Single nanoparticle counting platform based on Argonaute protein

Abstract

The invention belongs to the field of detection of analytical chemistry, and relates to the field of single-particle inorganic mass spectrum sensing, in particular to a single-nanoparticle counting platform based on Argonaute protein, and the single-nanoparticle counting platform comprises a nanoparticle labeling system, a Thermophilus Argonaute (TtAgo) cutting system and a single-particle inductively coupled plasma mass spectrum detection system. According to the invention, high-sensitivity miRNA detection can be realized through an exponential isothermal amplification reaction (EXPAR) and a double-circulation mechanism of TtAgo shearing, and due to the cooperation of the double-circulation mechanism of EXPAR and TtAgo shearing and the precise recognition capability of TtAgo and multiple detection advantages of isotope labeling, the platform shows extremely high sensitivity and specificity. Meanwhile, the platform shows good robustness in human serum and a cell extracting solution, and has a huge prospect in the clinical field.

Description

Technical Field

[0001] The present invention belongs to the field of analytical chemistry detection, relates to the field of single-particle inorganic mass spectrometry sensing, and particularly relates to a single-nanoparticle counting platform based on the Thermus thermophilus Argonaute protein for simultaneously detecting three colorectal cancer-related microRNAs. Background Art

[0002] Nucleic acids are indispensable molecules in living organisms, and their detection has important application values in many fields such as clinical diagnosis, food safety, and environmental monitoring. Especially microRNA (miRNA), the expression level of which can reflect numerous physiological and pathological information. The combined detection of multiple miRNAs can significantly improve the accuracy of early cancer diagnosis, staging, and prognosis monitoring. However, due to the short and highly homologous miRNA sequences, developing specific and highly sensitive multiplex detection methods faces many challenges. Programmable nucleases are a class of molecular tools widely used in gene editing. As one of the representatives of programmable nucleases, the CRISPR / Cas system has become the core technology in the field of nucleic acid detection due to its successful application in detection platforms such as SHERLOCK and DETECTR. However, the guide strand of the CRISPR / Cas system is RNA, which has a high cost and is easily degraded, and its recognition requires dependence on specific protospacer adjacent motifs (PAMs) or protospacer flanking sites (PFS). Although Gootenberg et al. proposed a multiplex detection method based on orthogonal CRISPR enzymes, the combined use of multiple enzymes and the preference for specific sequences undoubtedly limit the application potential of the CRISPR system in flexible multiplex biosensing.

[0003] Prokaryotic Argonautes (pAgos), as an emerging programmable nuclease, show the potential to overcome the challenges faced by the CRISPR / Cas system and are expected to become an efficient multiplex detection tool. Different from the CRISPR / Cas system, pAgos do not rely on protospacer adjacent motifs (PAMs), but precisely recognize target nucleic acids through base pairing with guide DNA (gDNA) or guide RNA (gRNA). pAgos can cleave complementary DNA or RNA targets at the junction between the 10th and 11th nucleotides, and after releasing the cleaved targets, re-cleave. This programmability, specificity, and multi-turnover characteristics have prompted the development of many pAgo-based multiplex detection methods, such as SPOT, MULAN, TEAM, and STAR. However, most of the existing pAgo-based detection methods use fluorescence intensity as the signal readout method. The high background signal and spectral overlap problems of traditional fluorescent probes limit the further expansion of the number of detection channels.

[0004] Single-particle inductively coupled plasma mass spectrometry (SP-ICPMS) has high sensitivity and high resolution for metal stable isotopes and does not face the spectral overlap difficulties of fluorescence probes. As a potential multi-analysis platform, it has attracted much attention in the field of biosensing. SP-ICPMS can provide the concentration and particle size information of nanoparticles through high-frequency signal acquisition. This mode of detecting single nanoparticles eliminates the signal differences caused by the differences in nanoparticle size and concentration, and due to the sufficient metal content in single nanoparticles, its sensitivity is significantly improved compared with the traditional integrated signal readout mode. In previous studies, SP-ICPMS has been successfully applied to biological assays such as proteins, nucleic acids, enzymes, and cells, further confirming its unique advantages. Given its great application potential as a multi-detection tool and combined with the specific cleavage ability of Ago protein, it is expected to construct a highly sensitive and selective multi-miRNA analysis platform.

[0005] Based on this, there is an urgent need to provide a TtAgo-mediated single-particle counting platform for the detection of multiple miRNAs based on ICPMS. Summary of the Invention

[0006] The purpose of the present invention is to provide a single-nanoparticle counting platform based on Argonaute protein, which realizes the simultaneous detection of three colorectal cancer-related microRNAs (miRNAs) by analyzing the frequencies of three noble metal nanoparticle hybrid probes (AuNPs, AgNPs, PtNPs) simultaneously.

[0007] The purpose of the present invention is achieved by the following technical solutions:

[0008] The present invention provides a single-nanoparticle counting platform based on Argonaute protein, and the single-nanoparticle counting platform includes a nanoparticle labeling system, a TtAgo cleavage system, and a single-particle inductively coupled plasma mass spectrometry detection system.

[0009] Furthermore, the nanoparticle labeling system includes three kinds of nanoparticles modified with probe DNA, and the nanoparticles include gold nanoparticles, silver nanoparticles, and platinum nanoparticles.

[0010] Furthermore, the specific modification method of the nanoparticles includes the following steps:

[0011] Add 1 nmol of report DNA treated with TCEP and 5'-thiol-modified polyT40 to the nanoparticle solution in proportion, mix well and freeze overnight, naturally melt at room temperature, centrifuge to remove excess DNA, and redisperse it in TN buffer to obtain.

[0012] Furthermore, the ratio of the reporting DNA to the polyT40 is 1:100. Among them, the reporting DNA that recognizes miR-141 is bound to gold nanoparticles; the reporting DNA that recognizes miR-31 is bound to silver nanoparticles; the reporting DNA that recognizes miR-21 is bound to platinum nanoparticles.

[0013] Furthermore, the TtAgo cleavage system includes 100 nM TtAgo, reaction buffer, and 0.75 μM MnCl2.

[0014] The present invention also provides a miRNA detection method for the single nanoparticle counting platform described above. The miRNA detection method includes steps of EXPAR pre-amplification, TtAgo cleavage reaction, and SP-ICPMS quantitative detection.

[0015] Furthermore, the reactants for the EXPAR pre-amplification are: templates targeting miR-141, miR-31, and miR-21, dNTP, Nt.BstNBI, DNA polymerase, ET SSB, reaction buffer, 0.5×NEBuffer TM r3.1, and miR-141, miR-31, and miR-21 targets; the reaction conditions for the EXPAR pre-amplification are to react at 55 °C for 20 min.

[0016] Furthermore, the TtAgo cleavage reaction includes adding 2 μL of the EXPAR reaction mixture to 10 μL of the TtAgo cleavage system, incubating at 80 °C for 15 minutes. After the reaction ends, adding the mixture to the washed streptavidin magnetic beads and performing magnetic separation to remove the captured nanoparticles.

[0017] Furthermore, the SP-ICPMS quantitative detection is, after the TtAgo cleavage reaction ends, aspirating 2 μL of the supernatant after magnetic separation, diluting it 10 4 times with ultrapure water, inserting the aspiration pump tube of the ICPMS into the diluted solution, setting the frequency mode to collect signals from the solution, with a dwell time of 100 μs and a detection time of 20 s.

[0018] The present invention also provides an application of the single nanoparticle counting platform described above in the preparation of products for colorectal cancer diagnosis. The products can simultaneously detect three colorectal cancer-related microRNAs.

[0019] Beneficial effects:

[0020] The present invention provides an analytical method capable of realizing multiplex detection of miRNAs. Using nanoparticles as cleavage signal probes, a single-particle counting platform is developed for simultaneous detection of miR-141, miR-31, and miR-21. Thanks to the dual-cycle mechanism of EXPAR and TtAgo cleavage and the cooperation of the precise recognition ability of TtAgo and the advantages of isotope-labeled multiplex detection, this platform exhibits extremely high sensitivity and specificity. At the same time, this platform shows good robustness in human serum and cell extracts and has great prospects in the clinical field. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It is a mechanism diagram of the analytical method of the present invention; among them, (A) Michaelis-Menten kinetic analysis of different probe cleavages mediated by TtAgo; (B) Schematic diagram of the dual-cycle mechanism of EXPAR and TtAgo cleavage; (C) Schematic diagram of the single-particle counting platform for multiplex detection of different miRNA targets;

[0023] Figure 2 It is a feasibility analysis diagram in the analytical method of the present invention, where (A) The relative frequencies detected by SP-ICPMS under different conditions; (B) ICPMS spectra of mixed nanoprobes with or without targets;

[0024] Figure 3 It is a time-resolved data diagram of SP-ICPMS with different concentrations of target substances in the analytical method of the present invention, where (A-C) The instantaneous signal of Au when different concentrations of miR-141 are spiked; (D-F) The transient signal of Ag when different concentrations of miR-31 are spiked; (G-I) The instantaneous signal of Pt when different concentrations of miR-21 are spiked; [[ID=】]

[0025] Figure 4 It is a specific selectivity analysis diagram and a linear diagram in the detection of three target substances in the analytical method of the present invention, where (A) The specificity of this counting platform for single-mismatch (SM) or triple-mismatch (TM) sequences of miR-141, miR-31, and miR-21; (B) The selectivity of the counting platform; Calibration curves for multiplex detection of miR-141 (C), miR-31 (D), and miR-21 (E); Detailed Embodiments

[0026] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms used in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0030] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0031] The chemical reagents, biochemical reagents, and materials used in the present invention can be obtained from commercial sources unless otherwise specified.

[0032] The principle of the present invention is as follows: applying nanoprobes to the double - cycle mechanism of EXPAR and TtAgo cleavage. After the target binds to the amplification template, it can be extended by DNA polymerase, and the endonuclease cleaves and releases a single - stranded DNA with a 5'-phosphate end through the recognition site (5′-GAGTC-3′). This single - stranded DNA can not only continue to complementarily pair with the amplification template for repeated amplification and cleavage, but also serve as a guiding DNA to activate TtAgo, specifically recognize and cleave the reporter DNA. After the reporter DNA is cleaved, the biotin at its 5' end also falls off from the nanoparticle surface, resulting in the inability of the nanoprobe to be captured by streptavidin magnetic beads. The uncaptured nanoprobes are present in the supernatant after magnetic separation. After dilution, quantitative detection of the target can be achieved through single - particle counting analysis. Adding the amplification template corresponding to each target and the nanoprobe (miR - 141 corresponds to AuNPs, miR - 31 corresponds to AgNPs, miR - 21 corresponds to PtNPs) into the system can realize the simultaneous detection of multiple miRNAs.

[0033] The present invention first constructs a double - cycle mechanism of EXPAR and TtAgo cleavage. The principle is as follows: after the target binds to the amplification template, it can be extended by DNA polymerase, and the endonuclease cleaves and releases a single - stranded DNA with a 5'-phosphate end through the recognition site (5′-GAGTC-3′). This single - stranded DNA can not only continue to complementarily pair with the amplification template for repeated amplification and cleavage, but also serve as a guiding DNA to activate TtAgo, specifically recognize and cleave the reporter DNA. After the reporter DNA is cleaved, the biotin at its 5' end also falls off from the nanoparticle surface, resulting in the inability of the nanoprobe to be captured by streptavidin magnetic beads. The uncaptured nanoprobes are present in the supernatant after magnetic separation. After dilution, quantitative detection of the target can be achieved through single - particle counting analysis. Adding the amplification template corresponding to each target and the nanoprobe (miR - 141 corresponds to AuNPs, miR - 31 corresponds to AgNPs, miR - 21 corresponds to PtNPs) into the system can realize the simultaneous detection of multiple miRNAs.

[0034] The detection process for nanoparticle counting is as follows: after magnetic separation, the probe with biotin dropped off after being cleaved by TtAgo is free in the supernatant. The number of probes in the supernatant is positively correlated with the concentration of the target; the higher the concentration, the more the number of probe particles. Finally, by introducing the diluted supernatant into ICPMS for single - particle counting analysis, with a residence time of 100 μs and a detection time of 20 s, precise quantification of the target miRNA can be achieved. The mechanism diagram of the analysis method in the present invention is as Figure 1 shown.

[0035] The specific preparation methods of AuNPs, AgNPs and PtNPs in the present invention are as follows:

[0036] (1) Preparation of AuNPs

[0037] (1.1) Take 1 mL of 1% chloroauric acid (w / v) and add it to 80 mL of ultrapure water. Place a magnetic stir bar in a three-necked flask and mix well. Heat under reflux in a heating mantle until boiling and maintain the temperature for 5 min;

[0038] (1.2) Add 0.6 - 2 mL of 1% sodium citrate reducing agent and keep refluxing with stirring for 20 min;

[0039] (1.3) Turn off the temperature control system of the heating mantle, keep the magnetic stir bar stirring, and wait for the solution to cool slowly to room temperature. Pour out the solution into a storage bottle to obtain AuNPs.

[0040] (2) Preparation of PtNPs

[0041] (2.1) Mix 0.72 mL of 1% chloroplatinic acid with 49.28 mL of ultrapure water in a three-necked flask and heat to boiling in a heating mantle and maintain for 15 min;

[0042] (2.2) Subsequently, add a mixed solution of 1.1 mL of 1% sodium citrate reducing agent and 0.05% citric acid, and then immediately add 0.55 mL of 1% sodium citrate reducing agent, a mixed solution of 0.05% citric acid and 0.08 - 0.11% KBH4;

[0043] (2.3) Turn off the heating system of the heating mantle and let the mixed solution cool slowly to room temperature. Take out the seed nanoparticle solution and make up the volume;

[0044] (2.4) Mix 56.226 mL of ultrapure water with 2 mL of the seed solution, add 1.864 mL of 1% chloroplatinic acid solution, and then add a mixed solution of 1 mL of 1% sodium citrate reducing agent and 1.25% ascorbic acid solution;

[0045] (2.5) Slowly heat to boiling and maintain for 30 min. Take out the seed solution of the second stage and make up the volume;

[0046] (2.6) Mix 56.226 mL of ultrapure water with 8 - 16 mL of the seed solution of the second stage, add 1.864 mL of 1% chloroplatinic acid solution, and then add a mixed solution of 1 mL of 1% sodium citrate reducing agent and 1.25% ascorbic acid solution;[[ID=XXX]] [[ID=XXX]]

[0047] (2.7) Slowly heat to boiling and maintain for 30 min. Take out the Pt nanoparticle solution and make up the volume for standby;

[0048] (3) Preparation of AgNPs

[0049] It should be noted that there are two tags and

[0047] in the original text which seem to be incomplete or incorrect in the numbering. I have left them as they are in the translation. If there are specific corrections or additional information for these parts, the translation can be adjusted accordingly.(3.1) 100 mL of ultrapure water containing 5 mM sodium citrate and 1 mM tannic acid was placed in a three-necked flask and heated to boiling.

[0050] (3.2) 1 mL of 25 mM AgNO3 was added, and the mixture was kept boiling and stirred for 45 min. After the solution was slowly cooled to room temperature, the solution was poured into a storage bottle to obtain AgNPs.

[0051] Example 1

[0052] In this example, the specific construction and detection method of the single nanoparticle counting platform based on Argonaute protein are as follows:

[0053] (1) Preparation of AuNPs

[0054] (1.1) 1 mL of 1% chloroauric acid (w / v) was added to 80 mL of ultrapure water. A magnetic stirrer was placed in the three-necked flask and mixed evenly. The mixture was refluxed and heated to boiling in a heating mantle and kept at this temperature for 5 min.

[0055] (1.2) 0.8 mL of 1% sodium citrate reducing agent was added, and the mixture was kept refluxing with stirring for 20 min.

[0056] (1.3) The temperature control system of the heating mantle was turned off, and the magnetic stirrer was kept stirring. After the solution was slowly cooled to room temperature, the solution was poured into a storage bottle to obtain AuNPs.

[0057] (2) Preparation of PtNPs

[0058] (2.1) 0.72 mL of 1% chloroplatinic acid was mixed with 49.28 mL of ultrapure water in a three-necked flask and heated to boiling in a heating mantle and kept at this temperature for 15 min.

[0059] (2.2) Subsequently, a mixed solution of 1.1 mL of 1% sodium citrate reducing agent and 0.05% citric acid was added, and then 0.55 mL of 1% sodium citrate reducing agent, a mixed solution of 0.05% citric acid and 0.08 - 0.11% KBH4 was added immediately.

[0060] (2.3) The heating system of the heating mantle was turned off, and the mixed solution was slowly cooled to room temperature. The seed nanoparticle solution was taken out and made up to a certain volume.

[0061] (2.4) 56.226 mL of ultrapure water was mixed with 2 mL of the seed solution, 1.864 mL of 1% chloroplatinic acid solution was added, and then a mixed solution of 1 mL of 1% sodium citrate reducing agent and 1.25% ascorbic acid solution was added.

[0062] (2.5) The mixture was slowly heated to boiling and kept at this temperature for 30 min. The seed solution in the second stage was taken out and made up to a certain volume.

[0063] (2.6) Mix 56.226 mL of ultrapure water with 12 mL of the second-stage seed solution, add 1.864 mL of 1% chloroplatinic acid solution, and then add 1 mL of a mixed solution of 1% sodium citrate reducing agent and 1.25% ascorbic acid solution;

[0064] (2.7) Slowly heat to boiling, maintain for 30 min, take out the Pt nanoparticle solution and make up the volume for standby;

[0065] (3) Preparation of AgNOs

[0066] (3.1) Place 100 mL of ultrapure water containing 5 mM sodium citrate and 1 mM tannic acid in a three-necked flask and heat to boiling;

[0067] (3.2) Add 1 mL of 25 mM AgNO3, keep boiling and stirring for 45 min, wait for the solution to cool slowly to room temperature, pour out the solution into a storage bottle, and obtain AgNPs.

[0068] (4) Labeling probe DNA with nanoparticles

[0069] (4.1) Add 1 nmol of TCEP-treated probe DNA and 5′-thiol-modified polyT40 in a ratio of (Reporter:T40 = 1:100) to 1000 μL of nanoparticles (141-R add AuNPs, 31-R add AgNPs, 21-R add PtNPs), and mix well;

[0070] (4.2) Place at -20 °C and freeze overnight;

[0071] (4.3) After natural melting at room temperature, remove the excess DNA by centrifugation three times (10 minutes, 4 °C, 10000 rpm), and redisperse it in TN buffer for further use.

[0072] (5) Exponential isothermal amplification reaction

[0073] (5.1) Mix three templates targeting miR-141, miR-31, and miR-21 at 100 nM, dNTP (250 μM), Nt.BstNBI (0.4 U μL -1 ), Vent(exo-) DNA polymerase (0.05 U μL -1 ), ET SSB (15 ng mL -1 ), reaction buffer and 0.5×NEBuffer TM r3.1 and three targets (miR-141, miR-31, and miR-21);

[0074] (5.2) React at 55 °C for 20 min.

[0075] (6) TtAgo cleavage reaction

[0076] (6.1) Add 2 μL of the EXPAR reaction mixture to a 10 μL TtAgo cleavage system, which includes 100 nM TtAgo, reaction buffer, 0.75 μM MnCl2, and reporter gene DNA-modified nanoparticles (AuNPs corresponding to miR-141, AgNPs corresponding to miR-31, PtNPs corresponding to miR-21);

[0077] (6.2) After vortexing the solution evenly, incubate it at 80 °C for 15 min;

[0078] (6.3) Add 2.5 μL of the reaction mixture to 7.5 μL of washed streptavidin magnetic beads and incubate at room temperature for 30 min.

[0079] (7) Quantitative detection by SP-ICPMS

[0080] (7.1) After the reaction, pipette 2 μL of the supernatant after magnetic separation and dilute it with ultrapure water by ~10 4 times;

[0081] (7.2) Insert the liquid suction pump tube of the ICPMS into the diluted solution;

[0082] (7.3) Set the frequency mode to collect signals from the solution, with a dwell time of 100 μs and a detection time of 20 s.

[0083] Example 2 Exploration of the feasibility of the analysis method of the present invention

[0084] This example tested the frequency signal changes of the platform in the present invention under different conditions to verify the feasibility ( Figure 2 A), when neither TtAgo nor the target was added, there were no significant pulse frequency signals of Au, Ag, or Pt. Only when both TtAgo and the target were present, the frequency signal increased significantly. At the same time, the characteristic spectra of ICPMS showed that regardless of the presence or absence of the target, there was no crosstalk between the isotope peaks of Au, Ag, and Pt, with excellent resolution, providing a solid foundation for multiplex miRNA detection ( Figure 2 B). Figure 3 The characteristic spectra of Au, Ag, and Pt in SP-ICPMS were shown when the target concentrations were 0, 100 aM, and 100 fM. The higher the target concentration, the more pulses, indicating the quantitative feasibility of the strategy based on single nanoparticle counting.

[0085] Example 3 Exploration of the specificity and selectivity of the analysis method of the present invention

[0086] For multiplex detection, due to the short length and high homology of miRNAs sequences, selectivity has become an important indicator for measuring miRNA detection methods. To evaluate the selectivity of this counting platform, this example tested the single-base mismatch and three-base mismatch sequences of three target miRNAs. It can be concluded from Figure 4 A that this method can clearly distinguish mismatch sequences, demonstrating that this counting platform has good selectivity.

[0087] To verify the feasibility of multiplex analysis of this counting analysis platform, the frequency signals of different combinations of three target miRNAs were tested. As Figure 4 shown in B, the signals of Au, Ag, and Pt increase with the presence of the corresponding target substances, and there is no crosstalk among them, confirming that this method can effectively avoid cross-reactions, has good specificity, and is a highly potential multiplex analysis method.

[0088] Example 4 explores the detection linearity of the analysis method of the present invention for three miRNAs related to colorectal cancer

[0089] This example explored the performance of the proposed analysis strategy for three miRNAs for colorectal cancer diagnosis; measured the pulse frequencies of nanoparticles containing target miRNAs at different concentrations to establish a calibration curve;

[0090] (1) Linearity of miR-141 detection by SP-ICPMS

[0091] According to the linear relationship between the logarithm of miR-141 concentration and the SP-ICPMS frequency signal, the linear range is 100 aM - 100 pM, and the detection limit is 85 aM;

[0092] (2) Linearity of miR-31 detection by SP-ICPMS

[0093] According to the linear relationship between the logarithm of miR-31 concentration and the SP-ICPMS frequency signal, the linear range is 100 aM - 100 pM, and the detection limit is 21 aM;

[0094] (3) Linearity of miR-21 detection by SP-ICPMS

[0095] According to the linear relationship between the logarithm of miR-21 concentration and the SP-ICPMS frequency signal, the linear range is 100 aM - 100 pM, and the detection limit is 25 aM;

[0096] Example 5 explores the detection of three colorectal cancer markers in actual samples by the analysis method of the present invention

[0097] To verify the robustness and accuracy of this counting platform, in this embodiment, three target substances with different concentrations were spiked into 100-fold diluted healthy human serum and HEK293 total RNA extracts respectively, and the recovery rates were calculated. The specific results are shown in Table 1 below.

[0098] Table 1 Recovery Rates and Relative Standard Deviations of miRNAs in Human Serum and HEK293 Extracts

[0099]

[0100]

[0101] As can be seen from Table 1, in human serum, when the target substance concentrations were 1 fM and 100 fM, the recovery rates were in the ranges of 92.5 - 110% and 81.8 - 110% respectively, and the relative standard deviations were in the ranges of 2.87 - 4.70% and 1.07 - 2.28% respectively; in HEK293 total RNA extracts, when the target substance concentrations were 1 fM and 100 fM, the recovery rates were in the ranges of 84.3 - 106% and 92.2 - 101% respectively, and the relative standard deviations were in the ranges of 1.96 - 9.81% and 2.53 - 9.16% respectively. The recovery rate results indicate that this method has good specificity, can effectively avoid matrix interference, and demonstrates its potential in clinical applications.

[0102] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A single nanoparticle counting platform based on Argonaute protein, characterized in that, The single nanoparticle counting platform includes a nanoparticle labeling system, a TtAgo cleavage system, and a single particle inductively coupled plasma mass spectrometry detection system.

2. The single nanoparticle counting platform according to claim 1, wherein The nanoparticle labeling system includes three types of probe DNA-modified nanoparticles, which include gold nanoparticles, silver nanoparticles, and platinum nanoparticles.

3. The single nanoparticle counting platform according to claim 2, wherein The specific modification method of the nanoparticles includes the following steps: 1 nmol of TCEP-treated reporter DNA and 5'-end thiol-modified polyT40 are added to the nanoparticle solution in proportion, mixed evenly and frozen overnight, naturally melted at room temperature, centrifuged to remove excess DNA, and redispersed in TN buffer to obtain the product.

4. The single nanoparticle counting platform according to claim 3, characterized in that, The ratio of the reporter DNA to the polyT40 is 1:

100. Among them, the reporter DNA that recognizes miR-141 is bound to gold nanoparticles; the reporter DNA that recognizes miR-31 is bound to silver nanoparticles; the reporter DNA that recognizes miR-21 is bound to platinum nanoparticles.

5. The single nanoparticle counting platform according to claim 1, characterized in that, The TtAgo cleavage system includes 100 nM TtAgo, 1×ThermoPol® reaction buffer, and 0.75 μM MnCl2.

6. A method for miRNA detection using the single nanoparticle counting platform according to any one of claims 1-5, characterized in that, The miRNA detection method includes steps of EXPAR pre-amplification, TtAgo cleavage reaction, and SP-ICPMS quantitative detection.

7. The miRNA detection method according to claim 6, wherein, The reactants for the EXPAR pre-amplification are: templates targeting miR-141, miR-31, and miR-21, dNTP, Nt.BstNBI, DNA polymerase, ET SSB, 1×ThermoPol® reaction buffer, 0.5×NEBuffer™ r3.1, and miR-141, miR-31, and miR-21 targets; the reaction conditions for the EXPAR pre-amplification are to react at 55°C for 20 min.

8. The miRNA detection method according to claim 6, wherein The TtAgo cleavage reaction includes adding 2 μL of the EXPAR reaction mixture to 10 μL of the TtAgo cleavage system, incubating at 80°C for 15 minutes. After the reaction ends, the mixture is added to the washed streptavidin magnetic beads for magnetic separation to remove the captured nanoparticles.

9. The miRNA detection method according to claim 6, wherein The SP-ICPMS quantitative detection is as follows: after the TtAgo cleavage reaction ends, 2 μL of the supernatant after magnetic separation is aspirated and diluted 10 4 times with ultrapure water. The liquid suction pump tube of the ICPMS is inserted into the diluted solution, and the signal of the solution is collected in the set frequency mode. The residence time is 100 μs and the detection time is 20 s.

10. Use of the single nanoparticle counting platform according to any one of claims 1-5 in the preparation of a product for colorectal cancer diagnosis, characterized in that, The product can simultaneously detect three colorectal cancer-related microRNAs.

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