High-robustness and high-signal-intensity digital immunodetection kit and method

Abstract

The invention relates to the field of quarantine detection, and discloses a high-robustness and high-signal-intensity digital immunodetection kit and method.The kit comprises an inert nucleic acid sealing agent and a nucleic acid embedding dye; the inert nucleic acid sealing agent is coated on the surface of the solid phase carrier or contained in the rolling circle amplification reaction liquid; the nucleic acid intercalation dye is contained in the rolling circle amplification reaction liquid, and the emission spectrum of the nucleic acid intercalation dye is compatible with the emission spectrum of the sequence specific probe with the fluorescence label. Strong and uniform signals can be converted into accurate quantitative results only by depending on accurate single magnetic bead counting. And meanwhile, the flow design difficulty is also reduced by clear signal separation, so that the single magnetic bead analysis in the presence of the sealing agent is simpler and more reliable. Through the combined action of the two, the kit is especially suitable for stable and accurate quantification of the extremely low abundance protein marker in a complex clinical sample.

Description

Technical Field

[0001] This invention relates to the field of quarantine and testing, and more particularly to a highly robust digital immunoassay kit and method with high signal intensity. Background Technology

[0002] In the prior art, the ultra-sensitive digital immunoassay method of rolling circle amplification (RCA) and flow cytometry captures single target protein molecules on antibody-coated magnetic beads, amplifies the signal of each binding event in situ via RCA to generate long single-stranded DNA tandems, hybridizes with sequence-specific fluorescent probes, and finally performs digital counting using flow cytometry.

[0003] However, when applying this technology to clinical practice, two key issues affecting its detection performance and reliability were discovered: 1. Magnetic bead aggregation problem: RCA amplification generates single-stranded DNA tandem repeats of thousands of bases. These long nucleic acids easily induce non-specific aggregation of immunomagnetic beads through base pairing, electrostatic interactions, and hydrophobic interactions. Magnetic bead aggregation leads to abnormal forward / lateral scattering signals during flow cytometry, decreased single-particle gating efficiency, significantly increased coefficient of variation (CV), quantitative result deviation, and even flow cytometer tubing blockage, seriously affecting the stability, repeatability, and clinical applicability of the detection method.

[0004] Signal intensity and uniformity issues: When relying solely on sequence-specific probes for hybridization labeling, the signal intensity may be affected by probe hybridization efficiency, local folding of RCA products, or steric hindrance. Clausson et al. (2015) pointed out that RCA products (RCP) may exhibit irregular signal focal points or even be dispersed into multiple signal spots due to the spontaneous folding of single-stranded DNA. This can affect the uniformity and intensity of the fluorescence signal of a single magnetic bead in flow cytometry, thereby affecting the signal-to-noise ratio and interpretation accuracy of low-concentration sample detection. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the problems of (1) magnetic bead aggregation caused by RCA long chain products and (2) insufficient or uneven RCA product signal intensity on a single magnetic bead in the application of existing digital immunoassay methods based on RCA amplification in IVD kits, thereby comprehensively improving the robustness, accuracy and sensitivity of the kit detection.

[0006] To address the aforementioned technical problems, this invention provides a highly robust digital immunoassay kit with high signal intensity, comprising: A solid-phase carrier coated with trapping molecules; Detection molecules with first affinity tags; A circular DNA template with a second affinity tag, wherein the first affinity tag and the second affinity tag can specifically bind; Rolling circle amplification enzyme; Rolling circle amplification reaction solution, wherein the rolling circle amplification reaction solution contains deoxynucleoside triphosphate and a sequence-specific probe labeled with fluorescence; The kit also includes an inert nucleic acid blocking agent and a nucleic acid intercalation dye; The inert nucleic acid blocking agent is coated on the surface of the solid-phase support or contained in the rolling circle amplification reaction solution; The nucleic acid intercalation dye is contained in the rolling circle amplification reaction solution, and the emission spectrum of the nucleic acid intercalation dye is compatible with the emission spectrum of the sequence-specific probe with fluorescent label.

[0007] This invention also provides a highly robust digital immunoassay method with high signal intensity, comprising the following steps: Step S1: Mix and incubate the sample to be tested with a solid support coated with the capturing molecules; Step S2: Add the detection molecule with the first affinity tag to the reaction system, incubate and wash, then add the circular DNA template with the second affinity tag, incubate and wash, the first affinity tag and the second affinity tag can specifically bind; Step S3: Add rolling circle amplification enzyme and rolling circle amplification reaction solution to the reaction system to perform rolling circle amplification reaction and wash. The rolling circle amplification reaction solution contains deoxynucleoside triphosphate and a sequence-specific probe with fluorescent label. Step S4: Resuspend the solid support after the reaction in step S3, input it into a flow cytometer for detection, collect forward scattered light signal, side scattered light signal and fluorescence channel signal, calculate the signal value AMP, and substitute the signal value AMP into the calibration curve to calculate the concentration of the target in the sample to be tested. The surface of the solid-phase support is pre-coated with an inert nucleic acid blocking agent, or an inert nucleic acid blocking agent is added to the rolling circle amplification reaction solution in step S3. In step S3, a nucleic acid intercalation dye is added to the rolling circle amplification reaction solution, and the emission spectrum of the nucleic acid intercalation dye is compatible with the emission spectrum of the sequence-specific probe with fluorescent label. In step S3, the fluorescently labeled sequence-specific probe hybridizes with the single-stranded DNA product generated by rolling circle amplification, and the nucleic acid intercalation dye intercalates into both the double-stranded and single-stranded regions of the single-stranded DNA product for dual fluorescent labeling.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows: Completely solves the problem of magnetic bead aggregation and improves detection reliability: The sealing agent ensures that the magnetic beads are accurately analyzed by the flow cytometer in a monodisperse state, fundamentally eliminating counting errors caused by aggregation and improving the precision and upper limit of the dynamic range of the detection.

[0009] Significantly enhanced and homogenized single magnetic bead signals, improving sensitivity and signal-to-noise ratio: The intercalating dye and specific probe work synergistically to dual-label RCA products. The intercalating dye can bind extensively to any double-stranded and single-stranded region of the RCA product, greatly amplifying the fluorescence signal intensity of each "open" magnetic bead while making the signal distribution more uniform. This directly improves the signal-to-noise ratio for detecting low-concentration samples, potentially further reducing the detection limit, and making the fluorescence signals of the magnetic beads more clearly distinguishable.

[0010] Synergistic effects enhance robustness: Two improvements complement each other. Strong and homogeneous signals require accurate single-bead counting to translate into accurate quantification. Simultaneously, clear signal separation simplifies flow cytometry design, making single-bead analysis in the presence of a blocking agent simpler and more reliable. Together, these improvements make this kit particularly suitable for the robust and accurate quantification of extremely low-abundance protein biomarkers in complex clinical samples. Attached Figure Description

[0011] Figure 1 This is a schematic diagram of the comparative example of the present invention without the addition of inert nucleic acid blocking agent and nucleic acid intercalation dye; Figure 2 This is a schematic diagram illustrating the addition of an inert nucleic acid blocking agent and a nucleic acid intercalation dye in an embodiment of the present invention. Detailed Implementation

[0012] The terms “first,” “second,” “third,” “fourth,” etc. (if present) in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms “comprising” or “having,” and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0013] A highly robust digital immunoassay kit with high signal intensity, comprising: A solid-phase carrier coated with trapping molecules; Detection molecules with first affinity tags; A circular DNA template with a second affinity tag, wherein the first affinity tag and the second affinity tag can specifically bind; Rolling circle amplification enzyme; Rolling circle amplification reaction solution, wherein the rolling circle amplification reaction solution contains deoxynucleoside triphosphate and a sequence-specific probe labeled with fluorescence; The kit also includes an inert nucleic acid blocking agent and a nucleic acid intercalation dye; The inert nucleic acid blocking agent is coated on the surface of the solid-phase support or contained in the rolling circle amplification reaction solution; The nucleic acid intercalation dye is contained in the rolling circle amplification reaction solution, and the emission spectrum of the nucleic acid intercalation dye is compatible with the emission spectrum of the sequence-specific probe with fluorescent label.

[0014] The inert nucleic acid blocking agent is selected from at least one of naturally derived nucleic acids and artificially synthesized nucleic acids; the naturally derived nucleic acids include at least one of salmon sperm DNA and calf thymus DNA; the artificially synthesized nucleic acids include at least one of poly(A) adenine nucleotides and random sequence single-stranded DNA.

[0015] The nucleic acid intercalation dye is a fluorescent dye capable of binding to double-stranded DNA and single-stranded DNA, and the nucleic acid intercalation dye is selected from at least one of SYBR Green I, SYBR Green II, SYBR Gold and EvaGreen.

[0016] The solid-phase support is selected from magnetic beads, microspheres, or chips; the capture molecule is a capture antibody, the detection molecule is a detection antibody, and the kit is used to detect protein, nucleic acid, or exosome targets.

[0017] The first affinity tag is biotin, and the second affinity tag is streptavidin; the fluorescent substance labeled by the sequence-specific probe with fluorescent label is fluorescein isothiocyanate (FITC); the rolling circle amplification enzyme is phi29 enzyme.

[0018] A highly robust digital immunoassay method with high signal intensity includes the following steps: Step S1: Mix and incubate the sample to be tested with a solid support coated with the capturing molecules; Step S2: Add the detection molecule with the first affinity tag to the reaction system, incubate and wash, then add the circular DNA template with the second affinity tag, incubate and wash, the first affinity tag and the second affinity tag can specifically bind; Step S3: Add rolling circle amplification enzyme and rolling circle amplification reaction solution to the reaction system to perform rolling circle amplification reaction and wash. The rolling circle amplification reaction solution contains deoxynucleoside triphosphate and a sequence-specific probe with fluorescent label. Step S4: Resuspend the solid support after the reaction in step S3, input it into a flow cytometer for detection, collect forward scattered light signal, side scattered light signal and fluorescence channel signal, calculate the signal value AMP, and substitute the signal value AMP into the calibration curve to calculate the concentration of the target in the sample to be tested. The surface of the solid-phase support is pre-coated with an inert nucleic acid blocking agent, or an inert nucleic acid blocking agent is added to the rolling circle amplification reaction solution in step S3. In step S3, a nucleic acid intercalation dye is added to the rolling circle amplification reaction solution, and the emission spectrum of the nucleic acid intercalation dye is compatible with the emission spectrum of the sequence-specific probe with fluorescent label. In step S3, the fluorescently labeled sequence-specific probe hybridizes with the single-stranded DNA product generated by rolling circle amplification, and the nucleic acid intercalation dye intercalates into both the double-stranded and single-stranded regions of the single-stranded DNA product for dual fluorescent labeling.

[0019] The capture molecule is a capture antibody, the detection molecule is a detection antibody, the first affinity tag is biotin, the second affinity tag is streptavidin, and the rolling circle amplification enzyme is phi29 enzyme; The specific operating conditions for step S2 are as follows: after adding the detection molecule with the first affinity tag, incubate at 37°C with shaking for 1 hour; after adding the circular DNA template with the second affinity tag, incubate at 37°C with shaking for 15 minutes. The specific operating conditions for step S3 are as follows: after adding the rolling circle amplification enzyme and the rolling circle amplification reaction solution, incubate at 37°C with shaking for 1 hour.

[0020] In step S4, the step of substituting the signal value AMP into the calibration curve to calculate the concentration of the target in the sample to be tested specifically includes: The signal values ​​AMP were calculated after the calibrators of different known concentrations were detected by the flow cytometer. The calibration curve is generated by fitting a four-parameter or five-parameter model with the known concentration of the calibrator as the X-axis and the corresponding signal value AMP as the Y-axis. The signal value AMP obtained after detecting the sample to be tested is substituted into the calibration curve to calculate the concentration of the target in the sample to be tested.

[0021] The method further includes the following steps for calculating the minimum detection limit: The zero-concentration calibrator was used as a sample for repeated measurements to obtain the signal value AMP corresponding to the measurement results. Calculate the average value M and standard deviation SD of the signal value AMP corresponding to the measurement result, and obtain the target signal value corresponding to the value M+2.5SD; A linear equation is derived by performing a two-point regression fitting based on the concentration and signal value AMP results between the zero-concentration calibrator and the adjacent concentration calibrators. Substitute the target signal value into the linear equation to calculate the corresponding concentration value as the limit of detection.

[0022] The following is the experimental part of this invention: Example 1:

[0023] 1. Add 10 μL of CEA capture antibody-coated magnetic beads to the reaction tube, then add 100 μL of the CEA antigen to be tested, and mix well; 2. Add 10 μL of biotin-labeled CEA detection antibody to the reaction tube, incubate at 37°C with shaking for 1 hour; wash 3 times with 200 μL PBST buffer and discard the supernatant; 3. Add 100 μL of streptavidin-circular DNA template conjugate to the reaction tube and incubate at 37°C with shaking for 15 minutes; wash three times with 200 μL PBST buffer and discard the supernatant; 4. Add 59 μL of RCA reaction solution (containing salmon sperm DNA and SYBR Green I and SYBR Green II dyes) and 1 μL of phi29 enzyme to the reaction tube, mix well, and incubate at 37°C with shaking for 1 hour. Wash 3 times with 200 μL of PBST buffer and discard the supernatant. The sample was resuspended in 5.50 μL of PBS buffer and then introduced into a flow cytometer for detection. The signals of the FSC, SSC, and FITC channels were acquired and the signal value AMP was calculated. The calibrator concentration was used as the X-axis and the AMP calculated from the calibrator detection was used as the Y-axis. A calibration curve was fitted using four or five parameters. The sample concentration was calculated by substituting the AMP of the sample into the calibration curve.

[0024] 6. Using the zero-concentration calibrator as the sample, perform the test 20 times, obtaining the AMP values ​​of the 20 measurements. Calculate the mean (M) and standard deviation (SD) of these AMP values, and then determine the AMP value corresponding to M + 2.5SD. Perform a two-point regression fitting based on the concentration-AMP values ​​between the zero-concentration calibrator and adjacent concentration calibrators to obtain a linear equation. Substitute the AMP value of M + 2.5SD into the equation to calculate the corresponding concentration value, which is the limit of detection. Data are shown in Table 1.

[0025] 7. Perform 10 repetitions each on quality control sample 1 and quality control sample 2, and calculate the mean M and standard deviation SD of the 10 measurements. Divide M by SD to obtain the coefficient of variation CV, which represents repeatability. Data are shown in Table 2. Example 2:

[0026] The detection steps are similar to those in Example 1 and Example 2, except that the RCA reaction solution does not contain SYBR Green I or SYBR Green II dyes.

[0027] The minimum detection limit data are shown in Table 1, and the repeatability data are shown in Table 2. Example 3:

[0028] Similar to the steps in Example 1 and Example 3, the difference is that the RCA reaction solution does not contain salmon sperm DNA.

[0029] The minimum detection limit data are shown in Table 1, and the repeatability data are shown in Table 2.

[0030] Example for comparison: The detection process was similar to that of Example 1, except that the RCA reaction solution did not contain salmon sperm DNA or SYBR Green I and SYBR Green II dyes.

[0031] The minimum detection limit data are shown in Table 1, and the repeatability data are shown in Table 2.

[0032] Table 1. Results of CEA minimum detection limit detection Table 2 CEA Repeatability Test Results like Figure 1-2 As shown, Figure 1 In the reaction system, no inert nucleic acid blocking agent or nucleic acid intercalation dye was added. Figure 2 In this kit, an inert nucleic acid blocking agent and a nucleic acid intercalation dye are added to the reaction system. The inert blocking agent can avoid the problem of magnetic bead aggregation and improve the reliability of detection. The nucleic acid intercalation dye can synergistically label RCA products with specific probes to enhance and homogenize the signal, improve sensitivity and signal-to-noise ratio. The synergistic effect of these two improvements makes the kit suitable for robust and accurate quantification of protein biomarkers with extremely low abundance in complex clinical samples.

[0033] As shown in Table 1, the limit of detection (LOD) is an important indicator of the sensitivity of immunological detection methods; a lower value indicates higher sensitivity. Examples 2 and 3 showed higher sensitivity than the control example, while Example 1 showed higher sensitivity than Examples 2 and 3. Adding an inert nucleic acid blocking agent or a nucleic acid intercalation dye to the reaction system can increase detection sensitivity; the combined use of both significantly increases the detection sensitivity.

[0034] As shown in Table 2, repeatability is an important indicator of the robustness of immunological detection methods; a lower value indicates better robustness. Examples 2 and 3 showed higher robustness than the control example, while Example 1 showed higher robustness than Examples 2 and 3. Adding an inert nucleic acid blocking agent or a nucleic acid intercalation dye to the reaction system can increase detection robustness; the combined use of both significantly enhances detection robustness.

[0035] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A highly robust digital immunoassay kit with high signal intensity, characterized in that, include: A solid-phase carrier coated with trapping molecules; Detection molecules with first affinity tags; A circular DNA template with a second affinity tag, wherein the first affinity tag and the second affinity tag can specifically bind; Rolling circle amplification enzyme; Rolling circle amplification reaction solution, wherein the rolling circle amplification reaction solution contains deoxynucleoside triphosphate and a sequence-specific probe labeled with fluorescence; The kit also includes an inert nucleic acid blocking agent and a nucleic acid intercalation dye; The inert nucleic acid blocking agent is coated on the surface of the solid-phase support or contained in the rolling circle amplification reaction solution; The nucleic acid intercalation dye is contained in the rolling circle amplification reaction solution, and the emission spectrum of the nucleic acid intercalation dye is compatible with the emission spectrum of the sequence-specific probe with fluorescent label.

2. The digital immunoassay kit according to claim 1, characterized in that, The inert nucleic acid blocking agent is selected from at least one of naturally derived nucleic acids and artificially synthesized nucleic acids; the naturally derived nucleic acids include at least one of salmon sperm DNA and calf thymus DNA; the artificially synthesized nucleic acids include at least one of poly(A) adenine nucleotides and random sequence single-stranded DNA.

3. The digital immunoassay kit according to claim 1, characterized in that, The nucleic acid intercalation dye is a fluorescent dye capable of binding to double-stranded DNA and single-stranded DNA, and the nucleic acid intercalation dye is selected from at least one of SYBR Green I, SYBR Green II, SYBR Gold and EvaGreen.

4. The digital immunoassay kit according to claim 1, characterized in that, The solid-phase support is selected from magnetic beads, microspheres, or chips; the capture molecule is a capture antibody, the detection molecule is a detection antibody, and the kit is used to detect protein, nucleic acid, or exosome targets.

5. The digital immunoassay kit according to claim 1, characterized in that, The first affinity tag is biotin, and the second affinity tag is streptavidin; the fluorescent substance labeled by the sequence-specific probe with fluorescent label is fluorescein isothiocyanate (FITC); the rolling circle amplification enzyme is phi29 enzyme.

6. A highly robust digital immunoassay method with high signal intensity, characterized in that, Includes the following steps: Step S1: Mix and incubate the sample to be tested with a solid support coated with the capturing molecules; Step S2: Add the detection molecule with the first affinity tag to the reaction system, incubate and wash, then add the circular DNA template with the second affinity tag, incubate and wash, the first affinity tag and the second affinity tag can specifically bind; Step S3: Add rolling circle amplification enzyme and rolling circle amplification reaction solution to the reaction system to perform rolling circle amplification reaction and wash. The rolling circle amplification reaction solution contains deoxynucleoside triphosphate and a sequence-specific probe with fluorescent label. Step S4: Resuspend the solid support after the reaction in step S3, input it into a flow cytometer for detection, collect forward scattered light signal, side scattered light signal and fluorescence channel signal, calculate the signal value AMP, and substitute the signal value AMP into the calibration curve to calculate the concentration of the target in the sample to be tested. The surface of the solid-phase support is pre-coated with an inert nucleic acid blocking agent, or an inert nucleic acid blocking agent is added to the rolling circle amplification reaction solution in step S3. In step S3, a nucleic acid intercalation dye is added to the rolling circle amplification reaction solution, and the emission spectrum of the nucleic acid intercalation dye is compatible with the emission spectrum of the sequence-specific probe with fluorescent label. In step S3, the fluorescently labeled sequence-specific probe hybridizes with the single-stranded DNA product generated by rolling circle amplification, and the nucleic acid intercalation dye intercalates into both the double-stranded and single-stranded regions of the single-stranded DNA product for dual fluorescent labeling.

7. The digital immunoassay method according to claim 6, characterized in that, The inert nucleic acid blocking agent is selected from at least one of naturally derived nucleic acids and artificially synthesized nucleic acids; the naturally derived nucleic acids include at least one of salmon sperm DNA and calf thymus DNA; the artificially synthesized nucleic acids include at least one of poly(A) nucleotides and random sequence single-stranded DNA; the nucleic acid intercalation dye is a fluorescent dye capable of binding to double-stranded DNA and single-stranded DNA, and the nucleic acid intercalation dye is selected from at least one of SYBR Green I, SYBR Green II, SYBR Gold, and EvaGreen.

8. The digital immunoassay method according to claim 6, characterized in that, The capture molecule is a capture antibody, the detection molecule is a detection antibody, the first affinity tag is biotin, the second affinity tag is streptavidin, and the rolling circle amplification enzyme is phi29 enzyme; The specific operating conditions for step S2 are as follows: after adding the detection molecule with the first affinity tag, incubate at 37°C with shaking for 1 hour; after adding the circular DNA template with the second affinity tag, incubate at 37°C with shaking for 15 minutes. The specific operating conditions for step S3 are as follows: after adding the rolling circle amplification enzyme and the rolling circle amplification reaction solution, incubate at 37°C with shaking for 1 hour.

9. The digital immunoassay method according to claim 6, characterized in that, In step S4, the step of substituting the signal value AMP into the calibration curve to calculate the concentration of the target in the sample to be tested specifically includes: The signal values ​​AMP were calculated after the calibrators of different known concentrations were detected by the flow cytometer. The calibration curve is generated by fitting a four-parameter or five-parameter model with the known concentration of the calibrator as the X-axis and the corresponding signal value AMP as the Y-axis. The signal value AMP obtained after detecting the sample to be tested is substituted into the calibration curve to calculate the concentration of the target in the sample to be tested.

10. The digital immunoassay method according to claim 9, characterized in that, The method further includes the following steps for calculating the minimum detection limit: The zero-concentration calibrator was used as a sample for repeated measurements to obtain the signal value AMP corresponding to the measurement results. Calculate the average value M and standard deviation SD of the signal value AMP corresponding to the measurement result, and obtain the target signal value corresponding to the value M+2.5SD; A linear equation is derived by performing a two-point regression fitting based on the concentration and signal value AMP results between the zero-concentration calibrator and the adjacent concentration calibrators. Substitute the target signal value into the linear equation to calculate the corresponding concentration value as the limit of detection.

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