferrocene labels that can be prepared using conventional DNA synthesis techniques, such as those described in application PCT / US08 / 82666 (International Publication No. 2009 / 061941 and U.S. Patent No. 7,820,391), owned by the same applicant, which is incorporated herein by reference in its entirety. U.S. Patent No. 9,891,215 is incorporated herein by reference in its entirety. [Figure 9] A flowchart illustrating a method for correlating signals from electrochemical and optical detection is shown. [Figure 10] This shows optical labeling, or fluorophores, directly labeled onto DNA bound to a capture probe, i.e., without a signal probe. Item 1 is the surface or electrode. [Figure 11] This demonstrates that fluorescence (generated by exciting fluorophores with voltage) is predicted to be dependent on the concentration of fluorophores in the solution. The excitation is detected by an optical detector. [Figure 12] This demonstrates that the hybridization complex can be detected by optical detection after applying a voltage to excite the optical signaling portion. The excitation is detected by an optical detector. [Figure 13] The detection electrode / gold pad exhibits bright-field reflectance and autofluorescence at different emission wavelengths (blue, green, red). [Figure 14] This shows bright-field reflection and far-red (and fusion of both) nucleic acid staining on the electrodes. [Figure 15] This demonstrates that a specific target, in this case influenza A, can be detected on an electrode array in contrast to a negative control. [Figure 16] This demonstrates that a specific target, in this case influenza A, can be differentially detected on the electrode array, where only arrays 10 and 12 have influenza A capture probes and can therefore bind to signal probes having fluorescent markers. [Figure 17] This demonstrates that the distribution of capture probes can be visualized by hybridizing them with a signal probe containing a fluorescent marker. [Modes for carrying out the invention]

[0024] While aspects of the subject matter of this disclosure can be embodied in various forms, the following description and accompanying drawings are intended to disclose only some of these forms as specific examples of the subject matter. Accordingly, the subject matter of this disclosure is not intended to be limited to the forms or embodiments described and illustrated.

[0025] Unless otherwise defined, all technical terms or specialized terms used herein have the same meaning as those generally understood by those skilled in the art to which this disclosure pertains. All patents, applications, published applications and other publications referenced herein are incorporated by reference in their entirety. If any definition set forth in this section contradicts or is inconsistent with any definition set forth in any patent, application, published application or other publication incorporated herein by reference, the definition set forth in this section shall prevail over the definition set forth in any patent, application, published application or other publication incorporated herein by reference.

[0026] Overview A system and method for generating a detectable signal (current) by positioning a fluorophore near an electrode surface are disclosed herein, wherein the excitation of the fluorophore is caused by a voltage, and the excitation of the fluorophore is detected by an optical reader, an electrochemical reader, or both.

[0027] This specification discloses a system and method for generating a detectable signal by positioning an ETM near an electrode surface, wherein the excitation of the ETM is caused by a light / laser beam, and the excitation of the ETM is detected by an optical reader, an electrochemical reader, or both an optical reader and an electrochemical reader.

[0028] Systems and methods for generating detectable signals by positioning fluorophores and ETMs near an electrode surface are disclosed herein, wherein the excitation of the fluorophores and ETMs is caused by voltage or light / laser beam, and the excitation of the fluorophores and ETMs is detected by an optical reader, an electrochemical reader, or both an optical reader and an electrochemical reader.

[0029] Methods for detecting fluorescent (emission, chemiluminescence, phosphorescence) signatures based on excitation from an electrical signal are disclosed herein. Typically, fluorescent or emission signatures are excited by light. Alternatively, in embodiments disclosed herein, electrically excited fluorophores are detected by optical detectors such as photomultiplier tubes (PMTs), charge-coupled devices (CCDs), complementary metal-oxide-semiconductor (CMOS) devices, cameras, microscopes, etc., and / or electrochemical sensors such as ammeters. In some embodiments, the electrically excited optical signaling portion is detected by both optical detectors and electrochemical sensors (such as ammeters).

[0030] This specification discloses a method for detecting ETM (e.g., oxidized moiety, reduced moiety, redox moiety, and / or transition metal complex) signatures based on excitation from a light or laser beam source. Typically, ETM signatures are thought to be excited by current / voltage. Instead, in embodiments disclosed herein, the redox reaction is detected by an optical detector such as a PMT (photomultiplier tube) or CCD (charge-coupled device), camera, microscope, and / or an electrochemical sensor such as an ammeter, and the redox reaction is facilitated by irradiation of the ETM with a light or laser beam.

[0031] The concept of electrical excitation of the fluorescence signaling moiety is a major breakthrough in fluorescence spectroscopy and its applications. The concept of photoexcitation of the ETM is a major breakthrough in electrochemistry and its applications. The combination of electrical excitation of the fluorescence signaling moiety and photoexcitation of the ETM is crucial.

[0032] definition As used herein, “amplification” refers to any in vitro method for increasing the copy number of a nucleotide sequence using polymerase. Nucleic acid amplification results in the incorporation of nucleotides into a nucleic acid molecule (e.g., DNA) or primer, thereby forming a new nucleic acid molecule complementary to the nucleic acid template. The formed nucleic acid molecule and its template can be used as templates for synthesizing further nucleic acid molecules.

[0033] As used herein, “amplicon” refers to a nucleic acid molecule comprising a primer or a portion of a primer and a newly synthesized chain that is a complement to the sequence downstream of the primer binding site.

[0034] As used herein, “analyze” means to measure, detect, or determine the presence or composition of something.

[0035] As used herein, “analyte” refers to an entity capable of selectively binding to a capture-binding ligand. The analyte may be natural, biological, or synthetic, for example, one of the synthetic or other molecules used in drug discovery that exhibits an unusually good or specific binding affinity to the “capture-binding ligand.” Both the analyte and the capture-binding ligand may consist of one or more distinct domains. Those skilled in the art will understand that a complementary orientation between the analyte and the capture-binding ligand is necessary. In one embodiment, the analyte may be environmental pollutants (including insecticides, pesticides, toxins, etc.); chemical substances (including solvents, organic materials, etc.); therapeutic molecules (including therapeutic and abuse drugs, antibiotics, etc.); biomolecules (including hormones, cytokines, proteins, lipids, carbohydrates, cell membrane antigens and receptors (nerve, hormone, nutrient, and cell surface receptors) or their ligands, etc.); whole cells (including prokaryotic cells (including pathogenic bacteria, etc.) and eukaryotic cells (including mammalian tumor cells)); viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, etc.); and spores, etc.

[0036] Suitable nucleic acid target analytes include orthomyxoviruses (e.g., influenza virus), paramyxoviruses (e.g., respiratory syncytial virus, mumps virus, measles virus), adenoviruses, rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g., rubella virus), parvoviruses, poxviruses (e.g., smallpox virus, vaccinia virus), enteroviruses (e.g., poliovirus, coxsackievirus), hepatitis viruses (including types A, B, and C), and herpesviruses (e.g., herpes simplex virus, varicella-zoster virus, cytomegalovirus). Any number of viruses, including Russ, Epstein-Barr virus, rotavirus, Norwalk virus, hantavirus, arenavirus, rhabdovirus (e.g., rabies virus), retrovirus (including HIV, HTLV-I and -II), papovavirus (e.g., papillomavirus), polyomavirus, and picornavirus, etc.), and bacteria (Bacillus; Vibrio, e.g., Vibrio cholerae; Escherichia, e.g., enterotoxin-producing Escherichia coli). E. coli), Shigella (e.g., S. dysenteriae); Salmonella (e.g., Salmonella typhi); Mycobacterium (e.g., Mycobacterium tuberculosis, Mycobacterium leprae); Clostridium (e.g., Clostridium botulinum, Clostridium tetanus, Clostridium difficile, Clostridium perfringens) C. perfringens); Corynebacterium, e.g., diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae; Staphylococcus, e.g., Staphylococcus aureus; Haemophilus, e.g., Haemophilus influenzae (H.This includes, but is not limited to, nucleic acids of a wide variety of pathogenic and non-pathogenic prokaryotes of interest, including influenzae; Neisseria species, e.g., meningitidis, gonorrhoeae; Yersinia species, e.g., Giardia lamblia, Yersinia pestis; Pseudomonas species, e.g., Pseudomonas aeruginosa, P. putida; Chlamydia species, e.g., C. trachomatis; Bordetella species, e.g., Bordetella pertussis; Treponema species, e.g., T. palladium) (collectively, "bacterial and viral targets").

[0037] Suitable nucleic acid target analytes are not limited to, but include: Bacillus cereus group, Bacillus subtilis group, Corynebacterium, Cutibacterium acnes, Propionibacterium acnes, Enterococcus, Enterococcus faecalis, Enterococcus faecium, Lactobacillus, Listeria, Listeria monocytogenes, Micrococcus, and Staphylococcus aureus. Examples include nucleic acids of any number of Gram-positive organisms, including *Streptococcus aureus*, *Staphylococcus epidermidis*, *Staphylococcus lugdunensis*, *Streptococcus*, *Streptococcus agalactiae* (GBS), the *Streptococcus anginosus* group, *Streptococcus pneumoniae*, *Streptococcus pyogenes* (GAS), and resistance genes mecA, mecC, vanA, or vanB (collectively, "Gram-positive targets").

[0038] Suitable nucleic acid target analytes include, but are not limited to, Acinetobacter baumannii, Bacteroides fragilis, Citrobacter, Cronobacter sakazakii, Enterobacter (non-cloacae complex), Enterobacter cloacae complex, Escherichia coli, Fusobacterium nucleatum, Fusobacterium necrophorum, Haemophilus influenzae, Klebsiella oxytoca, and Klebsiella pneumoniae. This includes nucleic acids of any number of Gram-negative organisms, including the pneumoniae group, Morganella morganii, Neisseria meningitidis, Proteus, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella, Serratia, Serratia marcescens, Stenotrophomonas maltophilia, resistance genes, CTX-M, IMP, KPC, NDM, OXA (OXA-23 and OXA-48), or VIM (collectively, "Gram-negative targets").

[0039] Suitable nucleic acid target analytes are not limited to, but include Candida albicans, Candida auris, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus gattii, and Cryptococcus neoformans. This includes nucleic acids of any number of fungi, including neoformans, Fusarium, or Rhodotorula (collectively referred to as "fungal targets").

[0040] In some embodiments, the target is a human-specific infectious pathogen or target, and the marker or target is a nucleic acid marker. In some embodiments, the target is a human disease such as cancer and neurodegenerative disease.

[0041] As used herein, “array” refers to a group of distinct sites having different capture-binding ligands. In some embodiments, an array is “addressable” insofar as the individual sites optionally have predetermined or determinable positions relative to each other, with the help of electronic connectors and / or software.

[0042] As used herein, "autofluorescence" refers to fluorescence that is naturally emitted by biological materials.

[0043] As used herein, “capture-binding ligand” is synonymous with “capture probe” or “capture-binding probe,” and is a compound that exhibits relatively strong or specific affinity to another compound, enabling it to separate that compound from a group of other compounds in a mixture of compounds. A capture-binding ligand may be a protein, carbohydrate, nucleic acid, small molecule, or any combination thereof.

[0044] As used herein, “chemiluminescent label” refers to a portion of a photogenerative reaction that participates in the presence of a triggering factor (here, a voltage applied to an electrode) or a cofactor. Suitable examples of chemiluminescent labels include, but are not limited to, peroxidases, bacterial luciferases, firefly luciferases, functionalized iron-porphyrin derivatives, luminals, isoluminols, acridinium esters, and sulfonamides. A preferred chemiluminescent label is xanthine oxidase using hypoxanthine as a substrate. Triggering agents include perborates, Fe-EDTA complexes, and luminols. The selection of a particular chemiluminescent label depends on several factors, including the cost of preparing the labeled member, the method used for covalent bonding to the detector molecule, and the size of the detector molecule and / or the chemiluminescent label. Correspondingly, the selection of a chemiluminescent triggering agent will depend on the specific chemiluminescent label used.

[0045] As used herein, a "dual-labeled signal probe" comprises a first signaling portion and a second signaling portion, wherein the first and second signaling portions are of different modalities (for example, one signaling portion is an ETM and the other signaling portion is an OSM).

[0046] As used herein, “electrode” refers to a composition that, when connected to an electronic device, can sense an electric charge or voltage and convert it into a signal. Electrodes are known in the art and are not limited to certain metals and their oxides, including gold, platinum, palladium, silicon, and aluminum; metal oxide electrodes, including platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo2O6), tungsten oxide (W03), and ruthenium oxide; and carbon (including glassy carbon electrodes, graphite, graphene, and carbon paste).

[0047] As used herein, “electrochemical detection” refers to the use of at least two electrodes to apply an electric potential and measure the current produced by a chemical reaction. “Electrochemical detection” excludes (i) electrochemiluminescence and (ii) detection of conductivity, impedance, or capacitance of a droplet, a portion of a droplet, or the contents of a droplet by optical means.

[0048] As used herein, “electrochemical sensor” or “electrochemical detector” or “ETM detector” refers to the detection of redox / oxidation reactions and includes potentiometric, current-measuring, conductivity-measuring, and / or impedance-measuring sensors. “Electrochemical detector” (etc.) excludes (i) electrochemical optic detectors and (ii) optical detectors that detect the conductivity, impedance, or capacitance of a droplet, a portion of a droplet, or the contents of a droplet.

[0049] As used herein, the terms “electron donor moiety,” “electron acceptor moiety,” “electron transfer moiety,” and “redox activity label,” or their grammatical equivalents herein, refer to molecules capable of transferring electrons under specific conditions. It should be understood that the electron donor and acceptor capabilities are relative; that is, a molecule that can lose electrons under one experimental condition may accept electrons under a different experimental condition. The number of possible electron donor and acceptor moieties is so large that those skilled in the art of electron transfer compounds can utilize several compounds, and the selection of these compounds is within the scope of the art. One advantage of redox-mediated electron detection is the variety of different electron transfer moiety labels, each having its own distinct potential that can be selectively measured or filtered. Some electron transfer moieties include, but are not limited to, transition metal complexes, organic electron transfer moieties, electrodes, metallocenes such as ferrocene, and ferrocene derivatives, methylene blue, and osmium. Electron transfer moieties further include oxidation moieties, reduction moieties, redox moieties, and / or transition metal complexes. Redox activity labels can be varied in many ways, as can the presence of dyes of different colors and chemically viscous compounds.

[0050] As used herein, "fluorescence" refers to the radiative transition of a molecule from its lowest excited singlet state (S1) to its singlet ground state (S0).

[0051] As used herein, “fluorescent moiety” refers to an electron-containing entity that can absorb photons and enter an excited state for a short time, after which it either disperses energy non-radiatively or emits energy as a photon, but with lower energy, i.e., longer wavelength (wavelength and energy are inversely proportional). Fluorescent moieties as described herein can readily enter an excited state after a voltage is applied to the fluorescent moiety, after which it either disperses energy non-radiatively or emits energy as a photon, but with lower energy, i.e., longer wavelength (wavelength and energy are inversely proportional). Fluorescent moieties include fluorophores, reactive dyes, quantum dots, and fluorescent proteins, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), or cyan fluorescent protein (CFP), and other optical signaling moieties as defined herein.

[0052] As used herein, “fluorophore” may include both exogenous and endogenous fluorophores. A fluorophore (or fluorescent dye, similar to a chromophore) is a fluorescent chemical compound that can re-emit photons when excited (by light or voltage / current). Fluorophores typically consist of several bonded aromatic groups, or planar or cyclic molecules having several π bonds.Representative fluorophores include Alexa Fluor® 350, dansyl chloride (DNS-Cl), 5-(iodoacetamida)fluoroceine (5-IAF); fluoroceine 5-isothiocyanate (FITC), tetramethylrhodamine 5-(and 6-)isothiocyanate (TRITC), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), 7-nitrobenzo-2-oxa-1,3-diazole-4-yl chloride (NBD-Cl), ethidium bromide, Lucifer Yellow, 5-carboxyrhodamine 6G hydrochloride, lysamine rhodamine B sulfonyl chloride, and Texas Red (trademark), sulfonyl chloride, BODIPY (trademark), naphthalamine sulfonic acid including but not limited to 1-anilinonaphthalene-8-sulfonic acid (ANS) and 6-(p-toluidinyl)naphthalene-2-sulfonic acid (TNS), anthroyl fatty acid, DPH, parinal acid, TMA-DPH, fluorenyl fatty acid, fluorescein-phosphatidylethanolamine, Texas Red-phosphatidylethanolamine, pyrenyl-phosphatidylcholine, fluorenyl-phosphotidylcholine, merocyanine 540, 1-(3-sulfonatopropyl)-4-[-β-[2 [(di-n-butylamino)-6-naphthyl]vinyl]pyridinium betaine (naphthylstyryl), 3,3'-dipropylthiadicarbocyanine (di-C3-(5)), 4-(p-dipentylaminostyryl)-1-methylpyridinium (di-5-ASP), Cy-3 iodoacetamide, Cy-5-N-hydroxysuccinimide, Cy-7-isothiocyanate, Rhodamine 800, IR-125, thiazole orange, azure B, Nile blue, Al phthalosia Examples include, but are not limited to, nin, oxaxine 1, 4',6-diamidino-2-phenylindole (DAPI), Hoechst33342, TOTO, acridine orange, ethidium homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinolinium (MQAE), Fura-2, calcium green, carboxy SNARF-6, BAPTA, coumarin, phytofluor, coronene, green fluorescent protein, and metal-ligand complexes.

[0053] Representative endogenous fluorophores include, but are not limited to, organic compounds having aromatic ring structures, including, NADH, FAD, tyrosine, tryptophan, purines, pyrimidines, lipids, fatty acids, nucleic acids, nucleotides, nucleosides, amino acids, proteins, peptides, DNA, RNA, sugars, and vitamins. Further suitable fluorophores include enzyme cofactors; lanthanides, green fluorescent proteins, yellow fluorescent proteins, red fluorescent proteins, or their variants and derivatives.

[0054] As used herein, the terms “hybridization” and “hybridize” refer to the pairing of two complementary single-stranded nucleic acid molecules (RNA and / or DNA) that give rise to a double-stranded molecule. As used herein, two nucleic acid molecules can hybridize, but their base pairings are not perfectly complementary. Therefore, mismatched bases do not prevent the hybridization of two nucleic acid molecules if appropriate conditions known in the art are used.

[0055] As used herein, the term “to immobilize” or its derivatives includes affixation, association, or bonding, whether covalent or noncovalent.

[0056] As used herein, the term “label” refers to an entity that signals or can be stimulated to signal the presence of an event or a molecule or complex of molecules. Labels may include, for example, dyes, radioactive atoms or molecules, redox-active compounds, enzymes, enzyme substrates, nucleic acids, and their derivatives. Labels may be immobilized on a detector molecule (signal probe) or a capture molecule.

[0057] As used herein, “light” is used in its broadest sense, meaning electromagnetic radiation, which propagates through space and includes not only visible light but also infrared and ultraviolet radiation.

[0058] As used herein, the terms “monolayer,” “self-assembled monolayer,” or “SAM” mean a relatively ordered assembly of molecules spontaneously chemisorbed onto a surface, where the molecules are oriented nearly parallel to each other and nearly perpendicular to the surface. Each molecule includes a functional group that adheres to the surface and a portion that interacts with adjacent molecules in the monolayer to form a relatively regular arrangement. A “mixed” monolayer includes a heterogeneous monolayer, i.e., one in which at least two different molecules constitute the monolayer.

[0059] As used herein, the terms “multimodal” or “different modalities” refer to a system incorporating two or more modalities / techniques. Multimodal detection may be defined as any combination of electrochemical, optical, fluorescence, surface plasmon resonance, nanoplasmonic sensors, biolayer interferometry, chemiluminescence, spectroscopy, or a combination of two or more of these colorimetric detections. Signals may be generated and detected by signal generation, either by conventional methods of each technique or by one of the other modalities, and detected by a single detector. For example, a multimodal system may combine optical, electrochemical, or radioactive labeling or detectors. Those skilled in the art will understand that optical detection / signaling / imaging is a different modality compared to electrochemical detection / signaling / imaging.

[0060] As used herein, “optical detection” refers to the recognition of an object via the electromagnetic spectrum, including but not limited to the visible and / or ultraviolet and / or infrared portions of the electromagnetic spectrum.

[0061] As used herein, “optical signaling molecule,” “optical signaling moiety,” or “excited molecule” refers to electromagnetic signals; fluorophores, quantum dots (Q dots); autofluorescence; chemiluminescent alkaline phosphatases and other chemiluminescent labels; fluorospheres, i.e., fluorosspheres and transfluospheres; polymer beads doped with one or more fluorescent labels; fluorescent microspheres; silicon nanoparticles; silica and silicate-doped materials; semiconductor materials; E-type fluorescent emitters; P-type fluorescent emitters; Fluo-3 and Fluo-4 calcium indicators; calcium green indicators; fluorinine zinc indicators; Phen Green for detection of a wide range of ions including Cu2+, Cu+, etc.; Newport Green for detection of Zn2+; lead and cadmium green dyes for measurement of lead and cadmium; magnesium green for electrical detection of free magnesium; Mag-fluo-2 and Mag-indo-1 for magnesium detection; Mag-fluo-4 for detection of both calcium and magnesium in both free solution and intercellular environments; phycobiliproteins (many different forms); buckyballs, C 60etc.; carbon nanotubes; cardiolian green / indocyanine green fluorescent indicators; metal colloids and mixed metal colloids such as Ag, Au, Pt, Fe, Pd, Cu, Zn, Rh, Cr, Pb; pH indicators such as SNARF-1, SNARF-4F, SNARF-5F, dextran BCECF, etc.; 6-chloro-9-nitro-5-oxo-5H-benzo(a)phenoxazine (CNOB) for detection of nitroreductase and nitrate reductase activity; SYTOX dead cell stains such as SYTOX Blue, green, orange, red, etc.; DAPI and propidium iodide labeling; ethidium bromide, Picogreen, Syber This may include, but is not limited to, probes for double-stranded DNA detection such as green; dyes in the Alexa fluorophore range; BODIPY and related structural dyes; cell lamps and organelle lamps (genetically encoded proteins); green fluorescent protein (GFP) and its analogues; coumarin dyes; prodan and related structural dyes; voltage-sensitive probes such as DisBAC4(3) and CC2-DMPE; and / or Ncode miRNA-labeled fluorophores.

[0062] As used herein, “optical biosensor,” “optical sensor,” or “optical detector” typically has the ability to detect light in a specific range of the electromagnetic spectrum (ultraviolet, visible, and infrared). The sensor detects either the wavelength, frequency, or polarization of light and converts it into an electrical signal by the photoelectric effect. Optical detectors include fluorescent biosensors, colorimetric biosensors, surface-enhanced Raman scattering (SERS) biosensors, surface plasmon resonance (SPR), photonic crystal-based, optical resonator-based, optical fiber-based, or optical wavelength-based sensors. Optical detectors convert optical signals into electrical signals. Optical sensors typically have the ability to detect light in a specific range of the electromagnetic spectrum (ultraviolet, visible, and infrared). The sensor detects either the wavelength, frequency, or polarization of light and converts it into an electrical signal by the photoelectric effect. “Optical detector” (etc.) excludes electrochemical detectors.

[0063] As used herein, “nucleic acid” or “oligonucleotide” or its grammatical equivalent means at least two nucleotides covalently linked. Nucleic acids generally contain phosphodiester bonds, but in some cases include nucleic acid analogs that may have alternative skeletons, such as phosphoramides, as outlined below. Nucleic acids can be DNA, both genome and cDNA, RNA, or hybrids, and include any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, such as uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, isoguanine, etc. As used herein, the term “nucleoside” includes nucleotides as well as nucleosides and nucleotide analogs, as well as modified nucleosides such as amino-modified nucleosides. Furthermore, “nucleoside” includes analog structures that do not exist in nature. Thus, for example, individual units of peptide nucleic acids, each containing a base, are referred to herein as nucleosides.

[0064] As used herein, the term “nucleotide” means, as used herein, a nucleoside-5'-oligophosphate compound or a structural analog of a nucleoside-5'-oligophosphate that can act as a substrate or inhibitor of nucleic acid polymerase. Exemplary nucleotides include, but are not limited to, nucleoside-5'-triphosphates (e.g., dATP, dCTP, dGTP, dTTP, and dUTP); nucleosides having a 5'-oligophosphate chain that is a phosphate of length 4 or more (e.g., 5'-tetraphosphorate, 5'-pentaphosphorate, 5'-hexaphosphorate, 5'-heptaphosphorate, 5'-octaphosphorate) (e.g., dA, dC, dG, dT, and dU); and structural analogues of nucleoside-5'-triphosphates that may have a modified base moiety (e.g., a substituted purine or pyrimidine base), a modified sugar moiety (e.g., an O-alkylated sugar), and / or a modified oligophosphate moiety (e.g., an oligophosphate including a thiophosphate, methylene, and / or other crosslinks between phosphates).

[0065] As used herein, “polymerase” refers to an enzyme that catalyzes the replication process of nucleic acids. More specifically, DNA polymerase catalyzes the polymerization of deoxyribonucleotides together with the DNA strand that it “reads” and uses as a template. The newly polymerized molecule is complementary to the template strand and identical to the template partner strand.Polymerases (including DNA polymerases and RNA polymerases) include, but are not limited to, Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotoga maritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT®) DNA polymerase, Pyrococcus furious (Pflu) DNA polymerase, DEEPVENT DNA polymerase, Pyrococcus woosii (Pwo) DNA polymerase, and Bacillus stearthermophilus. sterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca) DNA polymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNA polymerase, Thermus flavus (Tfl / Tub) DNA polymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus (DYNAZYME) DNA polymerase, Methanobacterium thermoautotrophum Examples include thermoautotrophicum (Mth) DNA polymerase, mycobacterium DNA polymerase (Mtb.Mlep), and their mutants, variants, and derivatives. RNA polymerases such as T3, T5, and SP6, and their mutants, variants, and derivatives may also be used.

[0066] As used herein, the term “primer” refers to an oligonucleotide, whether natural or synthetic, that, when combined with a polynucleotide template to form a double helix, acts as a starting point for nucleic acid synthesis and can be extended along the template from its 3' end to form an extended double helix. Primer extension is typically carried out using a nucleic acid polymerase, such as DNA polymerase or RNA polymerase. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Primers are typically extended by DNA polymerase. Primers typically have lengths ranging from 14 to 40 nucleotides or from 18 to 36 nucleotides. Primers are used in various nucleic acid amplification reactions, such as linear amplification reactions using a single primer or polymerase chain reactions using two or more primers. Guidelines for selecting primer lengths and sequences for specific applications are well known to those skilled in the art, as evidenced by the following references, which are incorporated herein by reference in their entirety: Dieffenbach, editor, PCR Primer: A Laboratory Manual, 2 nd Edition (Cold Spring Harbor Press, New York, 2003). As used herein, “probe” refers to a synthetic or biologically produced nucleic acid (DNA or RNA) containing a specific nucleotide sequence that, by design or selection, enables specific (i.e., preferential) hybridization to a target nucleic acid sequence under a given stringency. Like primers, probes typically have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not so long as to degrade fidelity during synthesis. Oligonucleotide probes are generally 15 to 30 nucleotides long (e.g., 16, 18, 20, 21, 22, 23, 24, or 25).

[0067] A primer (and / or probe) as defined herein may be chemically modified, i.e., a primer and / or probe may contain a modified nucleotide or a non-nucleotide compound. Thus, the probe (or primer) is a modified oligonucleotide. A “modified nucleotide” (or “nucleotide analog”) is different from a natural “nucleotide” by some modification, but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or phosphate-like moiety, or a combination thereof. For example, a “label” can be attached to the base portion of a “nucleotide” to obtain a “modified nucleotide.” The natural base in a “nucleotide” can also be replaced, for example, with 7-desazapurine, to similarly obtain a “modified nucleotide.” The terms “modified nucleotide” and “nucleotide analog” are used interchangeably in this application. A “modified nucleoside” (or “nucleoside analog”) is different from a natural nucleoside by some modification, as outlined above for “modified nucleotide” (or “nucleotide analog”).

[0068] As used herein, “redox-active” compound or part means one that can transfer, return, or receive electrons from another redox-active compound or electrode. Some redox-active compounds include ferrocene and its derivatives, electrodes containing methylene blue or osmium, and metallocenes.

[0069] As used herein, terms such as “sample” or “sample solution” refer to any sample containing biomolecules (such as proteins, peptides, nucleic acids, lipids, carbohydrates, or combinations thereof) obtained from any organism, including viruses. Other examples of organisms include mammals (veterinary animals such as humans, cats, dogs, horses, cattle, and pigs, as well as laboratory animals such as mice, rats, and primates), insects, annelids, arachnids, marsupials, reptiles, amphibians, bacteria, and fungi. Biological samples include tissue samples (such as tissue sections and needle biopsies), cell samples (such as cytological smears, like Pap smears or blood smears, or cell samples obtained by microdissection), or cell fractions, fragments, or organelles (such as those obtained by lysing cells and separating their components by centrifugation). Other examples of biological samples include blood, serum, urine, semen, feces, cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus, biopsy tissue (e.g., obtained by surgical biopsy or needle biopsy), nipple aspirate, earwax, milk, vaginal fluid, saliva, swabs (such as oral swabs), or any material containing biomolecules derived from the initial biological sample. In certain embodiments, the term “biological sample” as used herein refers to a sample (such as a homogenized or liquefied sample) prepared from a tumor or a portion thereof obtained from the subject.

[0070] As used herein, “signal probe” means a probe molecule having at least one label of some kind that can bind to an analyte and signal the presence of the analyte.

[0071] As used herein, “sensor” is an object that detects signals from its surrounding environment and converts them into meaningful or quantifiable information.

[0072] As used herein, “solid support” or “support” refers to any material or matrix suitable for attaching oligonucleotides / capture probes. Such oligonucleotides and / or capture probes may be attached to or bound to the support (covalently or non-covalently) by any technique or any combination of techniques known in the art. The support may be anything other than an aqueous phase at room temperature and may include, for example, beads, gels, columns, column matrices, multi-titer plates, paper, membranes, printed circuit boards, or other array surfaces or supports.

[0073] As used herein, the terms “target analyte,” “target nucleic acid,” or “target,” or their grammatical equivalents, refer to the nucleic acid sequence to be amplified or detected. These include any of the following strands: the original nucleic acid sequence to be amplified, its complementary second strand, and the copy of the original sequence produced by replication or amplification.

[0074] The target sequence may be part of a gene, regulatory sequence, genomic DNA, cDNA, mRNA, and RNA including rRNA. Under the understanding that longer sequences are more specific, it can be of any length. As will be understood by those skilled in the art, complementary target sequences can take many forms. For example, it may be contained within a larger nucleic acid sequence, i.e., in all or part of a gene or mRNA, or within a restriction fragment of a plasmid or genomic DNA. The presence or absence of the target sequence in a sample is determined by hybridizing the probe to the target sequence, as will be more thoroughly outlined below. The target sequence may also consist of different target domains; for example, the first target domain of the sample target sequence may hybridize to a capture probe or part of a capture probe, and the second target domain may hybridize to part of a different capture probe. The target domains may be adjacent or separated. The terms “first” and “second” do not imply any orientation of the sequence relative to the 5'-3' orientation of the target sequence. For example, assuming a 5'-3' orientation for complementary target sequences, the first target domain could be located either 5' relative to the second domain or 3' relative to the second domain. A target refers to a nucleic acid molecule that a particular primer or probe can preferentially hybridize.

[0075] As used herein, “target sequence” or “target analyte” refers to a nucleic acid sequence within a target molecule that a particular primer or probe can preferentially hybridize.

[0076] As used herein, the term “template” as used herein refers to a double-stranded or single-stranded molecule that is amplified, synthesized, or sequenced.

[0077] As used herein, "transducer" is a general device for converting energy from one form to another. Electrochemical sensors and optical sensors are types of transducers.

[0078] The terms used herein for recombinant DNA technology and other terms used in the fields of molecular biology and cell biology will generally be understood by those skilled in the art in the applicable field.

[0079] General explanation This disclosure provides systems and methods for nucleic acid detection. In some embodiments, the device includes an electrochemical detector, an optical detector, or both an electrochemical and an optical detector. In some embodiments, the systems of this disclosure may correspond to electrical excitation of an optical signaling portion rather than excitation of an optical signaling portion via light (wavelength). In other embodiments, the systems of this disclosure may correspond to optical (wavelength) excitation of an ETM rather than excitation of an ETM via voltage.

[0080] This disclosure also provides one or more reagents, such as one or more reagents suitable for detecting one or more target nucleic acids in a sample. In some embodiments, the reagents include signal probes, such as signal probes comprising an ETM (e.g., ferrocene, a derivative of ferrocene, or methylene blue) and an OSM or optical label (e.g., fluorescein).

[0081] The Disclosure also provides a method for detecting one or more target nucleic acid molecules in a sample, comprising hybridizing a target nucleic acid molecule (e.g., DNA or a fragment of DNA) to a signal probe, for example, a signal probe having an electron transfer portion (ETM) and an optical signaling portion (OSM), in order to form a signal probe-target complex. In some embodiments, the signal probe-target complex is hybridized to a capture probe attached to an electrode surface. In some embodiments, the signal probe-target complex is not hybridized to a capture probe attached to an electrode surface. In some embodiments, the method further comprises applying a voltage to the system and detecting signals from the ETM and OSM. In some embodiments, the voltage excites the ETM but not the OSM. In some embodiments, the voltage excites both the ETM and the OSM.

[0082] In some embodiments, electrically excited ETMs are detected by an electrochemical detector, and electrically excited OSMs are detected by an electrochemical detector. In some embodiments, electrically excited ETMs are detected by an electrochemical detector, and electrically excited OSMs are detected by an optical detector. In some embodiments, electrically excited ETMs are detected by an optical detector, and electrically excited OSMs are detected by an optical detector. In some embodiments, a wavelength of light is applied to the system, and signals from the ETMs and OSMs are detected. In some embodiments, the wavelength excites the optical signaling portion but not the electron transfer portion. In some embodiments, the wavelength excites both the electron transfer portion and the optical signaling portion. In some embodiments, photo-excited ETMs are detected by an electrochemical detector, and photo-excited OSMs are detected by an electrochemical detector. In some embodiments, photo-excited ETMs are detected by an electrochemical detector, and photo-excited OSMs are detected by an optical detector. In some embodiments, photo-excited ETMs are detected by an optical detector, and photo-excited OSMs are detected by an optical detector.

[0083] In contrast to signal probes that include two signaling portions using the same modality, the signal probes of this disclosure use a first signaling portion that can be detected by a first detection modality and a second signaling portion that can be detected by a second detection modality, where the first and second detection modalities are different (e.g., electrochemical detection as the first detection modality and optical detection as the second detection modality). The applicant considers this difference important because, prior to this disclosure, it was not known whether an electrochemical signal could be detected in the presence of an optical / fluorescent signal. Similarly, the applicant considers it not known whether an optical / fluorescent signal could be detected in the presence of an electrochemical signal. The applicant further considers it not known whether an optical / fluorescent signal inhibits an electrochemical signal or vice versa. This disclosure provides a multiplexed clinical diagnostic device that utilizes a combination of two different modalities that are known to be complementary to each other.

[0084] Some embodiments of this disclosure do not use two excitation methods. In some embodiments, a single excitation modality, such as electricity or light, is used to excite both the ETM and the OSM. In some embodiments, a single excitation method is used, but two or more different detection modalities are used, namely, a first detection modality (such as having a first detector) for a first signaling portion (e.g., the ETM) and a second detection modality (such as having a second detection tree, such as an optical detector) for a second optical signaling portion. In some embodiments, a single excitation method is used, and a single detection modality is used (e.g., a first detector for the ETM and the optical signaling portion).

[0085] In some embodiments, two excitation methods are used, namely, electricity is used to excite the ETM and light is used to excite the FM. In some embodiments, two excitation methods are used and two or more detection modalities are used, namely, a first detector is used for the ETM and a second optical detector is used for the second optical signaling portion. In some embodiments, two excitation methods are used and a single detection modality is used, namely, the first detector is used for both the ETM and the optical signaling portion.

[0086] In some embodiments, the optical label is detected directly from the PCR product (i.e., not hybridized to a signal probe or capture probe), and the ETM is then detected by hybridization to a capture probe and application of voltage through an electrode.

[0087] When developing an assay utilizing a sandwich assay (a capture probe bound to an amplicon bound to a signal probe), it can be difficult to find a segment of the target organism sequence that is highly conserved (i.e., detectable across many variants) but uniquely identifies the target organism of interest. The highly conserved but unique identifier must be long enough to allow both capture and signal probe binding, and this becomes even more complex if the signal probe has two signaling segments instead of one.

[0088] General background on the value of nucleic acid testing Labeling probes with ferrocene provides a relatively sensitive means to facilitate the detection of probe hybridization. Several patents deal with the electrochemical detection of nucleic acids; for example, U.S. Patent No. 10,001,476 (which is incorporated in its entirety by reference) discloses detection by various capture and signal probe configurations and combinations of configurations that can facilitate accurate and efficient multiplex analyte detection. U.S. Patent No. 10,001,476 discloses single-stranded DNA on an electrode (capture probe) that binds to a target. Such a system requires that the ferrocene label be held in close proximity to the electrode for it to function. Sandwich assays have been used to achieve proper orientation of the ferrocene label relative to the electrode. As mentioned above, sandwich assays require an amplicon that can bind to both the capture probe and the signal probe. The development of probes that can bind to both the capture probe and the signal probe is particularly complex when the probe must hold two signaling regions, rather than one, of different modalities, at proper orientation and distance from the electrodes that are excited and detected by their respective detectors. In a multiplex response, the system must also be able to distinguish between two signals of the same modality (and other signals of different modalities on the pad).

[0089] Electrochemical detection The systems and methods of the present disclosure include forming one or more hybridization complexes on a detection electrode under conditions in which a signal probe binds to a target nucleic acid. The hybridization complex of the signal probe and the target nucleic acid then binds to a capture probe and can hold two different signaling moieties in close proximity to the detection electrode for excitation and detection. In some embodiments, the signal probe comprises two different signaling moieties, each of which has a different detection modality ("dual-labeled signal probe"). In some embodiments, the two different signaling moieties may be excited using a single detection method and detected using two different detectors having different modalities, or in some embodiments, using a single detector. In some embodiments, the first signaling moiety of the two different signaling moieties is an ETM, and the second signaling moiety of the two different signaling moieties is optically labeled or an OSM.

[0090] In some embodiments, a dual-labeled signal probe can be electrically excited by the application of a voltage to generate a first signal of a detectable wavelength and a second signal that moves electrons to the electrode. In some embodiments, a dual-labeled signal probe can be optically excited by the application (i.e., irradiation) of light or a laser beam to generate a first signal of a detectable wavelength and a second signal that moves electrons to the electrode. In some embodiments, a dual-labeled signal probe not coupled to a capture probe diffuses outward from the electrode when the voltage / current is turned on and not detected. In some embodiments, the signal probe generates a measurable current when a first amperometric potential is applied.

[0091] In some embodiments, when multiple capture probes are used, each is specific to a different portion of the common nucleic acid sequence of interest. In some embodiments, when multiple capture probes are used, each is specific to a different target analyte of interest. In some embodiments, the nucleic acid sequence of interest may not be amplified by diffusion, or the nucleic acid may be amplified by, for example, PCR. In some embodiments, the electrode has a self-assembled monolayer ("SAM"). In some embodiments, the electrode has a mixture of two or more species of SAMs, each characterized by different chain lengths, number of conjugated bonds (if present), and / or substituents (if present).

[0092] In some embodiments, the first signaling portion of the dual-labeled signal probe is ferrocene or a derivative of ferrocene. In some embodiments, the ferrocene or ferrocene derivative is selected from N6, QW56, and / or QW80. See Figures 6, 7, and 8. In some embodiments, the ferrocene or ferrocene derivative is detected electrochemically, optically, or both electrochemically and optically. In some embodiments, the optical label of the dual-labeled signal probe may be detected electrochemically, optically, or both and optically.

[0093] In some embodiments, the captured oligonucleotides are immobilized on a gold surface. In some embodiments, the captured oligonucleotides are immobilized on carbon-based electrodes such as graphite, graphene, soft and hard carbon, or nanocarbon. In other embodiments, the captured oligonucleotides are immobilized on an electrode. In some embodiments, the electrode comprises an insulating self-assembled monolayer or a mixed monolayer.

[0094] In some embodiments, the captured oligonucleotides are immobilized on a substrate. In some embodiments, the substrate includes a printed circuit board (PCB). Generally, suitable substrates include, but are not limited to, glass fibers, Teflon®, ceramics, glass, silicon, mica, plastics (including acrylic, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polycarbonate, polyurethane, Teflon®, and their derivatives), GETEK (a blend of polypropylene oxide and glass fibers), etc.

[0095] In some embodiments, the detection chamber and electrodes are part of a cartridge that can be placed within a device containing electronic components (such as an AC / DC voltage source, ammeter, processor, readout display, temperature controller, light source, fluorescence detector, etc.). In this embodiment, the interconnections from each electrode are arranged such that when the cartridge is inserted into the device, connections are established between the electrodes and the electronic components.

[0096] Techniques for electrochemical detection are known to those skilled in the art. Generally, at least a first input signal (AC / DC voltage) is applied to the analytic complex, and an output signal (in units such as nanoamperes, uA, mA, etc.) is received. The output signal is then processed to detect the presence of the target analyte. Some embodiments utilize multiple assay hybridization complexes attached to different cells or pads of a detection array, respectively. This basic mechanism is described in U.S. Patents 5,591,578, 5,770,369, 5,705,348 and PCT US97 / 20014 (all incorporated herein by reference).

[0097] Optical detection Techniques for optical detection are known to those skilled in the art. Generally, at least a first input signal (light or laser beam) is applied to an assay complex, and an output signal (fluorescence) is received. The output signal is then processed to detect the presence of the target analyte. Conventional fluorescence analyzers include an optical unit for irradiating a sample with light and a detection unit for detecting the light emitted from the sample. The optical unit generally includes a light source, a dichroic mirror, an objective lens, and a sample holder. The detection unit includes an optical detector, such as a photomultiplier tube (PMT), and a filter that transmits light of a specific wavelength. The light source may include a variety of light sources such as halogen lamps, light-emitting diodes (LEDs), and lasers. Light emitted from the light source is reflected by the dichroic mirror and partially absorbed by the sample. Light emitted from the sample passes through the dichroic mirror and enters the detection unit. The light that enters the detection unit passes through the filter and therefore has a specific wavelength, and the optical detector detects the intensity of light of that specific wavelength. By changing the wavelength characteristics of a filter or light source, the analyte of a sample can be identified by analyzing the intensity of fluorescence emitted from the sample.

[0098] Nucleic acid testing using a single-use device Exemplary methods for nucleic acid testing are described herein. While various elements (steps) are described as a sequence of steps in a predetermined order, it should be understood that the processes are illustrative and not intended to be limiting. Those skilled in the art will recognize that many of the various elements (steps) can be performed in a different order than described herein, simultaneously or substantially simultaneously with other elements (steps), or can be omitted entirely. Therefore, the order of the elements (steps) described is not limiting.

[0099] Step 1: Load the sample. Step 2: Extract the DNA. Step 3: Combine the DNA with the amplification reagent. Step 4: Amplify the DNA to generate a double-stranded amplicon. Step 5: The double-stranded amplicon is incubated with exonuclease to form a single-stranded amplicon. Step 6: Combine the single-stranded amplicon with the signal probe. The signal probe has a first signaling portion and a second signaling portion, and the first and second signaling portions are of different modalities, namely electrochemical and optical, and form a signal probe-amplicon complex. Step 7: Combine the signal probe-amplicon complex with the capture probe. Step 8: The first signaling portion and the second signaling portion are excited with a voltage (AC / DC voltage). Step 9: The first signal transduction portion is detected by electrical sensor detection, and the second signal transduction portion is detected by optical detection. Step 10: Correlate the detected electrical sensor signal with the detected optical signal. Step 11: Generate detection results based on correlated electrical sensor signals and optical signals.

[0100] In some embodiments, before the electrical sensor signal and the optical signal are correlated, the electrical sensor signal and / or optical signal are processed by removing a subset of the signal or by filtering the signal. In some embodiments, the detection result is based on the correlated signal, the electrical sensor signal, and the optical signal. In some embodiments, the detection result is based on the correlated signal only. In some embodiments, the detection result is based on the correlated signal and either the electrical sensor signal or the optical signal.

[0101] In some embodiments, the excitation of a first signaling portion and a second signaling portion by voltage includes applying a first voltage to excite the first signaling portion and applying a second voltage to excite the second signaling portion. In some embodiments, the excitation of a first signaling portion and a second signaling portion by voltage includes applying a first voltage to excite the first signaling portion and a second signaling portion.

[0102] In some embodiments, the capture probe includes a third signaling portion, which is of the same type as the second signaling portion (i.e., a detection modality), and for example, both the second and third signaling portions are optical signaling portions.

[0103] In some embodiments, a first signal probe comprises two distinct signaling segments having different detection modalities (e.g., ETM and OSM), and a second signal probe comprises two distinct signaling segments having different detection modalities (e.g., ETM and OSM), and the signal from at least one of the two distinct signaling modalities of the first signal probe can be distinguished from the signal from at least one of the two distinct signaling modalities of the second signal probe (e.g., the two different OSMs may be distinguishable from each other).

[0104] In some embodiments, the first signaling portion is a ferrocene tag. The second signaling portion is an optical tag, and the first and second detectable portions undergo a detectable change in one or more properties upon application of voltage (or light). Non-limiting examples of the one or more properties that may change include signal intensity, electrochemical potential, and / or reaction constants.

[0105] In some embodiments, detection is performed without amplification, i.e., steps 3 and 4 are omitted. In some embodiments, detection is performed without a signal probe, i.e., the fluorescently labeled nucleic acid binds directly to the capture probe, i.e., step 6 is omitted. In some embodiments, the signal probe has only an OSM excited by the application of voltage (no ETM). In some embodiments, the signal probe has only an OSM (no ETM), and the OSM signal is detected by an electrochemical detector. In some embodiments, the signal probe has only an ETM excited by the application of light or a laser beam (no OSM). In some embodiments, the signal probe has only an ETM (no OSM), and the ETM signal is detected by an OSM detector. In some embodiments, in step 9, detection by an electrosensor detects the first signaling portion and the second signaling portion (no optical detection). In some embodiments, in step 9, detection by an optical detector detects the first signaling portion and the second signaling portion (no electrochemical detection).

[0106] PCR general Conventional PCR techniques are disclosed in U.S. Patents 4,683,202, 4,683,195, 4,800,159, and 4,965,188, the disclosures of which are incorporated herein by reference in their entirety. U.S. Patents 5,210,015, 5,487,972, 5,804,375, 5,804,375, 6,214,979, and 7,141,377 disclose real-time PCR and TaqMan® techniques, the disclosures of which are incorporated herein by reference in their entirety.

[0107] Polymerase chain reaction (PCR) is a relatively simple technique for amplifying a DNA template to produce a specific DNA fragment in vitro. A typical amplification reaction includes target DNA, thermostable DNA polymerase, two oligonucleotide primers (5' and 3'), deoxynucleotide triphosphate (dNTPs), reaction buffer, and magnesium chloride. Each cycle of PCR involves steps for template denaturation, primer annealing, and primer extension. The first step is to denaturate the target DNA by heating it. In the denaturation process, the two entangled DNA strands separate from each other, producing a single-stranded DNA template necessary for replication by thermostable DNA polymerase. In the next step of the cycle, the oligonucleotide primers form a stable association (annealing) with the denatured target DNA and the temperature is lowered so that they can function as primers for DNA polymerase. Finally, the synthesis of new DNA begins. An enzyme called "Taq polymerase" uses the original strand as a template to synthesize (construct) two new strands of DNA. This process results in a replication of the original DNA, with each new molecule containing one old strand and one new strand of DNA. Each of these strands can then be used to create two new copies, and so on. The cycle of denaturing and synthesizing new DNA is repeated 30 or even 40 times, resulting in over a billion exact copies of the original DNA segment. The PCR cycling process is typically automated using a thermocycler programmed to change the reaction temperature to allow for DNA denaturation and synthesis.

[0108] The amplicons disclosed herein can be generated by any amplification reaction, including PCR, 5-RACE, anchored PCR, "one-sided PCR," LCR, NASBA, SDA, RT-PCR, real-time PCR, quantitative PCR, quantitative RT-PCR, and other amplification systems known in the art. In some embodiments, the reaction is carried out at a single temperature (isothermal). These isothermal methods include cycling probe reactions, chain substitution, Invader®, SNPase, rolling circle reactions, and NASBA.

[0109] In some embodiments, in the absence of nucleic acid synthesis, there should be little to no electron transfer between the signal primer and the electrode. In some embodiments, the system binds the target analyte to the signal probe and is sensitive enough that amplification is not required to detect it.

[0110] Förster Resonance Energy Transfer (FRET) FRET technology (see, for example, U.S. Patents 4,996,143, 5,565,322, 5,849,489, and 6,162,603, each incorporated herein by reference) is based on the concept that when a donor fluorescence moiety and a corresponding acceptor fluorescence moiety are placed within a certain distance of each other, energy transfer occurs between two fluorescence moieties that can be visualized or otherwise detected and / or quantified. Typically, the donor, when excited by light emission of a suitable wavelength, transfers energy to the acceptor. Typically, the acceptor re-emits the transferred energy in the form of light emission of a different wavelength.

[0111] In one example, an oligonucleotide probe may contain a donor fluorescent moiety and a corresponding quencher, where the donor FM is excited by voltage, dissipating energy transferred in a form other than light or energy transferred in the form of wavelength. If the probe is intact, energy transfer typically occurs between the two fluorescent moieties, resulting in the quenching of fluorescence emission from the donor fluorescent moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplified product is cleaved, for example, by the 5'-to-3' exonuclease activity of Taq polymerase, so that the fluorescence emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Patents 5,210,015, 5,994,056, and 6,171,785, each incorporated by reference. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quenchers® (BHQ), (Biosearch Technologies, Inc., Novato, California), Iowa Black®, (Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry® Quencher 650 (BBQ-650), (Berry & Assoc., Dexter, Michigan).

[0112] Fluorescence analysis can be performed using, for example, a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters for monitoring fluorescence emission in a specific range), a photon-counting photomultiplier tube system, or a fluorophotometer. Excitation to initiate energy transfer can be performed using an argon-ion laser, a high-intensity mercury (Hg) arc lamp, an optical fiber light source, or other high-intensity light sources appropriately filtered for excitation in the desired range.

[0113] Fluorescent donor and corresponding acceptor portions are generally selected for (a) highly efficient Foerster energy transfer, (b) a large final Stokes shift (>100 nm), (c) a shift of emission to the red portion of the visible spectrum (>600 nm) as much as possible, and (d) a shift of emission to wavelengths higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, a donor fluorescence portion may be selected that has its maximum excitation wavelength near the laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap with the excitation spectrum of the corresponding acceptor fluorescence portion. A corresponding acceptor fluorescence portion may be selected that has a high extinction coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescence portion, and emission in the red portion of the visible spectrum (>600 nm).

[0114] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, Lucifer Yellow, β-phycoerythrin, 9-acridine isothiocyanate, Lucifer Yellow VS, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimidyl 1-pyrene butyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Typical acceptor fluorescence moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, lysamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate, or other chelates of lanthanide ions (e.g., europium or terbium), depending on the donor fluorescence moiety used. Donor and acceptor fluorescence moieties can be obtained, for example, from Molecular Probes (Junction City, Oregon) or Sigma Chemical Co. (St. Louis, Missouri).

[0115] The donor and acceptor fluorescent moieties can be bound to a suitable probe oligonucleotide via a linker arm. The length of each linker arm is important because it affects the distance between the donor and acceptor fluorescent moieties. For the purposes of this disclosure, the linker arm length is the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Generally, the linker arm is approximately 10 Å to approximately 25 Å. The linker arm may be of the type described in International Publication 84 / 03285. International Publication 84 / 03285 also discloses methods for binding the linker arm to a specific nucleotide base and for binding the fluorescent moiety to the linker arm.

[0116] Acceptor fluorescent moieties such as LC-Red640-NHS-ester can be combined with C6 phosphoamidites (available from ABI (Fosta City, California) or Glen Research (Sterling, Virginia)) to produce, for example, LC-Red640-phosphoamidites. Common linkers used to conjugate donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., fluorescein-CPG from Glen Research or ChemGene (Ashland, Massachusetts)), amide linkers (fluorescein-NHS-ester derived from fluorescein-CPG, such as BioGenex (San Ramon, California)), or 3'-amino-CPGs that require fluorescein-NHS-ester conjugation after oligonucleotide synthesis.

[0117] FRET can also cause a decrease in plasmon electricity.

[0118] A method for detecting DNA on an electrode surface using a voltage that excites a fluorescent donor is disclosed. In some embodiments, the excitation of the fluorescent donor is detected by an optical detector, an electrochemical detector, or both.

[0119] A signaling oligonucleotide is disclosed, comprising an ETM, a fluorescent donor, and a fluorescent acceptor.

[0120] A signal probe / target / capture probe hybridization complex is disclosed in which, upon excitation of the ETM and OSM on the signal probe via the application of voltage, energy is transferred to the electrode surface and a current is induced. In some embodiments, the current is a plasmon current. In some embodiments, the plasmon current is a direct measure of fluorescence, phosphorescence, or chemiluminescence signature. In some embodiments, the plasmon current is a direct measure of the target analyte in the sample, i.e., a direct measure of the hybridization event.

[0121] Probe synthesis, functionalization, and conjugation - general Design of synthetic oligonucleotides Regarding the design of synthetic oligonucleotides for use in amplification reactions, Rychlik et al. (1989, Nucleic Acids Research, vol 17(21):8543-8551) and Rychlik (1995, Molecular Biotechnology, vol 3:129-134) (both incorporated in their entirety by reference) describe selection criteria and computer programs for designing probes and primers. Both teach that probes should not generate secondary structures or exhibit self-hybridization. U.S. Patent No. 6,495,323 (incorporated in its entirety by reference) describes in detail the formation of probes attached to electron transfer moieties.

[0122] Capture probe synthesis U.S. Patent No. 10,001,476 discloses in more detail the synthesis, functionalization, and conjugation of probes, the entirety of which is incorporated by reference. The synthesis, functionalization, and conjugation of probes are all well known in the art.

[0123] Nucleic acid capture probes are typically designed to be complementary to a sequence of approximately 40–50 base pairs within the target. The capture probe sequence is usually complementary to the 3' region of the target (though the reverse 5' region may also be true) and designed to have a melting temperature (TM) of approximately 50°C. The capture probe may be modified at either its 3' or 5' end with a disulfide linker for covalent bonding to a gold electrode surface, as described, for example, in U.S. Patents 6,753,143 and 7,820,391, which are essentially co-owned and incorporated herein in their entirety by reference.

[0124] For example, capture probes containing nucleic acids can be bonded directly or indirectly, covalently or noncovalently, to electrodes or other substrate surfaces using various well-known techniques. See, for example, Ch.13, Chemically Modified Electrodes, Martin and Foss, pp.403-442, Laboratory Techniques in Electroanalytical Chemistry; 2d Ed., Kissinger and Heineman, Eds., MARCEL DEKKER, INC. (1996); Biochip Technology, Cheng and Kricka, Eds. George H. Buchanan Printing Company, Bridgeport, NJ (2001) (both are incorporated in their entirety by reference).

[0125] Signal probe synthesis The signal probe sequence is complementary to a specific region of the target and not to any region of the capture probe. The signal probe sequence is typically complementary to the 5' region of the target (but the reverse, 3', can also be true). If sequence identification is required, the sequence polymorphism must be as close as possible to the center of the amplicon sequence, and the TMs of the two amplicons must match as closely as possible. Ferrocene labels (typically 1-6 labels per probe) are added towards the 5' end of the signal probe sequence, near it, or at its location. Optical labels (typically 1-10 labels per probe) are added towards the 5' end and / or different locations of the signal probe sequence, near it, or at its location. Since hybridization must be performed at a single temperature, the TM values ​​of all signal probes should be within a 5°C range. Since all detection reactions must be performed in the same solution, the signal probe and capture probe must be designed to avoid any cross-hybridization, and the maximum ΔG0 value for cross-hybridization has been empirically established.

[0126] In some embodiments, one ferrocene or ferrocene derivative is added toward, near, or at the 5' end of the signal probe sequence, and one optical signaling label is added toward, near, or at the 5' end of the signal probe sequence, adjacent to the ferrocene or ferrocene derivative. In some embodiments, two ferrocene labels are added toward, near, or at the 5' end of the signal probe sequence, and two optical signaling labels are added toward, near, or at the 5' end of the signal probe sequence. In some embodiments, 1 to 10 ferrocene labels are added, three ferrocene labels are added to the 5' end of the signal probe sequence, 1 to 10 optical signaling labels are added, and three ferrocene labels are added to the 5' end of the signal probe sequence. In some embodiments, multiple ferrocene labels are attached to, near, or at the 5' end of the signal probe sequence, and multiple optical signaling labels are attached to, near, or at the 5' end of the signal probe sequence.

[0127] In one embodiment, the nucleic acid is modified with at least two detectable labels located at one location, i.e., adjacent to each other. In one embodiment, the nucleic acid is modified with at least two detectable labels located at two locations, i.e., not adjacent to each other. In one embodiment, the nucleic acid is modified with three or more detectable labels located at three or more locations, i.e., not adjacent to each other. In one embodiment, the nucleic acid is modified with multiple detectable labels located at multiple locations (adjacent or not adjacent to each other). For example, multiple detectable labels at multiple locations may be used to increase the signal obtained from the probe. For example, the detectable labels may be attached to both the 5' and 3' or any location in between. In one embodiment, the multiple electron transfer portions are the same, resulting in a uniform signal. Alternatively, each of the multiple detectable labels may be different (they may be from different modalities such as electrochemistry, optics, or radioisotopes).

[0128] In some embodiments, a detectable label on the signal probe is held one base pair away from the capture probe and still generates a detectable signal. In some embodiments, a detectable label on the signal probe is held at any location 1 to 10 base pairs away from the capture probe and still generates a detectable signal. In some embodiments, a detectable label on the signal probe is held at any location 1 to 50 base pairs away from the capture probe and still generates a detectable signal. In some embodiments, a detectable label on the signal probe is held at any location 1 to 100 base pairs away from the capture probe and still generates a detectable signal. In some embodiments, a detectable label on the signal probe is held at any location 36 to 72 base pairs away from the capture probe and still generates a detectable signal.

[0129] In some embodiments, the ETM is positioned such that there is a 0-base gap between the ETM and the optical label (e.g., OSM). In some embodiments, the sequence gap between the ETM and the optical label is 0 to 2 bases. In some embodiments, the ETM label is located about 5 to about 50 base pairs away from the optical label, for example, about 5 to 100 base pairs away from the optical label, or for example, about 5 to 500 base pairs away from the optical label.

[0130] In some embodiments, the methylene blue signaling moiety of ferrocene, a derivative of ferrocene, or a signal probe is located more than 5 base pairs away from the optical label, for example, more than 10 base pairs away, for example more than 50 base pairs away, for example more than 100 base pairs away, for example more than 200 base pairs away.

[0131] When synthesizing signal probes, electron transfer moieties and optical labels can be covalently attached to nucleic acids at various positions, namely the 5' end and middle of the signal probe (Figure 2a), the 3' end and middle of the sequence (Figure 2e), or both the 5' and 3' ends (Figure 2d). In one embodiment, binding is via attachment of the nucleoside to the base or binding to the nucleic acid backbone containing either the ribose or phosphate portion of the ribose-phosphate backbone. In embodiments, the composition is designed so that the electron transfer moiety is as close to a "π-way" as possible. Attachment of ferrocene tags and optical signaling labels should not disrupt the annealing of the amplicon to the signal probe.

[0132] Alternatively, the signal probe may contain extra terminal nucleosides at the ends of the nucleic acid (n+1 or n+2), which are used to covalently attach electron transfer moieties or optical labels but are not involved in base-pair hybridization to amplicons as shown in Figures 2j and 2l, where circles represent ETMs, squares represent optical moieties, and triangles represent colorimetric moieties.

[0133] Alternatively, it may be desirable to insert a linker arm that separates the electron transfer portion from the probe-amplicon coupling region. In Figures 2m to 2p, circles represent the ETM, squares represent the optical portion, and triangles represent the colorimetric portion.

[0134] Signal probes are synthesized using standard phosphoramidite chemistry and may contain any nucleotide or modified base suitable for DNA binding. The nucleic acid portion of the signal probe may be DNA or RNA, a chimeric mixture or derivative, or modified versions thereof. In addition to being labeled with electrochemically detectable and optical labels, signal probes may be modified at the base portion, sugar portion, or phosphate backbone and may contain other adducts or labels.

[0135] The signal probe can be of any suitable size but does not bind to the capture probe. In some embodiments, the signal probe is in the range of 10–30 nucleotides, 20–30 nucleotides, 15–30 nucleotides, or 10–25 nucleotides, but the signal probe may be longer or shorter as needed.

[0136] In some embodiments, the signal probe lacks enhancing groups. In some embodiments, the signal probe does not undergo any detectable change in any observable properties during hybridization to an amplicon or a capture probe. In some embodiments, the signal probe undergoes a detectable change only when a voltage is applied. In some embodiments, the signal probe undergoes a detectable change only when excited by light or a laser beam. In some embodiments, the signal probe undergoes a detectable change both when a voltage is applied and when excited by light or a laser beam.

[0137] The signal probe can be labeled (as described above) using any known labeling method. For example, the signal probe can be labeled by: (1) attachment of a phosphorothioate bond to the sulfur; (2) attachment at the 2'-amino group; (3) attachment at the 1' position using appropriately modified sugars, such as alkylamine-substituted carboxamides; (4) attachment at the 1' position using, for example, a base moiety and, for example, an alkyldiamine as a linker; (5) formation of a structure by reductive alkylation of an adduct formed between the alkyldiamine and the debase moiety; (6) incorporation using 4'-thio-2'-deoxyuridine or 4'-thiothymidine; (7) 4-thiothymidine or 4- (8) Attachment of thio-2'-deoxyuridine at the 2' position; (9) Attachment of deoxycytidine at the 4-amino position if the 4-amino group is derivatized with an alkylamine; (10) Attachment of adenine via the 6' position if the 6-amino group is derivatized with an alkylamino moiety; (11) Attachment of adenine at the N2 position if the 8' position is substituted with an alkylthioamine; (12) Attachment of aminoadenine at the N2 position if the N2 amino group is derivatized with an alkylamine.

[0138] All signal probes can be purified using techniques known in the art.

[0139] false positive result False positive results can lead to serious problems, including the unnecessary use of antibiotics, antibacterial agents, or antifungal treatments. To avoid false positive results, the signal probe should not be bound to the capture probe. For example, in Figure 1b, section 3b should not be bound to the capture probe (amplicon only) and therefore reduce false positives.

[0140] false negative result False negative results are rarer. Conventional systems cannot determine whether a negative signal is due to the absence of an amplicon or the failure of the signal probe / capture probe to form a sandwich. False negatives can be reduced by adding a second label on the signal probe.

[0141] composition Dual-labeled signal probe Signal probes, such as signal probes comprising a first signaling portion and a second signaling portion, are disclosed herein, for example, the first and second signaling portions being detectable using different detection modalities. In some embodiments, the first signaling portion is an ETM. In some embodiments, the second signaling portion is either an OSM or a FM.

[0142] Compositions comprising a first signal probe including a first signaling portion and a second signaling portion are also disclosed herein. In some embodiments, the first and second signaling portions are detectable using different detection modalities. In some embodiments, the first signaling portion is detected by a first transducer and the second signaling portion is detected by a second transducer, which are of different modalities than the first and second transducers. In some embodiments, the first signal probe may include a third signaling portion. In some embodiments, the composition further comprises a second signal probe (e.g., a second signal probe including at least one different signaling portion compared to the first signal probe) which is different from the first signal probe.

[0143] Appropriate signaling moieties include, but are not limited to, any detectable labels, including optically or electrochemically detectable labels. Appropriate signaling moieties include any molecules that can be detected via emission, fluorescence, chemiluminescence, phosphorescence, bioluminescence, electronic, electrochemical, radioactive, electrochemiluminescence, enzymes, Förster resonance energy transfer (FRET), surface plasmon resonance (SPR), photonic crystal-based, optical resonator-based, optical fiber-based, optical wavelength-based, and / or RAMAN technology. In some embodiments, signaling moieties include, but are not limited to, electron transfer moieties, fluorescence moieties, radioisotope moieties, optical dyes, RAMAN labels, etc.

[0144] In some embodiments, the first signaling portion is ETM and the second signaling portion is OSM. In some embodiments, the first signaling portion contains ferrocene and the second signaling portion is OSM. In some embodiments, the first signaling portion contains methylene blue and the second signaling portion is OSM.

[0145] In some embodiments, the first signaling portion is an ETM and the second signaling portion is a fluorescent portion (FM). In some embodiments, the first signaling portion contains ferrocene and the second signaling portion is a fluorescent portion. In some embodiments, the first signaling portion contains methylene blue and the second signaling portion is a fluorescent portion. In some embodiments, the fluorescent portion is fluorescein. In some embodiments, the ETM and the fluorescent portion are excited by voltage, and the fluorophore induces an enantiomer dipole in the metallic material, causing a flow of plasmon current.

[0146] In some embodiments, the signal probe includes a third signaling portion, such as a corresponding acceptor fluorescence portion, e.g., a quencher. In some embodiments, the signal probe may include a third signaling portion whose signal is distinguishable from another OSM of the signal probe. As an example, the signal probe may include three signaling portions, two of which are OSMs, and each of the two OSMs is distinct. In some embodiments, the probe may include at least one ETM, at least one FM, and at least one quencher portion. In some embodiments, the signal probe includes only an optical label, which is excited by the application of a voltage. In some embodiments, the signal probe has only an ETM, which is excited by the application of light or a laser beam.

[0147] Dual-labeled signal probe-nucleic acid complex A composition is disclosed comprising a signal probe comprising a first signaling portion and a second signaling portion, wherein the first and second signaling portions are of different detection modalities, and the signal probe is bound to a target nucleic acid. In some embodiments, the first signaling portion is detected by a first transducer, and the second signaling portion is detected by a second transducer, which are of different modalities. In some embodiments, the first signaling portion is a first electron transfer portion, and the second signaling portion is an optical signaling portion; in some embodiments, the second signaling portion is a fluorescent portion. In some embodiments, the first signaling portion is ferrocene or osmium-labeled, and the second signaling portion is a fluorescent portion. A composition is disclosed comprising a signal probe comprising at least one ETM and at least one FM, wherein the signal probe is bound to a target nucleic acid.

[0148] Capture probe-signal probe-nucleic acid complex A composition is disclosed comprising a first signal probe comprising a first signaling portion and a second signaling portion, wherein the signal probe is bound to a target nucleic acid ("signal probe-nucleic acid complex") and the signal probe-nucleic acid complex is bound to a capture probe. In some embodiments, the first signaling portion is detected by a first transducer and the second signaling portion is detected by a second transducer, which is of a different modality than the first and second transducers. A composition is disclosed comprising a signal probe comprising at least one ETM and at least one FM, wherein the signal probe is bound to a target nucleic acid and the signal probe-diffusion complex is bound to a capture probe.

[0149] In some embodiments, the first signal probe comprises ETM and OSM, the signal probe is bound to a target nucleic acid, and the signal probe-nucleic acid complex is bound to a capture probe. In some embodiments, the first signal probe comprises ETM and FM, the signal probe is bound to a target nucleic acid, and the signal probe-nucleic acid complex is bound to a capture probe. In some embodiments, the first signal probe comprises ferrocene or a derivative of ferrocene and a fluorescent moiety. In some embodiments, the first signal probe comprises methylene blue and a fluorescent moiety.

[0150] In some embodiments, the first signal probe includes an ETM and a fluorescein label. In some embodiments, the ETM is ferrocene or a derivative of ferrocene. In some embodiments, the ETM is methylene blue.

[0151] Capture probe-signal probe-nucleic acid system complex A composition is disclosed comprising a signal probe having a first signaling portion and a second signaling portion, wherein the signal probe is bound to a target nucleic acid ("signal probe-nucleic acid complex"), the signal probe-nucleic acid complex is bound to a capture probe ("capture probe-signal probe-nucleic acid complex"), and the capture probe-signal probe-nucleic acid complex is bound to an electrode ("capture probe-signal probe-nucleic acid system complex"). In some embodiments, the electrode comprises a self-assembled monolayer ("SAM"). In some embodiments, the electrode is a gold electrode, a silver electrode, or a platinum electrode.

[0152] The hybridization complexes described herein can be understood more fully by referring to the following numbered paragraphs.

[0153] Paragraph 1. A hybridization complex comprising an amplicon-bound capture probe, wherein the amplicon is bound to a signal probe, and the signal probe comprises a nucleic acid, a first detectable label, and a second detectable label, the first detectable label and the second detectable label being of different modalities.

[0154] Paragraph 2. A hybridization complex comprising a capture probe bound to an amplicon, wherein the amplicon is bound to a signal probe, and the signal probe comprises an electron transfer portion and an optical signal transduction portion or a radioactive portion.

[0155] Paragraph 3. A hybridization complex comprising an amplicon-bound capture probe, wherein the amplicon is bound to a signal probe, and the signal probe comprises (i) one of ferrocene, a ferrocene derivative, methylene blue, or osmium, and (ii) fluorescein or a radioactive moiety.

[0156] Paragraph 4. A hybridization complex comprising an amplicon-bound capture probe, wherein the amplicon is bound to a signal probe, and the signal probe comprises a nucleic acid, an electron transfer moiety, and an optical signal transduction moiety.

[0157] Paragraph 5. A hybridization complex comprising an amplicon-bound capture probe, wherein the amplicon binds to a signal probe, the signal probe comprises a nucleic acid, an electron transfer portion, and an optical signal transduction portion, the amplicon comprises a first portion and a second portion, and the capture probe can bind to the first portion but not to the second portion.

[0158] Paragraph 6. A hybridization complex comprising an amplicon-bound capture probe, wherein the amplicon binds to a signal probe, the signal probe comprises a nucleic acid, an electron transfer portion, and an optical signal transduction portion, the amplicon comprises a first portion and a second portion, and the capture probe can bind to the second portion but not to the first portion.

[0159] Paragraph 7. A hybridization complex comprising an amplicon-bound capture probe, wherein the amplicon binds to a signal probe, the signal probe comprises a nucleic acid, an electron transfer portion, and an optical signaling portion, the amplicon comprises a first portion and a second portion, and the capture probe can bind to the second portion and the first portion.

[0160] Paragraph 8. A hybridization complex comprising a capture probe bound to an amplicon, wherein the amplicon is bound to a signal probe, the signal probe comprises a nucleic acid, an electron transfer moiety, and an optical signal transduction moiety, the hybridization complex is bound to an electrode, and the electrode comprises a monolayer.

[0161] Signal probe for multiplex detection A composition is disclosed comprising a first signal probe including a first electron transfer portion and a second signaling portion, and a second signal probe including a first electron transfer portion and a third signaling portion, wherein the first electron transfer portion is of a different modality than the second signaling portion, and the second and third signaling portions are of the same modality, but the signals produced by the second and third signaling portions are distinguishable. In some embodiments, the second and third signaling portions are optical signaling portions. In some embodiments, the second and third signaling portions are fluorescent signaling portions. In some embodiments, the first signaling portion is ferrocene or a derivative of ferrocene, and the second and third signaling portions are fluorescent signaling portions. In some embodiments, the first signaling portion is methylene blue, and the second and third signaling portions are fluorescent signaling portions. In some embodiments, at least one of the second or third signaling portion is fluorescein. In some embodiments, the second signaling portion is fluorescein, and the third signaling portion is rhodamine-labeled. A composition is disclosed comprising a first signal probe comprising ferrocene or a ferrocene derivative and fluorescein, and a second signal probe comprising ferrocene or a ferrocene derivative and Alexa Fluor 405 labeling.

[0162] A composition is disclosed comprising: a first signal probe comprising at least one ETM and FM, wherein the signal probe is bound to a first portion of a target nucleic acid and the first signal probe-nucleic acid complex is bound to a capture probe; and a second signal probe comprising at least one ETM and FM, wherein the FM signal of the first signal probe is distinguishable from the FM signal of the second signal probe, the second signal probe is bound to a second portion of a target nucleic acid and the second signal probe-nucleic acid complex is bound to a capture probe.

[0163] In some embodiments, the first signaling portion is detected by a first transducer, the second and third signaling portions are detected by a second transducer, and the first and second transducers are of different modalities.

[0164] Fluorescently stained nucleic acids on electrodes A composition is disclosed comprising a fluorescently stained nucleic acid bound to a capture probe, wherein the capture probe is bound to an electrode.

[0165] In some embodiments, fluorescently stained nucleic acids are transported to an electrode, where they are bound by a capture probe to form a capture probe / stained DNA hybridization complex. In some embodiments, a voltage is applied to the capture probe / stained DNA hybridization complex. In some embodiments, the fluorescently stained nucleic acids produce a detectable signal in response to the applied voltage. In some embodiments, the detectable signal is detected by an optical detector. In some embodiments, the detectable signal is detected by an electrochemical detector. In this way, the fluorescently stained nucleic acids are measured by applying a voltage. In some embodiments, the detectable signal is a dipole moment, a plasmon current, and / or fluorescence.

[0166] Excitable probe or label In general, conventional fluorophores can be used as excitable molecular sources that emit energy detectable by an optical detector. Disclosed are excitable probes or labels that generate a signal detectable by an optical detector when the probe or label is excited by a voltage. Disclosed are voltage-excited fluorophores that emit energy to induce an enantiomer dipole moment within a metal surface. The applicant has found that excitable probe or label sources generate plasmon electricity when the probe or label is in near-field, i.e., in close proximity to a metal structure, and is excited by a voltage. In some embodiments, the FM generates a measurable plasmon current that can be detected by a current detector. In some embodiments, the FM generates a measurable plasmon current that can be detected by an optical detector.

[0167] Quantum dots (Q dots); chemiluminescent alkaline phosphatases and other chemiluminescent labels; fluorospheres, i.e., fluorosspheres and transfluospheres; polymer beads doped with one or more fluorescent labels; fluorescent microspheres; silicon nanoparticles; silica and silicate-doped materials; semiconductor materials; E-type fluorescent emitters; P-type fluorescent emitters; Fluo-3 and Fluo-4 calcium indicators; calcium green indicators; fluorine zinc indicators; Cu 2+ Cu + Phen Green for the detection of a wide range of ions, including Zn. 2+ Newport Green for detection; Lead and Cadmium Green dye for measurement of lead and cadmium; Magnesium Green for electrical detection of free magnesium; Mag-fluo-2 and Mag-indo-1 for magnesium detection; Mag-fluo-4 for detection of both calcium and magnesium in both free solution and between cells; Phycobiliproteins (many different forms); Buckyball, C 60etc; carbon nanotubes; cardiolian green / indocyanine green fluorescent indicators; metal colloids and mixed metal colloids such as Ag, Au, Pt, Fe, Pd, Cu, Zn, Rh, Cr, Pb; pH indicators such as SNARF-1, SNARF-4F, SNARF-5F, dextran BCECF; 6-chloro-9-nitro-5-oxo-5H-benzo{a}phenoxazine (CNOB) for detection of nitroreductase and nitrate reductase activity; SYTOX dead cell stains such as SYTOX Blue, green, orange, red; DAPI and propidium iodide labeling; ethidium bromide, Picogreen, SYBR Disclosed are excitable probes or labels that may include, but are not limited to, probes for double-stranded DNA detection such as green; dyes in the Alexa fluorophore range; BODIPY and related structural dyes; cell lamps and organelle lamps (genetically encoded proteins); green fluorescent protein (GFP) and its analogues; coumarin dyes; prodan and related structural dyes; voltage-sensitive probes such as DisBAC4(3) and CC2-DMPE; and / or Ncode miRNA-labeled fluorophores.

[0168] Fluorophores belong to several common chemical classes, including coumarin, fluorescein (or fluorescein derivatives and analogs), rhodamine, resorphine, luminophores, and cyanines. Additional examples of fluorescent molecules can be found in *Molecular Probes Handbook - A Guide to Fluorescent Probes and Labeling Technologies*, *Molecular Probes*, Eugene, OR, ThermoFisher Scientific, 11. thThis is described in the Edition. In other embodiments, the fluorophore is selected from xanthene derivatives, cyanine derivatives, squaline derivatives, naphthalene derivatives, coumarin derivatives, oxadiazole derivatives, anthracene derivatives, pyrene derivatives, oxazine derivatives, acridine derivatives, arylmethine derivatives, and tetrapyrrole derivatives. In other embodiments, the fluorescent portion may be CF dyes (available from Biotium), DRAQ and CYTRAK probes (available from BioStatus), BODIPY (available from Invitrogen), ALEXA FLUOR (available from Invitrogen), DYLIGHT FLUOR (e.g., DYLIGHT649) (available from Thermo Scientific, Pierce), Atto and Tracy (available from Sigma Aldrich), FLUOPROBES (available from Interchim), ABBERIOR Dyes (available from Abberior), DY and MEGASTOKES Dyes (available from Dyomics), SULFO CY dyes (available from Cyandye), HILYTE FLUOR (available from AnaSpec), SETA, SETAU and SQUARE Dyes (available from SETA BioMedicals), QUASAR and CAL FLUOR dyes (available from Biosearch Technologies), SURELIGHT Dyes (available from APC, RPEPerCP, Phycobilisomes) (Columbia Biosciences), as well as APC, APCXL, RPE, BPE (available from Phyco-Biotech, Greensea, Prozyme, Flogen) are selected. Other non-limiting examples of fluorescent dyes include FAM (5- or 6-carboxyfluorescein), VIC, NED, PET, fluorescein, FITC, IRD-700 / 800, CY3, CY5, CY3.5, CY5.5, CY7, HEX, TET, TAMRA, JOE, ROX, BODIPY TMR, Oregon Green, Rhodamine Green, Rhodamine Red, Texas Red, or Yakima Yellow.

[0169] Molecules capable of fluorescence include, but are not limited to, fluorophores, chromophores, lumophores, biomolecules, or any molecule or device that provides endogenous or exogenous luminescence activity.

[0170] In one embodiment, a bioassay system is disclosed that includes an electrode positioned near the electrode surface for enhancing the effect of a chemiluminescence-based reaction, wherein an excitable probe or label is excited by a voltage applied to the electrode, which can be measured using an optical detector, an electrochemical detector, or both. In another embodiment, a bioassay system is disclosed that includes an electrode positioned near the electrode surface for enhancing the effect of a chemiluminescence-based reaction using an ETM, wherein an excitable ETM probe or label is excited by light or a laser beam, which can be measured using an optical detector, an electrochemical detector, or both.

[0171] In some embodiments, electrical excitation may also include the use of microwave or sonic energy to increase any reaction rate in the assay detection system. In some embodiments, light or laser beam stimulation may also include the use of microwave or sonic energy to increase any reaction rate in the assay detection system.

[0172] In some embodiments, the assay system of the present disclosure does not include a light or laser beam source for directing an energy beam to any included fluorophore. In some embodiments, the assay system of the present disclosure includes a light or laser beam source for directing an energy beam to any included fluorophore to provide excitation energy. In some embodiments, the laser beam may be positioned adjacent to the system for directing the beam to the molecular component. In some embodiments, the laser may be any device capable of focusing an energy beam to a specific point on a solid or liquid source material for excitation, and the laser may transmit RF, infrared, microwave, or UV energy. Any light source known to those skilled in the art may be used, such as a laser that emits light, and light is used in its broad sense to mean electromagnetic radiation that propagates through space and includes not only visible light but also infrared and ultraviolet light. Thus, a single instrument positioned above the surface of the assay can be used to generate energy for exciting fluorescent molecules. The light may be emitted from the fiber continuously or intermittently, as desired. Furthermore, two-photon excitation can be used at approximately 375–900 nm using a laser diode source with continuous or short pulse width (<50 ps) and high repetition rate (>1 MHz). Various pulsed laser diode sources compatible with fluorophores are commercially available and can be used in conjunction with the present invention.

[0173] In some embodiments, voltage-induced fluorescence can be observed at a distance depending on the type of excitable molecule being detected. For example, if the fluorophore is positioned at approximately 5 nm to 200 nm relative to the electrode surface, fluorescence induction can be observed. In some embodiments, the distance is approximately 5 nm to 50 nm relative to the electrode surface, and in some embodiments, 10 nm to 30 nm. At this scale, there are few phenomena that offer new levels of detection, manipulation, and control opportunities. Furthermore, devices of this scale can dramatically reduce size, weight, and therefore cost, while offering dramatically improved performance, sensitivity, and reliability.

[0174] The applicant has discovered that fluorophores can be excited by the application of current and / or voltage to generate fluorescence. Interestingly, the induced signal increases correspondingly with increasing concentration of the fluorophores present. In some embodiments, more fluorophores present require a higher excitation voltage. Figure 11 shows the predicted degree of energy-induced fluorescence with respect to the concentration of fluorescein (fluorescent probe) in water. The fluorescence is 3 log of the tested fluorescent probe. 10 A significant increase is predicted over the concentration range. This result suggests that the more fluorophores present near the electrode, the greater the induced fluorescence. It is interesting to note that in conventional fluorescence-based immunoassays, the degree of detected fluorophores (usually fluorescence intensity) is directly related to the analyte concentration determined by the assay. Therefore, it is predicted that fluorescence-based immunoassays can be constructed on the electrode surface, and the concentration of the analyte (antigen) can be determined by voltage-induced fluorescence. Notably, even if the excitation is purely digital (voltage), the fluorescence is read indirectly (optically). The signal can be digitally converted and displayed. In contrast, fluorescence-based immunoassays existing today are not digitally excited (i.e., by applied voltage). Thus, this method represents a significant breakthrough in how fluorescence is generated, measured, and quantified. In some embodiments, the target concentration is based on or correlates with the plasmon current.

[0175] signal probe During detection, the signal probes are not directly coupled to the electrodes, but rather to amplicon / capture probes that hold them in place for detection.

[0176] In some embodiments, the signal probe consists of deoxyribonucleic acid, ribonucleic acid, peptide nucleic acid, PEG-modified nucleic acid, hexa-polyethylene glycol-modified nucleic acid, chimeric mixtures or derivatives, or modified versions thereof.

[0177] In some embodiments, the signal probe does not overlap with the capture probe. In some embodiments, the signal probe overlaps with the capture probe by approximately 1 to 10 base pairs.

[0178] A signal probe is shown in Figure 2(d-g), where ii is a detection subregion, e.g., a region containing one or more signal transduction subregions (e.g., ETM / optical signal transduction subregions), and i is an annealing region, e.g., a region coupled to amplicon detection. In some embodiments, the signal probe has two regions: a first region that can be coupled to the amplicon of item i in Figure 2a, and a second region that can generate the signal described in item ii in Figure 2a. In some embodiments, the signal probe includes three regions: a first region that can be coupled to the amplicon of item i in Figure 2p, a second region that can generate a signal during redox-mediated electron detection of item ii in Figure 2p, and a third region linking the first and second regions. See Figure 2l (linker symbolized as a rhombus).

[0179] The signal probes described herein can be understood more fully by referring to the following numbered paragraphs.

[0180] Paragraph 1. A signal probe comprising a nucleic acid, an electron transfer portion, and an optical signal transduction portion, wherein the nucleic acid comprises a first portion and a second portion, the first portion being able to bind to a first capture probe but not to a second capture probe, and the second portion being able to bind to a second capture probe but not to a first capture probe.

[0181] Paragraph 2. A signal probe comprising a nucleic acid, an electron transfer portion, and an optical signaling portion, wherein the nucleic acid comprises a first portion and a second portion, the first portion being capable of binding to a first capture probe and a second capture probe, and the second portion being capable of binding to the first capture probe but not to the second capture probe.

[0182] Paragraph 3. A signal probe comprising a nucleic acid, an electron transfer portion, and an optical signaling portion, wherein the nucleic acid comprises a first portion and a second portion, the first portion being able to bind to a first capture probe but not to a second capture probe, and the second portion being able to bind to both the first and second capture probes.

[0183] Paragraph 4. The signal probe according to paragraphs 1-3, wherein the electron transfer portion is attached toward, near, or at the 5' end of the signal probe, and the optical signal transduction portion is attached toward, near, or at the 5' end of the signal probe.

[0184] Paragraph 5. A signal probe comprising a capture probe binding region, a target analyte binding region, an electrochemically detectable labeling region, and an optically detectable labeling region.

[0185] The signal probes described herein can be better understood by the following numbered paragraphs.

[0186] Paragraph 1. A probe comprising nucleic acid, a first detectable label and a second detectable label, wherein the first detectable label and the second detectable label are of different modalities.

[0187] Paragraph 2. A probe comprising a nucleic acid, a first electron transfer moiety, and a second detectable label which is not a conventional electron transfer moiety.

[0188] Paragraph 2a. A probe comprising a nucleic acid, a first signaling moiety, and an optical signaling moiety, wherein the first signaling moiety is not a conventional optical signaling moiety.

[0189] Paragraph 3. A probe comprising a nucleic acid, ferrocene or a derivative of ferrocene, and a second detectable label that is not ferrocene-labeled.

[0190] Paragraph 3a. A probe comprising nucleic acid, methylene blue, and a second detectable label that is not ferrocene-labeled.

[0191] Paragraph 3b. A probe comprising a nucleic acid, a first signaling moiety, and a fluorophore, wherein the first signaling moiety is not a fluorophore.

[0192] Paragraph 4. A probe comprising a nucleic acid, an electron transfer portion, wherein the electron transfer portion is attached toward or near the 5' end of the probe, and a second optical label attached toward or near the 3' end of the probe.

[0193] Paragraph 5. A probe comprising a nucleic acid, a first electron transfer portion capable of generating a signal in response to the application of a first voltage, and a second label capable of generating an optical signal in response to the application of a first voltage, wherein the second label is not an electron transfer portion.

[0194] Paragraph 5a. A probe comprising a nucleic acid, a first electron transfer portion capable of generating an electrochemical signal in response to irradiation with light or a laser beam, and a second label capable of generating an optical signal in response to the application of light or a laser beam.

[0195] Paragraph 5b. A probe comprising a nucleic acid, a first electron transfer portion (e.g., ferrocene or methylene blue) capable of generating an optical signal in response to the application of light or a laser beam, and a second label capable of generating an optical signal in response to the application of light or a laser beam, wherein the first label is not an optical signaling portion.

[0196] Paragraph 6. Probes comprising nucleic acids, electrochemically detectable labels, and optically detectable labels.

[0197] Paragraph 7. A probe comprising a nucleic acid, an electron transfer moiety (e.g., ferrocene or methylene blue), wherein the electron transfer moiety is attached toward the 5' end of the probe, near or at that position, and a second optical label attached toward the center of the probe sequence, near or at that position.

[0198] Paragraph 8. A probe comprising a nucleic acid, a first electron transfer moiety (e.g., ferrocene or methylene blue), and a second detectable label that is not an electron transfer moiety, wherein both the first electron transfer moiety and the second detectable label are detectable after a voltage is applied to the probe.

[0199] Paragraph 9. A probe comprising a nucleic acid, a first electron transfer moiety (e.g., ferrocene or methylene blue), and a second detectable label that is not an electron transfer moiety, wherein the first electron transfer moiety is detectable after a first voltage is applied to the probe, and the second detectable label is detectable after a second voltage is applied to the probe, the first voltage and the second voltage being different.

[0200] Paragraph 10. A probe comprising a nucleic acid, a first electron transfer moiety (e.g., ferrocene or methylene blue), and a second detectable label that is not an electron transfer moiety, wherein both the first electron transfer moiety and the second detectable label are detectable after a voltage is applied to the probe, wherein the first electron transfer moiety is detected by an electrochemical sensor and the second detectable label is detected by a sensor that is not an electrochemical sensor.

[0201] Detection system Integrated nucleic acid test cartridge Integrated multiplex target analysis systems are known and described in U.S. Patent No. 10,864,522, the entire disclosure of which is incorporated herein by reference.

[0202] An integrated nucleic acid test cartridge capable of amplification and detection is disclosed. Generally, the integrated nucleic acid test cartridge is capable of receiving a sample, extracting DNA, combining the DNA with an amplification reagent, amplifying the DNA, incubating the amplicon with an exonuclease, combining a signal probe (the signal probe includes an ETM and an optical signaling portion) with a target, and forming a hybridization complex by combining the signal probe / target with a capture probe. In some embodiments, a voltage is applied to the hybridization complex, and electrosensor detection of the ETM and optical detection of the OSM are performed based on the applied voltage. In some embodiments, a voltage is applied to the hybridization complex, and optical detection of both signaling portions is performed based on the applied voltage. In some embodiments, a voltage is applied to the hybridization complex, and electrosensor detection of both signaling portions is performed based on the applied voltage. In some embodiments, light or a laser beam is applied to the hybridization complex, and electrosensor detection of the ETM and optical detection of the OSM are performed based on the applied light. In some embodiments, light or a laser beam is applied to the hybridization complex, and optical detection of both signaling portions is performed based on the applied light. In some embodiments, a light or laser beam is applied to the hybridization complex, and electrical sensor detection of both signaling portions is performed based on the applied light.

[0203] In some embodiments, an integrated cartridge for nucleic acid testing that operates in conjunction with at least one reader device is disclosed herein. Disclosed herein is an integrated cartridge for nucleic acid testing that operates in conjunction with a first reader device and a second reader device, wherein the first reader device and the second reader device are of different modalities. In some embodiments, the first reader device reads a first signaling portion, and the first reader device and the first signaling portion are of the same modality; the second reader device reads a second signaling portion, and the second reader device and the second signaling portion are of the same modality; the first reader device and the second reader device are of different modalities; and the first signaling portion and the second signaling portion are of different modalities.

[0204] In some embodiments, an integrated cartridge for nucleic acid testing is disclosed herein, which operates in conjunction with only one reader instrument to detect signals from two signaling segments of different modalities. In some embodiments, the reader instrument is an ETM reader, which reads signals from ETM and OSM. In some embodiments, the reader instrument is an optical reader, which reads signals from ETM and OSM.

[0205] Imaging of optical label after excitation from voltage A detection system, a. One or more capture probes on an electrode surface coupled to at least one fluorophore, wherein the fluorophore is capable of generating a detectable signal when a voltage is applied to the electrode, b. An amperometric energy source for exciting the fluorophore, c. Detector and A detection system comprising the above is disclosed.

[0206] In some embodiments, the fluorophore is positioned about 5 nm to about 50 nm from one or more capture probes. In some embodiments, the detector is an optical detector, an electrochemical detector, or both.

[0207] The present invention includes a fluorescent, luminescent, chemiluminescent, or phosphorescent component that has the ability to emit light energy when in contact with photons in the UV-IR range. In some embodiments, the detectable signal of the excitable probe is fluorescence.

[0208] Imaging of ETM labeling after excitation from a light or laser beam source. A detection system, a. A capture probe (e.g., ferrocene or methylene blue) on the electrode surface coupled to at least one ETM, wherein the ETM can generate a detectable signal when light or a laser beam is applied to the ETM, b. A light or laser beam source for exciting the ETM, c. Detector and A detection system comprising the above is disclosed.

[0209] In some embodiments, the ETM is positioned approximately 5 nm to 50 nm from one or more capture probes. In some embodiments, the detector is an optical detector, an electrochemical detector, or both.

[0210] In some embodiments, the electrodes are separated by a distance sufficient to provide optimal signal readings, with a separation of approximately 5 nm to 100 nm.

[0211] Imaging of fluorescently stained nucleic acids on electrodes Nucleic acids can be fluorescently labeled via binding, intercalation, or covalent modification. Generally, covalent fluorescent labels can be introduced directly into the nucleic acid of interest or via a two-step approach, meaning that a reactive handle is first installed to allow for subsequent post-synthesis functionalization using the click reaction described above. Direct chemical labeling via solid-phase synthesis, two-step chemical labeling via solid-phase synthesis, direct chemioenzyme labeling, and two-step chemioenzyme labeling are all known techniques for labeling nucleic acids. See Chem Soc Rev. 2020 Dec 7;49(23):8749-8773, which is incorporated herein in its entirety. In recent years, ribozymes and deoxyribozymes have become versatile tools for directly or in two-step labeling RNA with fluorescent tags.

[0212] In some embodiments, fluorescently stained nucleic acids are transported to an electrode, where they are bound by a capture probe to form a capture probe / stained DNA hybridization complex. A current / voltage is applied to the capture probe / stained DNA hybridization complex. The fluorescently stained nucleic acids produce a detectable signal in response to the applied voltage. In some embodiments, the detectable signal is detected by an optical detector. In this way, the fluorescently stained nucleic acids are measured by applying a voltage. In some embodiments, the detectable signal is detected by an electrochemical detector. Figure 10 shows fluorescently labeled (F) nucleic acids (A-C) bound to a capture probe (e) bound to an electrode (1). The fluorophores are excited when energy is applied to the electrode, generating a signal detectable by an optical detector, an electrochemical detector, or both.

[0213] A system for generating electric current A system for generating electric current, i. The substrate and, ii. A set of conductive electrodes that are communicatively coupled to a substrate, iii. An excitable probe that emits fluorescence, light emission, or a phosphorescent signature when excited by electromagnetic energy, wherein such excitation induces a signal detectable by an optical detector or both an optical detector and an electrochemical detector. A system comprising the above is disclosed.

[0214] Importantly, the current increases as the number of excitable probes increases, thereby providing an assay that delivers an electrical signal proportional to the amount of excitable probes bound to the target substance. In other words, the electrical signal is proportional to the amount of signal-target hybridization complex bound to the capture probe. Figure 12 shows that the hybridization complex is expected to be detected by optical detection after the application of a voltage to excite the optical signaling portion.

[0215] The above methods and systems can be used in multiple detection systems, including but not limited to immunoassays, hybridization assays, resonance energy transfer assays, polarization / anisotropy-based assays, chemiluminescence-based assays, luminescence-based assays, and enzyme-linked immunosorbent assays.

[0216] A system for measuring chemiluminescence A system for measuring chemiluminescence, i. A capture molecule having affinity for a desired molecule, wherein the capture molecule is positioned on a surface substrate, and the substrate is an electrode or connected to a set of electrodes, ii. A detector molecule having affinity for a desired molecule, wherein the detector molecule includes a chemiluminescent label, iii. A trigger component that reacts with a chemiluminescent label to generate a chemically induced electronically excited state when bound to the desired molecule and the captured molecule, wherein the chemically induced electronically excited state is measured by an optical detection device, and such a chemically induced electronically excited state is proportional to the amount of the desired molecule in the test sample, and the trigger component and A system comprising the above is disclosed.

[0217] In some embodiments, the capture molecule may include biotin that binds to avidin or streptavidin. In other embodiments, the capture molecule may include a thiolated molecule that binds to gold particles. In yet another embodiment, the capture molecule may include an amine-terminated molecule that binds to an NHS activating molecule. In some embodiments, the capture molecule includes an immobilized antibody that can be used to bind to a molecule containing or conjugated thereto a specific antigenic molecule. In other embodiments, the capture molecule includes an antigenic molecule that can be used to bind to an immobilized antibody. In some embodiments, the capture molecule includes an enzyme that can be used to bind to a molecule containing or conjugated thereto a specific enzyme substrate. In other embodiments, the capture molecule includes a substrate for the enzyme that can be used to bind to the enzyme. In some embodiments, the capture molecule includes a receptor that can be used to bind to a molecule containing or conjugated thereto a specific receptor ligand. In other embodiments, the capture molecule includes one or more receptor ligands that can be used to bind to a molecule containing the receptor. In some embodiments, the capture molecule includes a lectin that can be used to bind to a molecule containing or conjugated thereto a specific polysaccharide. In other embodiments, the capture molecule comprises one or more polysaccharides that can be used to bind to a molecule containing or conjugated to one or more lectins. In further embodiments, the capture molecule comprises one or more nucleic acid sequences that can be used to bind to a molecule containing or conjugated to complementary nucleotide sequences. In other embodiments, the capture molecule may comprise a tethered DNA / RNA aptamer that can specifically bind to a target analyte such as a small molecule, peptide, protein, or cell.

[0218] In some embodiments, chemically induced electronic excitation states can generate photons in the UV-IR range.

[0219] Furthermore, it is disclosed that the concept of the present invention is used to measure the excited state of a fluorophore using an optical detector, including a microscope, which can electronically excite the fluorophore using voltage and provide a visual image, but can also measure the flow of the induced plasmon current.

[0220] In some embodiments, an excitable probe or label generates a metal surface plasmon, induces an enantiomer dipole in the metal structure, generates an electric current in the solution, which is then detected.

[0221] Correlation between electrochemical signals and optical signals Figure 9 is a flowchart illustrating how the first signaling subsignal and the second signaling subsignal are correlated. The first transducer receives the first signal from the first signaling subsignal 901. The first transducer may demodulate, descramble, or decode the first signaling subsignal to generate the first modified signal 902. The second transducer receives the second signal from the second signaling subsignal 903. (Steps 901 and 903 or 902 and 903 may occur simultaneously). The second transducer may demodulate, descramble, or decode the second signaling subsignal to generate the second modified signal 904. (Steps 901 and 904 or 902 and 904 may occur simultaneously). In some embodiments, steps 902 and 904 are omitted, i.e., the signals are not modified before processing. The first and second modified signals may be sent to a processor (905). In some embodiments, the signals are not modified before correlation. The processor may further modify the first modified signal and the second modified signal (906). In some embodiments, the processor does not modify the first modified or unmodified signal and the second modified or unmodified signal, but simply correlates the signals. The processor may modify the first modified signal and the second modified signal by subtracting the first modified signal from the second modified signal (or vice versa) to determine the correlation coefficient. In some embodiments, if the correlation coefficient is above a predetermined threshold, the processor transmits a signal indicating that the target analyte has been detected (907). In some embodiments, only the first signal, or only the second signal, or both the first and second signals are modified before being received by the processor. In some embodiments, neither the first nor the second signal is modified before being received by the processor.In some embodiments, only the first signal, only the second signal, or both the first and second signals are modified by the processor to generate a correlation coefficient. In some embodiments, if the correlation coefficient is above a predetermined threshold, the processor transmits a signal indicating that the target analyte was detected at a predetermined concentration. In some embodiments, if the correlation coefficient is above a predetermined threshold, the processor transmits a signal indicating that the target analyte was detected within a predetermined concentration range. In some embodiments, if the correlation coefficient is below a predetermined threshold, the processor transmits a signal indicating that the target analyte was not detected at a predetermined concentration. In some embodiments, if the correlation coefficient is below a predetermined threshold, the processor transmits a signal indicating that the target analyte was not detected within a predetermined concentration range. In some embodiments, if the correlation value is below a predetermined threshold, no detection result is reported; instead, an error message is reported, i.e., the correlation value signals that the system did not properly process the sample.

[0222] In some embodiments, the first signaling partial signal and the second signaling partial signal have a 1:1 relationship. In some embodiments, the first signaling partial signal and the second signaling partial signal have a relationship of 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:50, or 1:100. In some embodiments, the first signaling partial signal and the second signaling partial signal have a relationship of 2:1, 2:3, 2:4, 2:5, 2:10, or 2:50. In some embodiments, the first signaling partial signal and the second signaling partial signal have a relationship of 3:1, 3:2, 3:4, 3:5, 3:10, 3:20, 3:50, or 3:100.

[0223] In some embodiments, a first signaling partial signal and a second signaling partial signal are organized to form a new data stream. The new data stream is analyzed to determine a correlation coefficient. If the correlation coefficient is above a predetermined threshold, the target analyte is detected. If the correlation coefficient is above a predetermined threshold, the target analyte is detected at a predetermined concentration. In some embodiments, the correlation coefficient is based on one or more parameters selected from the group which essentially includes / consists of / consists of the correlation coefficient (R²), scaled fitting error (EFT), standard fitting error (RFT), slope of the line created by the new data stream, or intercept of the line created by the new data stream.

[0224] The processor may compare a first signaling partial signal to a predetermined criterion and a second signaling partial signal to a second predetermined criterion in order to determine the correlation coefficient. The processor may also compare a first modified signal to a predetermined criterion and a second modified signal to a second predetermined criterion in order to determine the correlation coefficient.

[0225] In some embodiments, the results are based on correlation values ​​and electrochemical signal values. In some embodiments, the results are based on correlation values ​​and optical signal values. In some embodiments, the results are based on correlation values, electrochemical signal values ​​and optical signal values.

[0226] In some embodiments, the correlation is calculated by multiplying the two signals together and then summing the products. The result is a single number indicating the similarity between signal x[n] and signal y[n].

[0227] This specification provides a correlation-based detection system for correlating a first electrochemical signal with a second optical signal, comprising: a correlation calculator configured to receive the first electrochemical signal and the second optical signal and generate a correlation value; and a processor configured to evaluate the correlation value and determine whether or not a target analyte is present based on the correlation value and a predetermined value.

[0228] In some embodiments, for a detection result to be reported (detected or not detected), both the electrochemical signal and the optical signal must be above a predetermined threshold. In some embodiments, for a detection result to be reported (detected or not detected), the electrochemical signal, not the optical signal, must be above a predetermined threshold. In some embodiments, for a detection result to be reported (detected or not detected), the optical signal, not the electrochemical signal, must be above a predetermined threshold. In some embodiments, for a detection result to be reported (detected or not detected), the correlation signal (correlation value) must be above a predetermined threshold. In some embodiments, if the correlation signal (correlation value) is below a predetermined threshold, an error signal is generated. In some embodiments, if the correlation signal (correlation value) falls below a predetermined threshold, an error signal is generated.

[0229] 1. A method for generating detection results, The first detector receives the first signal from the first signal transduction portion, The second detector receives the second signal from the second signal transduction region, The first signal and the second signal are correlated, thereby generating a detection result. Methods that include...

[0230] 2. Correlation of a first signal and a second signal, the method according to Embodiment 1, comprising comparing the first signal to a predetermined criterion to generate a first correlation signal, and comparing the second signal to the first correlation signal.

[0231] 3. The method according to Embodiment 1, wherein correlating a first signal with a second signal includes comparing the first signal with a first predetermined criterion to generate a first correlation signal, comparing the second signal with a second predetermined criterion to generate a second correlation signal, and comparing the first correlation signal with the second correlation signal.

[0232] 4. Correlation of a first signal with a second signal, the method according to Embodiment 1, comprising adjusting the first signal based on an adjustment coefficient to generate a first correlation signal, and comparing the second signal with the first correlation signal.

[0233] 5. The method according to Embodiment 1, wherein correlating a first signal with a second signal includes adjusting the first signal based on a first adjustment coefficient to generate a first correlation signal, adjusting the second signal with respect to a second adjustment coefficient to generate a second correlation signal, and comparing the first correlation signal with the second correlation signal.

[0234] 6. The method according to Embodiment 5 or 6, wherein the adjustment coefficient comprises removing the highest and lowest signals, the adjustment coefficient is the slope of the signal line, the adjustment coefficient is the amplitude of the signal line, or the adjustment coefficient is an estimate of the peak or trough of the signal.

[0235] 7. The method according to Embodiments 1 to 6, wherein the first signaling portion and the second signaling portion are from different modalities.

[0236] 8. The method according to embodiments 1 to 7, wherein the first signal transduction partial detector and the second signal transduction partial detector are from different modalities.

[0237] method The signal probes disclosed herein may be used in diagnostic methods, and the signal probes are complementary to sequences (e.g., genomes or cDNAs) of infectious disease factors of human diseases, including but not limited to viruses (e.g., HIV, HPV, etc.), bacteria, parasites, and fungi, thereby diagnosing the presence of infectious factors in a sample from a patient. The type of target nucleic acid may be genome, cDNA, mRNA, or synthetic, or the source may be human, animal, fungal, or bacterial. In another embodiment where they can be used for the diagnosis or prognosis of a disease or disorder, the target sequence may be a wild-type human genomic DNA or RNA or cDNA sequence whose mutation is associated with the presence of a human disease or disorder, or a mutant sequence. In such embodiments, the same sample may be contacted with different sets of signal probes (e.g., differently labeled signal probes) that selectively identify wild-type sequences or mutants. For example, the mutation may be an insertion, substitution, and / or deletion, or translocation of one or more nucleotides. In another embodiment, the signal probes may be used in SNP analysis, pharmacogenomics, and toxicological genetics.

[0238] In certain embodiments, a method is disclosed for detecting or measuring the product of a nucleic acid amplification or synthesis reaction, comprising: (a) contacting a sample containing one or more target nucleic acid molecules with one or more signal probes (such probes being of different modalities and including two or more labels that can be labeled internally, and / or at or near the 3' end of the signal probe, and / or at or near the 5' end); and (b) detecting or measuring one or more targets by redox-mediated electron detection and optical detection. In some embodiments, the optical label is excited by voltage and the excitation is read by an optical reader. In some embodiments, the optical label is excited by voltage and the excitation is read by an electron transfer reader. In some embodiments, the ETM label is excited by voltage and the excitation is read by an electron transfer reader. In some embodiments, the ETM label is excited by photoexcitation (light or laser beam) and the excitation is read by an optical reader. In some embodiments, the ETM label is excited by optical excitation and the excitation is read by an electron transfer reader.

[0239] A method for detecting a target nucleic acid sequence is disclosed, comprising contacting a sample containing a mixture of nucleic acids with at least one oligonucleotide, wherein the oligonucleotide is capable of hybridizing with the target nucleic acid sequence and comprises at least two detectable portions (the detectable portions being of different modalities), wherein a first signaling portion undergoes a redox reaction after a voltage is applied thereto, and a second signaling portion undergoes an optical reaction after a voltage is applied thereto, wherein a change in redox potential indicates the presence of the target nucleic acid sequence, and a change in optical properties indicates the presence and / or concentration of the target nucleic acid sequence.

[0240] A method for detecting a target nucleic acid sequence is disclosed, comprising contacting a sample containing a mixture of nucleic acids with at least one oligonucleotide, wherein the oligonucleotide is capable of hybridizing with the target nucleic acid sequence and comprises at least two detectable portions (the detectable portions being of different modalities), wherein a first signaling portion undergoes a redox reaction after an optical signal (light or laser beam) is applied to it, and a second signaling portion undergoes an optical reaction after an optical signal (light or laser beam) is applied to it, wherein a change in redox potential indicates the presence of the target nucleic acid sequence, and a change in optical properties indicates the presence and / or concentration of the target nucleic acid sequence.

[0241] A method for determining the presence or absence of a target in a sample is disclosed. In some embodiments, the target sequence is a wild-type human genome or an RNA or cDNA sequence. In some embodiments, the target sequence is a mutant human genome or an RNA or cDNA sequence. The mutation is associated with the presence of human disease or disorder. In some embodiments, the signal probe amplifies the wild-type target, and in other embodiments, it amplifies the mutant target. For example, the mutation may be an insertion, substitution and / or deletion of one or more nucleotides, or a translocation. In another embodiment, the signal probe can be used in SNP analysis, pharmacogenomics, and toxicological genetics.

[0242] In a particular embodiment, a method for detecting the presence or absence of a target nucleic acid is disclosed, comprising: (a) contacting a sample containing one or more target nucleic acid molecules with one or more signal probes (such probes may contain two or more labels, at least two of which are of different modalities, but the others may be the same, and may be labeled internally and / or at or near the 3' end and / or the 5' end); and (b) detecting or measuring one or more target nucleic acid molecules by electrochemical detection and detecting or measuring one or more target nucleic acid molecules by optical detection.

[0243] In a particular embodiment, a method for detecting the presence or absence of a target nucleic acid is disclosed, comprising: (a) contacting a sample containing one or more target nucleic acid molecules with one or more signal probes (such probes may contain two or more labels, at least two of which are of different modalities, but the others may be the same, and may be labeled internally and / or at or near the 3' end and / or the 5' end); and (b) detecting or measuring one or more target nucleic acid molecules by a single detection method, where the single detection method is electrochemical detection or optical detection.

[0244] A method is disclosed for determining the absence of at least one specific target or template nucleic acid molecule in a sample, comprising: (a) contacting a sample containing one or more target nucleic acid molecules with one or more signal probes (such probes may contain two or more labels, at least two of which are of different modalities, but others may be the same, and may be labeled internally and / or at or near the 3' end of the signal probe and / or at or near the 5' end); and (b) failure to detect or measure one or more target nucleic acid molecules by electrochemical detection and failure to detect or measure one or more target nucleic acid molecules by optical detection. Such detection failures indicate the absence of at least one specific target or template nucleic acid molecule in the sample.

[0245] A method is disclosed for determining the absence of at least one specific target or template nucleic acid molecule in a sample, comprising: (a) contacting a sample containing one or more target nucleic acid molecules with one or more signal probes (such probes may contain two or more labels, at least two of which are of different modalities, but the others may be the same, and may be labeled internally and / or at or near the 3' end of the signal probe and / or at or near the 5' end); and (b) failure to detect or measure one or more target nucleic acid molecules by a single detection method, where the single detection method is electrochemical detection or optical detection. Such detection failure indicates the absence of at least one specific target or template nucleic acid molecule in the sample.

[0246] In a further embodiment, the present invention provides a method for detecting a target analyte in a sample. The method involves adding a sample to a composition as outlined above, such that the target analyte is bound to a transport composition (signaling probe) and a reporter composition (capture probe) to form an assay complex, and the presence or absence of a first signaling moiety (e.g., ETM, e.g., ferrocene, ferrocene derivatives, methylene blue, osmium signaling moiety) and a second signaling moiety (e.g., an optical signaling moiety, e.g., FM) is detected, and the first and second signaling moieties are of different modalities. In some embodiments, the detector is an ETM detector, an optical detector, or both. In some embodiments, the ETM and OSM are excited by applying a voltage. In some embodiments, the ETM and OSM are excited by a light or laser beam source. In some embodiments, the ETM is excited by a light or laser beam source and the OSM is excited by a voltage. In some embodiments, the OSM is excited by a light or laser beam source and the ETM is excited by a voltage.

[0247] More specifically, the target nucleic acid is extracted from a sample and amplified using polymerase chain reaction (PCR). The resulting double-stranded DNA is then digested by an exonuclease to produce single-stranded DNA. The single-stranded DNA is bound to a signal probe having a first signaling region (e.g., ETM, e.g., ferrocene, a ferrocene derivative, methylene blue, or osmium) and a second signaling region (e.g., an optical signaling region, e.g., FM), where the first and second signaling regions are of different modalities ("signal complex"). When the signal complex is bound to a capture probe ("hybridize complex"), the first signaling region (e.g., ETM, e.g., ferrocene) and the second signaling region (e.g., an optical signaling region, e.g., FM) are brought near the surface of an electrode (gold, silver, or platinum electrode). In some embodiments, a voltage specific to the first signaling region (e.g., an ETM such as ferrocene) is applied. In some embodiments, a voltage specific to a second signaling portion (e.g., an optical signaling portion such as an FM) is applied (i.e., a single voltage excites either the ETM or the optical signaling portion, or both, but not both). In some embodiments, a voltage specific to the hybridized complex is applied (i.e., a single voltage excites both the ETM and the optical signaling portion). An electrical signal specific to the first signaling portion (e.g., an ETM such as ferrocene) is generated as a byproduct of a reduction-oxidation reaction when a voltage is applied to the system (Figure 1a). A first detection instrument measures and interprets this electrical output (e.g., in units such as nanoamperes, nA, uA, mA) to determine the result for each target (detected or not detected). An optical / fluorescent signal specific to the second signaling portion (e.g., an optical signaling portion such as an FM) is generated as a byproduct when a voltage is applied to the system. A second detection instrument measures and interprets this fluorescence output (of wavelength) to determine the result for each target (detected, not detected, or target concentration).In some embodiments, a first detection instrument measures and interprets the fluorescence output from the OSM (in units such as nanoamperes, nA, uA, mA, etc.) to determine the result for each target (detection, non-detection, or target concentration).

[0248] In some embodiments, a light or laser beam specific to a first signaling portion (e.g., an ETM such as ferrocene) is applied. In some embodiments, a light or laser beam specific to a second signaling portion (e.g., an optical signaling portion such as an FM) is applied (i.e., a single wavelength excites either the ETM or the optical signaling portion, but not both). In some embodiments, a light or laser beam specific to a hybridized complex is applied (i.e., a single wavelength excites both the ETM and the optical signaling portion). An electrical signal specific to the first signaling portion (e.g., an ETM such as ferrocene) is generated as a byproduct of a reduction-oxidation reaction when a light or laser beam is applied to the system. A first detection instrument measures and interprets this electrical output (e.g., in units such as nanoamperes, nA, uA, mA) to determine the result (detection or non-detection) for each target. An optical / fluorescent signal specific to the second signaling portion (e.g., an optical signaling portion such as an FM) is generated as a byproduct when a light or laser beam is applied to the system. A second detection instrument measures and interprets this fluorescence output (in wavelength) to determine the result for each target (detected, not detected, or target concentration). In some embodiments, the second detection instrument measures and interprets the signal (in wavelength units) from the ETM to determine the result for each target (detected or not detected).

[0249] The use of a “signal probe” is disclosed which has a ferrocene tag (N6, QW56, or QW80) toward or near its 5' end, and a second label toward or near its 5' end. The use of a “signal probe” is disclosed which has a ferrocene tag (N6, QW56, or QW80) toward or near its 5' end, and a second label elsewhere. In some embodiments, the second label is a colorimetric label, an luminescent label, or an optical label such as a fluorescent label such as ethidium bromide, fluorescein, or green fluorescent protein. As shown in Figure 1b, the signal probe binds to a capture probe and carries the ferrocene label(s) and optical label(s)(s) toward the surface of the electrode to generate a first electrical signal and a second fluorescent signal when a voltage is applied. In some embodiments, the first electrical signal is detected by an electrochemical detector and the second fluorescent signal is detected by a fluorescence detector. In some embodiments, the first electrical signal is detected by an electrochemical detector, and the second fluorescent signal is detected by an electrochemical detector. In some embodiments, the first electrical signal is detected by a fluorescence detector, and the second fluorescent signal is detected by a fluorescence detector.

[0250] In some embodiments, a signal probe is bound to a capture probe, which carries ferrocene labels(or more) and optical labels(or more) near the electrode surface to generate a first electrical signal and a second fluorescence signal when light or a laser beam is applied. In some embodiments, the first electrical signal is detected by an electrochemical detector and the second fluorescence signal is detected by a fluorescence detector. In some embodiments, the first electrical signal is detected by a fluorescence detector and the second fluorescence signal is detected by an electrochemical detector. In some embodiments, the first electrical signal is detected by a fluorescence detector and the second fluorescence signal is detected by a fluorescence detector.

[0251] The methods described herein can be better understood by the following numbered paragraphs. Paragraph 1: A method for performing nucleic acid detection, (a) Combining reagents for polymerase chain reaction, DNA polymerase, target nucleic acid, and primers, (b) cycling the mixture of (a) to provide multiple copies of the amplicon, (c) Exposing the mixture from (b) to an exonuclease to produce a single-stranded amplicon, (d)(c) Exposure of the above mixture to a signal probe oligonucleotide complementary to the single-stranded amplicon, wherein the signal probe oligonucleotide comprises a first electrochemically detectable label and an optically detectable label, thereby hybridizing the signal probe oligonucleotide to the single-stranded amplicon; (e)(d) is exposed to a capture probe oligonucleotide complementary to the single-stranded amplicon, wherein the capture probe oligonucleotide is bound to the electrode surface, thereby hybridizing the mixture in (d) to the capture probe oligonucleotide. (f) Applying a voltage to the electrode surface, (g) To detect the electrochemically detectable label and the optically detectable label mentioned above. Methods that include...

[0252] Paragraph 2. The method according to Paragraph 1, wherein the target nucleic acid is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid, as well as modifications and derivatives thereof.

[0253] Paragraph 3. The method according to Paragraph 1, wherein the target nucleic acid is extracted from blood, oral swabs, tissues, bodily fluids, environmental samples, the surface of materials, plants, animals, bacteria, or fungi.

[0254] Paragraph 4. The method according to Paragraph 1, wherein the electrochemically detectable label is an electron transfer portion.

[0255] Paragraph 5. The method according to Paragraph 1, wherein the electrochemically detectable label is a ferrocene label.

[0256] Paragraph 6. The method according to paragraph 1, wherein the cycling is isothermal.

[0257] Paragraph 7. The method according to paragraph 1, wherein the capture probe oligonucleotide is immobilized on a gold surface or a carbon-based surface.

[0258] Paragraph 8. The method according to paragraph 1, wherein the detection is electrochemical.

[0259] Paragraph 9. The method according to paragraph 1, wherein the detection is optical.

[0260] Paragraph 10. The method according to paragraph 1, wherein the optical label is not exposed to a second label or a second moiety having a quencher.

[0261] The methods described herein may be better understood by the following numbered paragraphs. Paragraph 1. A method of performing nucleic acid detection, comprising: (a) exposing a sample to a first signal probe having a first oligonucleotide sequence, a first detectable label, and a second detectable label, wherein the first detectable label and the second detectable label are of different modalities; (b) exposing the sample to a second signal probe having a first oligonucleotide sequence, a second oligonucleotide sequence, a first detectable label, and a third detectable label, wherein the first detectable label and the second detectable label are of different modalities, the second detectable label and the third detectable label are of different modalities, and the first oligonucleotide sequence and the second oligonucleotide sequence are of different modalities; (c) exposing the mixture of (a) and (b) to a first capture oligonucleotide complementary to the first signal probe and a second capture oligonucleotide complementary to the second signal probe; (d) Hybridizing the above signal probe with the above capture oligonucleotide, (e) detecting the above-mentioned mark associated with the first detectable mark rather than the second or third detectable mark in (i), (ii) the first detectable mark and the second detectable mark rather than the third detectable mark, (iii) the first detectable mark and the third detectable mark rather than the second detectable mark, or (iv) the second detectable mark rather than the first detectable mark and the third detectable mark. Methods that include...

[0262] Methods for detecting target pathogens A method for detecting a target pathogen in a sample using a photodetector and an electrochemical detector, a. To provide a system, the system is i. An immobilized capture DNA sequence probe positioned on a surface substrate, wherein the substrate is an electrode or connected to an electrode, and the immobilized capture DNA sequence probe has a DNA sequence complementary to a known DNA sequence of a target pathogen. ii. A capture DNA sequence probe having a DNA sequence complementary to a known DNA sequence of a target pathogen, wherein the free capture DNA sequence probe has a fluorophore or equivalent and an electron transfer moiety (ETM) attached to it, and To provide a system that includes, b. Contacting an immobilized capture DNA sequence probe with a sample containing a target pathogen, wherein any DNA sequence of the target pathogen binds to the immobilized capture DNA sequence probe. c. Contacting a DNA sequence bound to a target pathogen with a free-captured DNA sequence probe, wherein the free-captured DNA sequence probe binds to the DNA sequence of the target pathogen, causing the fluorophore or its equivalent to be positioned approximately 5 nm to 50 nm from the surface substrate. d. Applying a voltage to an electrode, thereby exciting a fluorophore or its equivalent; e. Measuring the fluorescence of a fluorophore or its equivalent using a photodetector, and measuring the current of an ETM using a current detector, wherein the current is not proportional to the amount of ETM, and the fluorescence is proportional to the amount of the fluorophore or its equivalent. Methods including the following are disclosed.

[0263] The data demonstrates the absence of significant interference / quenching caused by the gold electrodes. See Figures 15 and 16.

[0264] The substrate may include any form of metal such as silver, gold, platinum, zinc, aluminum, indium, palladium, rhodium iron, nickel, copper, carbon, and combinations thereof, and more preferably the substrate is gold. The substrate may also include glass, quartz, cellulose, and / or polymer materials.

[0265] In some embodiments, at least a portion of each capture probe-signal probe-target hybridization complex is in contact with a polar solvent, or a dipolar aprotic solvent having a dipole moment and inducible such as water, other polar solvents including methanol or acetic acid, ionic salt solutions and / or acetone, ethylene acetate.

[0266] Therefore, the system requires post-amplification processing before detecting OSM.

[0267] Assay The assay is disclosed, and this method is a. To provide at least one container or housing, wherein a first electrode and a second electrode are positioned within the container or connected to the container in a manner that allows communication with the container. b. Introducing a capture probe into a container, wherein the container contains a polar solution, the capture probe is connected to the surface of the container, and is connected to communicate with a first electrode and a second electrode. c. Introducing a molecule that exhibits signal transduction when excited by voltage, and positioning such a molecule near a capture probe, such that the molecule is positioned at a predetermined proximity to the electrode surface. d. Measuring the signal when excited by voltage using an optical detector. Includes.

[0268] Real-time detection Real-time PCR (also known as quantitative PCR (qPCR)) offers the ability to detect amplification reactions in real time, in contrast to conventional PCR. Reaction kinetics can be monitored in the liquid phase while the amplification process is still underway. Real-time chemistry enables the detection of PCR amplification during early stages of the reaction. Based on the increase in fluorescence intensity from a specific dye, the concentration of a target can be determined even before the amplification reaches its plateau.

[0269] A method for measuring the amount of amplicons in PCR in real time is disclosed using a signal probe having one or more electroactivity indicators (oxidized moieties, reduced moieties, redox moieties, and / or transition metal complexes) and one or more optical activity indicators (e.g., ethidium bromide, fluorescein, or green fluorescent protein).

[0270] Accordingly, one aspect of this subject relates to a first signaling portion useful for electrochemically detecting amplified nucleic acids and a second signaling portion for quantifying the amplified nucleic acids in real time or after each PCR thermal cycle, comprising contacting an amplified sample containing a target nucleic acid with a dual-labeled signal probe that adds potential (labeled with at least one electroactive indicator and at least one optical indicator), and detecting or measuring the electrical signals generated by the electroactive indicator and the optical indicator in real time or after each PCR thermal cycle, thereby quantifying the amount of nucleic acid present in the sample. In some embodiments, no optical signal is applied to the complex to excite the OSM.

[0271] In one aspect, a real-time solid-phase method for electrochemically or electrically monitoring or quantifying the amount of nucleic acid(s) by forming polynucleic acid(s) generated by polymerase chain reaction (PCR), comprising: a. contacting a sample containing the target nucleic acid(s) with a polymerase chain reaction enzyme(s) under conditions effective for PCR amplification to occur; b. amplifying the target nucleic acid(s); c. dissociating the amplified target nucleic acid(s) to form single-stranded target nucleic acid; d. hybridizing the single-stranded target nucleic acid(s) with a signal probe to form a signal-target hybridization complex, wherein the signal probe comprises a first ETM and a second optical label; e. hybridizing the signal-target hybridization complex with a capture probe bound to an electrode; f. applying a potential to the sample and detecting or measuring in real time the signal(s) generated by the optical label; g. quantifying the amount of nucleic acid(s) present in the sample and the amount of polynucleic acid(s) generated by correlating the change(s) in the optical signal(s) over time with the formation of polynucleic acid(s). The electrode may comprise a surface including a solid support, which may include glass, and the surface of at least one electrode(s) patterned and incorporated into a microchip comprises indium tin oxide, gold, or platinum. The microchip may further comprise a temperature sensor(s) and a microheater(s) integrated therein. The temperature sensor(s) and the microheater(s) may also be off-chip. In some embodiments, the device is a portable device and / or a microdevice.

[0272]

[0273] ​ By combining electrochemical detection methods with optical detection methods, a superior method for detecting and quantifying target molecules such as nucleic acids or nucleic acid-binding molecules is disclosed that is less expensive, simpler, and more accurate than conventional methods.

[0274] The methods described herein can be understood more fully by referring to the following numbered paragraphs.

[0275] Paragraph 1. An amplification method for real-time analysis of a nucleic acid sample, comprising the steps of: contacting the sample with a nucleic acid primer or probe that can hybridize with a selected target nucleic acid under chain extension conditions; performing polymerase-mediated chain extension to produce a PCR product, wherein the PCR product does not have a detectable label; performing PCR post-treatment, wherein a detectable label is hybridized to the PCR product; and detecting the presence or absence of a detectable signal based on the presence or absence of a target nucleic acid in the sample.

[0276] Paragraph 2. The method according to Paragraph 1, wherein the PCR post-treatment comprises contacting the PCR product with an FM-labeled signal probe to generate a signal probe / PCR product complex.

[0277] Paragraph 3. The method according to Paragraph 2, further comprising contacting the signal probe / PCR product complex with a capture probe to generate a signal probe / PCR product / capture probe complex.

[0278] Paragraph 4. The method according to Paragraph 3, further comprising contacting the signal probe / PCR product complex with a capture probe to generate a signal probe / PCR product / capture probe complex.

[0279] Paragraph 5. The method according to Paragraph 4, further comprising applying a voltage to a signal probe / PCR product / capture probe complex, thereby detecting the presence or absence of a target nucleic acid in the sample.

[0280] The methods described herein can be understood more fully by referring to the following numbered paragraphs.

[0281] Paragraph 1. A method for detecting a target nucleic acid in a sample, comprising: providing a reaction mixture comprising a target nucleic acid, at least one oligonucleotide primer, and DNA polymerase; amplifying the target nucleic acid; providing at least one oligonucleotide probe wherein the probe comprises an optical signaling moiety; hybridizing the at least one oligonucleotide probe to the amplified target nucleic acid; and detecting the complex wherein the presence of the complex indicates the presence of the target nucleic acid in the sample.

[0282] Paragraph 2. The method according to Embodiment 1, wherein the target nucleic acid comprises at least one of DNA and RNA.

[0283] Paragraph 3. The method according to any of the preceding paragraphs, wherein the target nucleic acid is RNA, and the amplification of the target nucleic acid comprises the step of synthesizing at least one DNA copy of the above RNA using reverse transcriptase.

[0284] Paragraph 4. The method according to any of the preceding paragraphs, wherein multiple target nucleic acids are amplified and detected.

[0285] Paragraph 5. The method according to Embodiment 4, wherein at least one oligonucleotide primer and at least one oligonucleotide probe are provided for all target nucleic acids that are amplified and detected.

[0286] Paragraph 6. The method according to Embodiment 4, wherein two or more oligonucleotide primers are provided for all of the target nucleic acids that are amplified and detected.

[0287] Paragraph 7. The method according to any of the preceding paragraphs, wherein the detection of the target nucleic acid is performed after amplification.

[0288] Paragraph 8. A method according to any of the preceding paragraphs, wherein the detection of a target nucleic acid is performed in real time.

[0289] Paragraph 9. The method according to any of the preceding paragraphs, wherein detection comprises hybridizing the complex to a capture probe, the capture probe being bound to an electrode.

[0290] Paragraph 10. The method according to any of the preceding paragraphs, wherein detection comprises hybridizing a complex to a capture probe, the capture probe being immobilized on an electrode, and applying a voltage to the electrode.

[0291] Paragraph 11. The method according to any of the preceding paragraphs, wherein detection comprises hybridizing a complex to a capture probe, the capture probe being immobilized on an electrode, applying a voltage to the electrode, and detecting a signal via an optical detector.

[0292] Paragraph 12. The method according to any of the preceding paragraphs, wherein the oligonucleotide probe further comprises an ETM, and detection comprises hybridizing the complex to a capture probe, wherein the capture probe is bound to an electrode, applying a voltage to the electrode, and detecting a signal via an optical detector and an ETM detector.

[0293] Paragraph 13. The method according to any of the preceding paragraphs, wherein amplification and detection of the target nucleic acid are performed to measure the amount of the target nucleic acid in the sample.

[0294] Paragraph 14. Amplification of a target nucleic acid, including the use of isothermal amplification, as described in any of the preceding paragraphs.

[0295] Paragraph 15. The method according to any of the preceding paragraphs, wherein the optical signaling portion is fluorescently labeled.

[0296] Paragraph 16. The method according to any of the preceding paragraphs, wherein detection comprises detecting a detection signal, and the detection of the signal indicates the presence of a target nucleic acid in the reaction mixture.

[0297] Paragraph 17. The method according to any of the preceding paragraphs, wherein multiple target nucleic acids are amplified and detected, at least one of the above oligonucleotide primers is provided for all amplified target nucleic acids, and at least one of the above oligonucleotide probes is provided for all detected target nucleic acids.

[0298] Paragraph 18. The method according to any of the preceding paragraphs, wherein the optical label comprises at least one of a fluorescent label, a fluorescent polarizing label, or a FRET probe, wherein the FRET probe changes its fluorescence properties when it forms a complex with a target nucleic acid, and the change indicates the presence of the complex.

[0299] Paragraph 19. The method according to Embodiment 18, wherein the FRET probe includes a hybridization trigger FRET probe.

[0300] Paragraph 20. The hybridization trigger FRET probe according to Embodiment 19, comprising at least one of a Scorpion primer and a Beacon probe.

[0301] Paragraph 21. The method according to Embodiment 20, wherein the FRET probe includes at least one of a severable FRET probe and a severable FRET probe including a TaqMan probe.

[0302] Paragraph 22. Detection PCR is a method described in any of the preceding paragraphs, including quantitative PCR.

[0303] Determination of target concentration As mentioned above, electrochemical detection is an endpoint detection system. It cannot be used to determine the concentration of amplification in the target. By adding a second optical label to the signal probe that can be detected during the amplification cycle, the endpoint detection system is transformed into a real-time detection system.

[0304] A detection system for detecting the concentration of a target analyte is disclosed, comprising: a first measurement unit comprising a cartridge, the cartridge comprising an optical measuring instrument configured to measure the concentration of the target analyte, wherein the first measurement unit measures the concentration of the target analyte by an optical method after exciting a signaling portion with a voltage; and a second measurement unit comprising an electrochemical measuring instrument configured to measure the presence, rather than the concentration, of the target analyte, wherein the second measurement unit measures the presence of the target analyte by an electrochemical method after exciting an ETM with a voltage.

[0305] In some embodiments, the optical measuring instrument does not have an optical excitation mechanism (such as a light source) for exciting the optical signal transduction portion.

[0306] In some embodiments, the optical signaling portion / fluorescence portion is excited by the same voltage that excites the ETM. In some embodiments, the optical signaling portion / fluorescence portion is excited by a different voltage than that that excites the ETM.

[0307] A method for detecting the concentration of a target analyte is disclosed, comprising: binding the target analyte to a signal probe; obtaining a first measurement from an optical signaling portion, wherein the first measurement is obtained by measuring the concentration of the target analyte using an optical method; and obtaining a second measurement from the ETM, wherein the second measurement is obtained by measuring the presence of the target analyte using an electrochemical method, rather than by measuring the concentration of the target analyte using an optical method.

[0308] A method for quantifying a target nucleic acid molecule is disclosed, comprising: contacting a sample containing a mixture of nucleic acids including the target nucleic acid molecule with at least one oligonucleotide containing a first signaling moiety and a second signaling moiety, wherein the first signaling moiety undergoes a detectable redox reaction upon application of charge and the second signaling moiety undergoes a detectable optical reaction upon application of charge; and observing the reaction, wherein the observable reaction is proportional to the amount of the target nucleic acid molecule in the sample.

[0309] Multiplex composition A multiplex assay is a type of assay that measures multiple analytes simultaneously in a single experiment. The multiplexing capability of the detection system is increased by adding an optical signaling portion to the signal probe.

[0310] Aspects of this disclosure relate to a method for determining which of several different target nucleic acids is present in a test sample. In some embodiments, different capture probes with different binding affinities are attached to different electrodes so that different targets are detected on different electrodes (see Figures 4a and 4b). Furthermore, multiplexing can be enhanced by using an optical signaling moiety for control and an ETM for detection. In some embodiments, the ETM is used for control and the optical signaling moiety is used for detection.

[0311] In some embodiments, the method involves contacting a sample with a plurality of different signal probes, where the first signal probe includes an ETM and a first optical signaling portion, and the second signal probe includes an ETM and a second optical signaling portion, and the first and second optical signaling portions are of different modalities and can be distinguished from each other. See Figure 5.

[0312] Paragraph 1. A device for multiple detection of target analytes, a) A first signal probe including an ETM and a first optical signaling portion, b) A second signal probe including the ETM and a second optical signaling portion, c) Capture probe and, d) A first detector capable of detecting the signal generated by the ETM, e) A second detector capable of distinguishing the first optical signal generated by the first optical signaling portion from the second optical signal generated by the second optical signaling portion, thereby detecting a target. A device equipped with the following features.

[0313] Paragraph 2. A method for multiple detection of target analytes in a sample, a) Exposing the sample to a first signal probe including the ETM and a first optical signaling portion, b) Exposing the sample to a second signal probe including the ETM and a second optical signaling portion, c) To generate a first signal-target hybridization complex, the first signal probe is hybridized to a first portion of the sample, d) To generate a second signal-target hybridization complex, the second signal probe is hybridized to a second portion of the sample, e) Hybridizing the first signal-target hybridization complex to the first capture probe in order to form the first capture probe hybridization complex, f) Hybridizing the second signal-target hybridization complex with the second capture probe in order to form a second capture probe hybridization complex, g) Applying a voltage to the first capture probe hybridization complex and the second capture probe hybridization complex, d) Detecting optical signals from the first capture-probe hybridization complex and the second capture-probe hybridization complex, e) Detecting electrochemical signals from a first capture probe hybridization complex and a second capture probe hybridization complex, thereby detecting a target analyte in the sample. Methods that include...

[0314] SNP analysis by fluorescence detection Methods, kits, and signal probes for detecting single nucleotide polymorphisms (SNPs) in target nucleic acids in a sample are described herein. Compared to other methods, the increased sensitivity of real-time PCR for detecting SNPs in target nucleic acids, as well as the improved functionality of real-time PCR, including sample containment and real-time detection of amplified products, makes the implementation of this technique for routine diagnosis and detection of SNPs in target nucleic acids in clinical laboratories feasible. The methods provided avoid problems of sample contamination, false negatives, and false positives.

[0315] This method may include performing at least one cycling step, which involves amplifying one or more portions of a target nucleic acid molecule in a sample, such as a gene target containing a SNP of interest to be detected, using one or more primers or one or more primer pairs. The primers anneal specifically to the nucleic acid sequence target and initiate synthesis from the nucleic acid sequence target under appropriate conditions. Each primer anneals to a region within or adjacent to the respective target nucleic acid molecule such that at least a portion of each amplification product contains the nucleic acid sequence corresponding to the respective target and, if present, the SNP. The amplification products are produced, provided that the target nucleic acid is present in the sample, regardless of whether the SNP of interest is present in the target nucleic acid molecule.

[0316] The method may also include a hybridization step, which involves contacting the amplification product with an SNP-specific signal probe containing a nucleic acid sequence complementary to the SNP-containing region of the amplification product. The SNP-specific signal probe may include a first detectable label and a second detectable label (the first and second detectable labels being of different modalities). The signal probe may be designed to include a non-naturally occurring nucleic acid region that may contain one or more altered nucleotides that are not part of a naturally occurring sequence, or one or more additional non-naturally occurring nucleotides that are nucleotides added to a naturally occurring sequence.

[0317] To detect whether a SNP of interest is present within a nucleic acid target in a sample, an amplification product is detected by activating a detectable label on a signal probe. If the amplification product is detected by the SNP-specific signal probe, the presence of the SNP is indicated. Conversely, if the amplification product is not detected by the SNP-specific signal probe, the presence of the SNP is not indicated. Therefore, the presence of an amplification product indicates the presence of the SNP in the target nucleic acid, and the absence of an amplification product indicates the absence of the SNP in the target nucleic acid.

[0318] To detect SNPs in a target nucleic acid sequence, primers and probes for amplifying the target nucleic acid sequence can be prepared. Functional variants can also be evaluated for specificity and / or sensitivity by those skilled in the art using routine methods. Typical functional variants may include, for example, one or more deletions, insertions, and / or substitutions in the primers and / or probes disclosed herein. For example, substantially identical variants of a primer or probe can be provided, having at least, for example, 80%, 90%, or 95% sequence identity with respect to a single original primer and probe or its complement.

[0319] Functionally active variants of either the primer and / or probe can be identified that provide similar or higher specificity and sensitivity in the methods, kits, or signal probes described herein, compared to their respective original sequences.

[0320] As described herein, amplification products can be detected using labeled signal probes that utilize FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of amplification products, and therefore the presence or absence of SNPs in the target nucleic acid. TaqMan® technology utilizes a single-strand hybridization signal probe labeled with two fluorescent sites. When the first fluorescent site is excited with light of an appropriate wavelength, the absorbed energy is transferred to the second fluorescent site according to the FRET principle. The second fluorescent site is typically a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is degraded during the subsequent extension phase by the 5'-to-3' exonuclease activity of Taq polymerase. As a result, the excited fluorescent site and the quencher site are spatially separated from each other. Consequently, when the first fluorescent site is excited in the absence of the quencher, fluorescence emission from the first fluorescent site can be detected. For example, the ABI PRISM® 7700 sequence detection system (Applied Biosystems) is suitable for carrying out the methods described herein for detecting the presence or absence of SNPs in a target nucleic acid, using TaqMan® technology. Roche's Lightcycler or COBAS can also detect SNPs in a target.

[0321] Generally, the presence of FRETs indicates the presence of SNPs in the target nucleic acid in the sample, and the absence of FRETs indicates the absence of HSV-1 and / or HSV-2 in the sample. However, insufficient sample collection, delayed transport, inappropriate transport conditions, or the use of certain collection swabs (calcium alginate or aluminum shafts) are all conditions that can affect the success and / or accuracy of the test results. Using the method disclosed herein, for example, detection of FRETs within the 45 cycling step indicates the presence of SNPs in the target nucleic acid in the sample.

[0322] Typical biological samples that can be used to implement the methods of the present invention include, but are not limited to, skin swabs, nasal swabs, wound swabs, blood cultures, and skin and soft tissue infections. Methods for collecting and storing biological samples are known to those skilled in the art. Biological samples can be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release target nucleic acids, or in some cases, the biological samples can be brought into direct contact with PCR reaction components and appropriate oligonucleotides.

[0323] While each thermocycler is running, control samples can be cycled similarly. Positive control samples can amplify target nucleic acid control templates (other than the amplified product of the described target gene) using, for example, control primers and control probes. Positive control samples can also amplify plasmid constructs containing, for example, the target nucleic acid molecule. Such plasmid controls can be amplified internally (e.g., within the sample) or in a separate sample run alongside the patient sample. Each thermocycler run can also include negative controls lacking, for example, target template DNA. Such controls are indicators of the success or failure of amplification, hybridization, and / or FRET reactions. Thus, control reactions can easily determine, for example, the ability of primers to anneal and initiate extension by sequence specificity, as well as the ability of probes to hybridize by sequence specificity and for FRET to occur.

[0324] In one embodiment, the method includes a step to avoid contamination. For example, an enzymatic method utilizing uracil-DNA glycosylase is described in U.S. Patents No. 5,035,996, No. 5,683,896, and No. 5,945,313 for reducing or eliminating contamination between one thermocycler operation and the next.

[0325] The method of the present invention can be implemented using conventional PCR methods combined with FRET technology. In one embodiment, a LightCycler® instrument is used. The following patent applications describe real-time PCR used with LightCycler® technology: International Publication No. 97 / 46707, International Publication No. 97 / 46714, and International Publication No. 97 / 46712.

[0326] LightCycler® can be operated using a PC workstation and can utilize the Windows NT operating system. The signal from the sample is obtained as the machine sequentially places the capillary onto the optical unit. The software can display the fluorescence signal in real time immediately after each measurement. After each cycling step, a quantitative display of fluorescence versus cycle count can be continuously updated for all samples. The generated data can be saved for further analysis.

[0327] It is understood that embodiments of the present invention are not limited to the configuration of one or more commercially available devices.

[0328] Paragraph 1. A method for detecting the presence or absence of single nucleotide polymorphisms (SNPs) in target nucleic acids in a sample, If the target nucleic acid is present in the sample, the amplification step includes contacting the sample with a primer containing a first nucleic acid sequence to produce an amplification product containing a region containing an SNP. A hybridization step is performed which includes adding an SNP-specific signal probe containing a second nucleic acid sequence complementary to the SNP-containing region of the amplified product to a sample, and, if the target nucleic acid is present in the sample, contacting the amplified product with the SNP-specific signal probe, wherein the SNP-specific signal probe contains an ETM and a fluorescent moiety, and the hybridization step is performed. The detection of the presence or absence of amplification products, where the presence of amplification products indicates the presence of SNPs in the target nucleic acid. Methods that include...

[0329] Paragraph 2. A kit for detecting single nucleotide polymorphisms (SNPs) in target nucleic acids in a sample, If the target nucleic acid is present in the sample, at least one primer containing a first nucleic acid sequence specific to produce an amplification product containing a region containing an SNP, An SNP-specific signal probe comprising a second nucleic acid sequence complementary to the SNP-containing region of the amplification product, wherein the SNP-specific signal probe comprises an ETM and a fluorescent moiety. A kit that includes the following:

[0330] Paragraph 3. A single nucleotide polymorphism (SNP) specific signal probe comprising a nucleic acid sequence complementary to the SNP-containing region of an amplification product, the SNP-specific signal probe comprising an ETM and a fluorescent moiety.

[0331] Paragraph 4. The method, kit, or signal probe according to paragraphs 1-3, wherein the donor fluorescence portion comprises a donor fluorescence portion and an acceptor portion of the donor fluorescence portion.

[0332] Paragraph 5. The method, kit, or signal probe according to Paragraph 4, wherein the acceptor portion is located internally within the SNP-specific hydrolysis probe.

[0333] Paragraph 6. The method, kit, or signal probe according to Paragraph 4, wherein the ETM is at the 5' end, the donor fluorescent moiety is at the 5' end, and the acceptor moiety is within 5 nucleotides of the donor fluorescent moiety on the signal probe.

[0334] Paragraph 7. The method, kit, or signal probe according to Paragraph 4, wherein the acceptor portion is the quencher.

[0335] Paragraph 8. The method according to Paragraph 1, wherein the amplification step utilizes a polymerase enzyme having 5'-3' exonuclease activity.

[0336] Paragraph 9. The method, kit, or signal probe according to any of the preceding paragraphs, wherein the first nucleic acid sequence of the primer and / or the second nucleic acid sequence of the signal probe comprises at least one modified nucleotide.

[0337] Paragraph 10. The method, kit, or signal probe according to any of the preceding paragraphs, wherein the first nucleic acid sequence of the primer and / or the second nucleic acid sequence of the signal probe have 40 or fewer nucleotides.

[0338] Measuring the location of the capture probe Variations in manufacturing can lead to false positives or false negatives. Furthermore, sample-to-answer systems with variable detection limits may detect pathogens in some cases but not in others. Therefore, the ability to monitor and improve quality control during manufacturing is both a requirement and best practice.

[0339] The detectable label on the capture probe may be an ETM or an optical signaling moiety. In some embodiments, a first plurality of detectable labels have ETMs, and a second plurality of capture probes have optical labels. The capture probe bound to the detectable label may be specific to the analyte of interest. The capture probe bound to the detectable label may function as a control and may contain its own signal that is distinguishable from that bound to the signal probe. In other words, the labeled nucleic acid capture probe may have the same or a different redox potential as the label on the signal probe. In other words, the labeled nucleic acid capture probe may have the same or a different modality as the label on the signal probe. In some cases, the signal probe may and may not be able to bind to the labeled capture probe. In some embodiments, the label on the capture probe and the label on the signal probe are identical, but they do not have to be identical. In some embodiments, they are both energy transfer moieties. In some embodiments, they are both optical signaling moieties. In some embodiments, they are ferrocene systems, ferrocene derivative compounds, methylene blue, or osmium. As shown in Figure 3a, the detectable label, which may be an optical label, is located at the free end of the capture probe. As shown in Figure 3b, the detectable marker, which may be an optical marker, is closer to the electrode surface and connected to the electrode surface via a linker.

[0340] For example, Figure 13 shows the background fluorescence of the detection electrode / gold pad. Almost no fluorescence is detected. In contrast, Figure 14 shows nucleic acid staining on the electrode. Fluorescence is detected. Finally, Figure 17 shows that the distribution of the capture probe can be visualized by hybridizing it with a signal probe containing a fluorescent marker.

[0341] Paragraph 1. An electrode sensor device configured to evaluate a capture probe deposited on an electrode surface in order to determine whether the electrode is within a first defined parameter and / or whether the capture probe spotting device is within a second defined parameter, A spotting device with a detection probe, A capture probe including an optical signaling portion, An electrode having at least one capture probe spotted and configured to receive a voltage, An optical detector configured to detect an optical signaling portion when excited by voltage, A processor for analyzing the optical signaling portion when excited by voltage. An electrode sensor device comprising the above features.

[0342] Paragraph 2. A method for peeling off a detection electrode when it satisfies defined parameters, The process involves spotting a capture probe onto a detection electrode, wherein the capture probe includes an optical signaling portion. Applying voltage to the capture probe, The detection of a signal generated by an optical signaling portion, wherein if the signal generated by the optical signaling portion is within a predetermined threshold, the detection electrode satisfies defined parameters and can be incorporated into a detection cartridge. Methods that include...

[0343] Paragraph 3. A method for removing a detection electrode spotting device when it meets defined criteria, The process involves spotting a capture probe onto a detection electrode, wherein the capture probe includes an optical signaling portion. Applying voltage to the capture probe, The detection of a signal generated by an optical signaling portion, wherein if the signal generated by the optical signaling portion is within a predetermined range, the detection electrode spotting device meets defined criteria and can be used to manufacture a detection electrode. Methods that include...

[0344] Paragraph 4. The method according to Paragraph 2 or 3, wherein a predetermined threshold and / or range includes the location, density, concentration, or pattern of the capture probe.

[0345] Paragraph 5. The method according to any one of paragraphs 2 to 4, further comprising determining the concentration of a capture probe spotted on the electrode surface.

[0346] Paragraph 6. The method according to Paragraph 5, further comprising adjusting the detection signal generated by the optical signaling portion based on the concentration of a capture probe spotted on the electrode surface.

[0347] Paragraph 7. The method according to any one of paragraphs 2 to 6, further comprising the step of repositioning the electrode surface with the capture probe if the location, density, concentration, or pattern of the capture probe falls below a predetermined threshold and / or range.

[0348] Paragraph 8. The electrode sensor according to Paragraph 1, further comprising a surface regenerator for cleaning the electrode surface if the location, density, concentration, or pattern of the capture probe falls below a predetermined threshold and / or range.

[0349] Probe location for filtering and removing probes in detection analysis Therefore, methods are developed that can achieve the detection of spatial information (e.g., capture probe location and / or distribution).

[0350] In some embodiments, certain probe signals, such as peripheral probes, probes too close to each other, or probes that are not uniformly spotted, may be filtered out. The filtering process examines the probe location and disqualifies certain probes from detection analysis.

[0351] Paragraph 1. A method for determining whether a target analyte is present in a sample, comprising: acquiring and storing location information of each capture probe on an electrode; determining, based on the stored locations, whether a first probe should be filtered out of detection analysis; selecting a subset of capture probes corresponding to probes having desired locations; and determining, based on the selected subset of capture probes, whether a target analyte is present.

[0352] Paragraph 2. The method according to Paragraph 1, wherein the determination step includes determining the location of the probe relative to the electrode and / or other probes on the electrode.

[0353] Paragraph 3. A method according to any of the preceding paragraphs, wherein the step of selecting a subset includes the step of selecting all probes within a defined area.

[0354] Paragraph 4. The method of any of the preceding paragraphs, further comprising the step of filtering the selected subset to remove outlier probe values ​​before the determination step.

[0355] Paragraph 5. The method according to any of the preceding paragraphs, wherein the filtering step includes removing any probes that have signals of two standard deviations from a normal distribution.

[0356] Paragraph 6. The method according to any of the preceding paragraphs, wherein the filtering step includes determining the average signal of the probes and removing probes having a signal exceeding a predetermined amount from the determined average signal.

[0357] Paragraph 7. A system for determining whether a target analyte is present in a sample, comprising: a location fetcher for acquiring and storing location information of a plurality of capture probes; a location processor for determining the location of each of the plurality of capture probes on an electrode; and a mapping engine for selecting a subset of the plurality of capture probes corresponding to probes having a desired location on the electrode.

[0358] Paragraph 8. The system further comprises a filter for removing captured probe signals having signal ratings outside of the specified ratings.

[0359] Method for calibrating a sensor assembly The sensor assembly can measure various characteristics of the capture probes deposited on the electrode surface, including the position, density, and pattern of the capture probes. The sensed information is used to ensure that the electrode is properly manufactured and that the capture probe deposition values ​​are within a target range (parameter), thereby allowing the detection signal to be reliably provided with detection results. The sensor assembly can also measure various metrics used to assess the status or "health" of the electrode. The position, density, or pattern of the capture probes may indicate problems related to the manufacturing of the electrode or the capture probe droplet generator (i.e., the device that deposits the capture probes onto the electrode).

[0360] In some embodiments, the electrochemical signal and the optical signal are transmitted simultaneously. In some embodiments, the electrochemical signal is received by the electrochemical sensor at the same time that the optical signal is received by the optical sensor. In some embodiments, the correlation calculator receives the electrochemical signal from the electrochemical sensor at the same time that the optical signal is received from the optical sensor.

[0361] In some embodiments, the electrochemical signal and the optical signal are transmitted continuously. In some embodiments, the electrochemical signal is received by the electrochemical sensor before or after the optical signal is received by the optical sensor. In some embodiments, the correlation calculator receives the electrochemical signal from the electrochemical sensor before or after the optical signal is received from the optical sensor.

[0362] kit Kits for detecting nucleic acid molecules in a sample are disclosed. Such kits may also be designed to detect target nucleic acid molecules during or after a nucleic acid amplification reaction. Such kits may be diagnostic kits in which the presence of nucleic acids correlates with the presence or absence of a disease or disorder. Kits for carrying out the amplification reactions described herein and kits for preparing the compositions described herein are disclosed.

[0363] In certain embodiments, the kit comprises one or more dual-labeled signal probes as defined herein. The kit may further comprise additional components for carrying out a detection assay or other method. Such a kit may comprise one or more additional components selected from the group comprising, consisting of, or essentially comprising, one or more polymerases (e.g., DNA polymerase and reverse transcriptase), one or more nucleotides, one or more buffer salts (including nucleic acid amplification buffer), one or more control nucleic acid target molecules (to act as a positive control for testing the assay), instructions for carrying out the method, etc.

[0364] Kit for detecting SNPs In another embodiment, a kit is provided for detecting SNPs in a target nucleic acid in a sample, comprising: at least one primer comprising a first nucleic acid sequence specific for generating an amplification product of the target nucleic acid; and an SNP-specific double-labeled signal probe comprising a second nucleic acid sequence complementary to the SNP-containing region of the amplification product, wherein the SNP-specific double-labeled signal probe comprises a first detectable label and a second detectable label (where the first and second detectable labels are of different modalities). The kit may also comprise a polymerase enzyme having 5'-3' exonuclease activity, and a capture probe comprising a nucleic acid sequence complementary to the SNP-containing region of the amplification product.

[0365] All references cited herein are incorporated herein by reference in their entirety.

Claims

1. A method for detecting the presence or absence of a target in a sample, a. Hybridizing a target analyte to a first signal probe in order to form a first signal probe-target hybridization complex, wherein the first signal probe has a first signaling portion and a second signaling portion, and the first signaling portion and the second signaling portion are detectable using different detection modalities. b. Hybridizing the formed first signal probe-target hybridization complex to the first capture probe in order to form a first capture probe hybridization complex, c. By applying a voltage to the sample, the first signal transduction portion and the second signal transduction portion are excited, d. Detecting the presence or absence of the target in the sample using electrochemical detection and optical detection. Methods that include...

2. The method according to claim 1, wherein the first signal transduction portion includes an electron transfer portion, and the second signal transduction portion includes an optical signal portion.

3. The method according to any one of claims 1 to 2, wherein the first signal transduction portion of the signal probe includes electron transfer partial labeling at its 5' end, 3' end, or both the 5' end and the 3' end.

4. The method according to any one of claims 1 to 3, wherein the second signal transduction portion of the signal probe includes an optical label at the 5' end, the 3' end, or both the 5' end and the 3' end.

5. The method according to claim 4, wherein the signal probe includes an optical label inside the signal probe.

6. The method according to any one of claims 1 to 5, wherein the signal probe includes a plurality of electron-mobility partial markers located at at least two different locations.

7. The method according to any one of claims 1 to 6, wherein the signal probe comprises a first portion that can hybridize to the target analyte, a second portion that cannot bind to the capture probe, and a third portion including the first signal transduction portion and the second signal transduction portion.

8. The method according to any one of claims 1 to 6, wherein the signal probe comprises a first portion that can hybridize to the target analyte, a second portion including a linker, and a third portion including the first signaling portion and the second signaling portion, the linker linking the first portion and the third portion.

9. The method according to any one of claims 1 to 8, wherein the first signal transduction portion is detected via electrochemical detection.

10. The method according to any one of claims 1 to 9, wherein the second signal transduction portion is detected via optical detection.

11. The method according to any one of claims 1 to 10, wherein the first signal transduction portion is ferrocene, a ferrocene derivative, or osmium.

12. The method according to any one of claims 1 to 10, wherein the first signal transduction portion is methylene blue.

13. The method according to any one of claims 1 to 12, wherein the optical signal portion is a fluorophore.

14. The method according to claim 13, wherein the fluorophore is fluorescein.

15. A method for detecting the presence or absence of a target in a sample, a. Hybridizing a target analyte to a first signal probe in order to form a signal transduction complex, wherein the first signal probe has a first signal transduction portion and a second signal transduction portion, and the first signal transduction portion and the second signal transduction portion are of different modalities. b. Hybridizing the signal transduction complex with the first capture probe in order to form a capture probe hybridization complex, c. Exciting the first signal transduction portion and the second signal transduction portion by applying light or a laser beam, d. Detecting the presence or absence of the target in the sample using electrochemical detection and optical detection. Methods that include...

16. The method according to claim 15, wherein the first signal transduction portion is an electrochemically detectable signal transduction portion, and the second signal transduction portion is an optical signal transduction portion.

17. The method according to any one of claims 15 to 16, wherein the first signal probe cannot be coupled to the first capture probe.

18. The method according to any one of claims 15 to 16, wherein the signal transduction complex is unable to bind to the first capture probe.

19. The method according to any one of claims 15 to 16, wherein a first portion of the signal transduction complex binds to the first capture probe, and a second portion of the signal transduction complex binds to the second capture probe.

20. The method according to any one of claims 15 to 19, wherein the first signal transduction portion is ferrocene, a ferrocene derivative, or osmium.

21. The method according to any one of claims 15 to 19, wherein the first signal transduction portion is methylene blue.

22. The method according to any one of claims 15 to 21, wherein the optical signal portion is a fluorophore.

23. The method according to claim 22, wherein the fluorophore is fluorescein.

24. A method for detecting the presence or absence of a target in a sample, a. Hybridizing the target analyte to the first signal probe in order to form a signal probe-target hybridization complex, b. Hybridizing the signal hybridization complex with the first capture probe in order to form a capture probe hybridization complex, c. Exciting the first signal probe by applying light or a laser beam and voltage, d. Detecting the presence or absence of the target in the sample using electrochemical detection and optical detection. Methods that include...

25. The method according to claim 24, wherein the first signal probe includes a first signal transduction portion which is an electron transfer portion and a second signal transduction portion which is a GFP.

26. The method according to any one of claims 24 to 25, wherein the first signal transduction portion is detected via electrochemical detection.

27. The method according to any one of claims 24 to 26, wherein the second signal transduction portion is detected via optical detection.

28. The method according to any one of claims 24 to 27, wherein the first signal transduction portion is not excited by the application of voltage.

29. A signal probe comprising a first signaling portion and a second signaling portion, wherein the first signaling portion and the second signaling portion are detectable by different detection modalities.

30. The signal probe according to claim 29, wherein the first signaling portion is ferrocene, a ferrocene derivative, or osmium.

31. The signal probe according to claim 29, wherein the first signal transduction portion is methylene blue.

32. The signal probe according to any one of claims 29 to 31, wherein the second signal transduction portion is a fluorophore.

33. The signal probe according to claim 32, wherein the fluorophore is fluorescein.

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