CN116203094A - Electrochemical staged detection method for single base mutant of multiple helicobacter pylori - Google Patents

Electrochemical staged detection method for single base mutant of multiple helicobacter pylori Download PDF

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CN116203094A
CN116203094A CN202211618727.7A CN202211618727A CN116203094A CN 116203094 A CN116203094 A CN 116203094A CN 202211618727 A CN202211618727 A CN 202211618727A CN 116203094 A CN116203094 A CN 116203094A
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dna
cas9
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胡善文
杨欢
黄冠泽
邓媛
叶为民
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Abstract

The invention belongs to the technical field of biological detection, and relates to an electrochemical staged detection method for a single base mutant of multiple helicobacter pylori. Electrochemical sensors employed in the methods include sensing electrodes, helper DNA, sgrnas, and Cas9 nucleases; the sensing electrode is composed of a conductive matrix, gold nanoparticles, probe DNA and nano CdS, wherein both ends of the probe DNA are respectively connected with the gold nanoparticles and the nano CdS, and the conductive matrix is connected with the gold nanoparticles; the probe DNA, the auxiliary DNA and the target DNA can be hybridized with each other to form a Y-shaped structure; the sgrnas and the Cas9 nucleases are capable of forming Cas9/sgRNA complexes, and the Cas9/sgRNA complexes are capable of disrupting the Y-structure, allowing separation of nano CdS from the Y-structure. The invention realizes the comprehensive evaluation of helicobacter pylori infection status and drug resistance status by detecting the total nucleic acid and single base mutant.

Description

Electrochemical staged detection method for single base mutant of multiple helicobacter pylori
Technical Field
The invention belongs to the technical field of biological detection, and relates to an electrochemical staged detection method for a single base mutant of multiple helicobacter pylori.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Helicobacter pylori (h.pyrri) is a gram-negative bacillus whose infection is closely related to many digestive system diseases. Currently, H.pyri infection control faces the problems of increased infection rate and increased drug resistance due to single nucleotide mutants (SNVs). Common detection techniques in H.pyrri clinical diagnosis include gastroscopy, 13C/14C urea breath, serum, fecal and urine tests. Currently, the detection technologies for SNV mainly include: melting curve analysis for SNV detection, SNV detection using enhanced hybridization probes, and protein-assisted SNV detection. However, there has been no method for simultaneously analyzing the total amount of nucleic acid and detecting a single genotype. Therefore, further research on a novel method for detecting helicobacter pylori DNA and single base mutant with high flux and high selectivity is important.
Disclosure of Invention
In order to solve the defects and actual demands of the prior art, the invention aims to provide an electrochemical detection method of a single base mutant of multiple helicobacter pylori, and comprehensive evaluation of helicobacter pylori infection status and drug resistance status is realized through detection of total nucleic acid and the single base mutant.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
in one aspect, an electrochemical sensor comprises a sensing electrode, an auxiliary DNA, a sgRNA, and a Cas9 nuclease;
the sensing electrode is composed of a conductive matrix, gold nanoparticles, probe DNA and nano CdS, wherein the probe DNA is of a hairpin structure, the two ends of the probe DNA are respectively connected with the gold nanoparticles and the nano CdS, and the conductive matrix is connected with the gold nanoparticles;
the probe DNA, the auxiliary DNA and the target DNA can be hybridized with each other to form a Y-shaped structure;
the sgrnas and the Cas9 nucleases are capable of forming Cas9/sgRNA complexes, which Cas9/sgRNA complexes are capable of disrupting the Y-structure, allowing separation of nano CdS from the Y-structure.
In another aspect, a method for electrochemical detection of multiple helicobacter pylori single base mutants provides the above electrochemical sensor; the method comprises the following steps:
adding auxiliary DNA and target DNA to be detected into a sensing electrode for incubation, and detecting ECL signals;
pre-incubating the sgrnas and Cas9 nucleases to obtain Cas9/sgRNA complexes;
adding the obtained Cas9/sgRNA complex to the incubated sensing electrode, continuing incubation, and detecting ECL signals;
detecting target DNA to be detected through ECL signal difference;
the target DNA is helicobacter pylori single base mutant.
In a third aspect, an electrochemical detection kit for helicobacter pylori single base mutants comprises the electrochemical sensor and a buffer solution.
Since the probe DNA is in a hairpin structure, the gold nanoparticles and the nano CdS are close to generate a resonance energy transfer effect, so that ECL signals are absent at the moment. When the target DNA exists, the target DNA can be hybridized with the auxiliary DNA and the probe DNA to form a Y-shaped structure, and the change of the space conformation breaks the resonance energy transfer effect and generates ECL signals. The signal intensity is positively correlated with the nucleic acid concentration, and the efficiency of single base mismatch binding in long-chain reactions is limited, so that in the first stage, both mutants and non-mutants can form Y-shaped structures to generate signals, and the detection of helicobacter pylori total nucleic acid can be completed through the low-specificity mode. In the second stage, cas9 endonucleases with high sequence specificity were used to detect mutants. The sgRNA and the Cas9 are combined to form a Cas9/sgRNA complex, nucleic acid with specific genotype is excised, Y-shaped structure is destroyed, and different single base mutants are detected by a signal difference method. All mutant phenotypes were detected by the specific genotype.
The beneficial effects of the invention are as follows:
1. the invention firstly completes the detection of the total amount of helicobacter pylori nucleic acid, and the target sequence breaks the resonance energy transfer effect of the hairpin probe through forming a Y-shaped DNA structure to generate an Electrochemiluminescence (ECL) signal, and the signal intensity is positively correlated with the nucleic acid concentration. In the second stage, through the high-specificity recognition cleavage of Cas9, nucleic acid with specific genotype is excised by using corresponding guide RNA, and different single base mutants are detected by a signal difference method. The helicobacter pylori infection status and the drug resistance status were comprehensively evaluated by detection at two different specific stages.
2. The invention can integrate electrode array on chip by taking Indium Tin Oxide (ITO) conductive glass as substrate, and can improve detection flux. Each electrode unit can complete analysis of the sample, and the transparent glass substrate is favorable for visual detection, so that visual detection of nucleic acid is realized.
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The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
Fig. 1 is a schematic diagram of a detection principle according to an embodiment of the present invention.
FIG. 2 is a high power transmission electron microscope image of an embodiment of the present invention. a is a transmission electron microscope photo of AuNPs prepared by the embodiment of the invention; b is a transmission electron microscope photo of the CdS prepared by the embodiment of the invention; c is a high-power transmission electron microscope photograph of CdS combined with AuNPs prepared by the embodiment of the invention.
FIG. 3 is a representation of gel electrophoresis of an embodiment of the present invention. a is a gel electrophoresis chart formed by verifying a Y-shaped structure in the embodiment of the invention; fig. 3b is a gel electrophoresis diagram of an embodiment of the present invention validating CRISPR/Cas9 digestion Y-type structures.
FIG. 4 is a graph of the results of conditional optimizations according to an embodiment of the present invention, aFor different Na + Concentration and ECL signal change curve, b is the same as Mg 2+ Concentration versus ECL signal profile, c is the ECL signal profile over time with different buffers, d is a histogram of the variation of Help chain concentration versus ECL signal intensity.
FIG. 5 is a graph of the log linear relationship between the concentration of target DNA and ECL signal for different phase series. The sensitivity detection result of the embodiment of the invention. a is a correction curve of different target DNA concentration logarithms and ECL signals in the first stage; FIG. b shows the calibration curve of ECL signal versus log concentration of different target DNA in the second phase;
FIG. 6 is a graph of ECL signal at the first stage corresponding to the mixed DNA sample of the wtDNA and mutDNA at different molar ratios according to the example of the present invention; b is a graph of ECL signal at the second stage corresponding to the mixed DNA sample of wtDNA and mutDNA at different molar ratios according to the examples of the present invention.
FIG. 7 shows the results of specific assays according to embodiments of the present invention.
FIG. 8 is a schematic diagram of a high throughput ITO array electrode chip template prepared according to the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
In view of the fact that there is currently no method capable of simultaneously analyzing the total amount of nucleic acid and detecting a single genotype, the present invention proposes an electrochemical detection method of a single base mutant of multiple helicobacter pylori.
In an exemplary embodiment of the invention, an electrochemical sensor is provided that includes a sensing electrode, an auxiliary DNA, an sgRNA, and a Cas9 nuclease;
the sensing electrode is composed of a conductive matrix, gold nanoparticles, probe DNA and nano CdS, wherein the probe DNA is of a hairpin structure, the two ends of the probe DNA are respectively connected with the gold nanoparticles and the nano CdS, and the conductive matrix is connected with the gold nanoparticles;
the probe DNA, the auxiliary DNA and the target DNA can be hybridized with each other to form a Y-shaped structure;
the sgrnas and the Cas9 nucleases are capable of forming Cas9/sgRNA complexes, which Cas9/sgRNA complexes are capable of disrupting the Y-structure, allowing separation of nano CdS from the Y-structure.
In some embodiments, the probe DNA is modified at one end with a thiol group and the gold nanoparticle is attached to the probe DNA via an Au-S bond.
In some embodiments, the probe DNA is modified at one end with an amine group and the nano CdS is attached to the probe DNA via an amide bond.
In some embodiments, the conductive substrate is a glassy carbon electrode or conductive glass. Such as ITO conductive glass.
In some embodiments, the probe DNA is shown in SEQ ID NO. 2.
In some embodiments, the helper DNA is as set forth in SEQ ID NO. 3.
In some embodiments, the target DNA is set forth in SEQ ID nos. 1, 4, and/or 5.
In some embodiments, the sgrnas are set forth in SEQ ID nos. 11, 12, and/or 13.
In another embodiment of the invention, an electrochemical detection method of a single base mutant of multiple helicobacter pylori is provided, and the electrochemical sensor is provided; the method comprises the following steps:
adding auxiliary DNA and target DNA to be detected into a sensing electrode for incubation, and detecting ECL signals;
pre-incubating the sgrnas and Cas9 nucleases to obtain Cas9/sgRNA complexes;
adding the obtained Cas9/sgRNA complex to the incubated sensing electrode, continuing incubation, and detecting ECL signals;
detecting target DNA to be detected through ECL signal difference;
the target DNA is helicobacter pylori single base mutant.
In some embodiments, probe DNA and gold nanoparticles are added to the surface of a conductive substrate, and the probe DNA is immobilized on the surface of the conductive substrate using Au-S bonds.
In one or more embodiments, the cadmium sulfide is activated using an EDC/NHS mixed solution, and the activated cadmium sulfide is amidated with probe DNA immobilized on the surface of the conductive substrate.
When working curves were drawn, both wild-type and mutant target DNA were used and detection was performed using only one sgRNA that was perfectly matched to the wild-type. In performing phased detection, the reason for using array detection is that each array uses a different sgRNA (which is perfectly matched to DNA of a different genotype, respectively), so that when perfectly matched, a cut occurs, a large signal difference occurs, and detection of a particular genotype is accomplished.
In a third embodiment of the invention, an electrochemical detection kit for helicobacter pylori single base mutants is provided, which comprises the electrochemical sensor and a buffer solution.
Specifically, the buffer is a PBS buffer.
In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the technical scheme of the present invention will be described in detail with reference to specific embodiments.
Examples
The detection principle of the embodiment is shown in figure 1, firstly, nano gold is synthesized, the nano gold can be marked on DNA chains with mercapto groups and amino groups respectively modified at two ends, auNPs are modified on the surface of an electrode through Au-S bonds, cdS is further connected through amide reaction, then target chains and auxiliary DNA are modified on the electrode, a Y-shaped structure is formed through assembly, and the change of the space conformation breaks through the resonance energy transfer effect, and ECL signals are generated. The sgRNA can be combined with Cas9 to form an activated Cas9/sgRNA complex, nucleic acid with specific genotype is excised, Y-shaped structure is destroyed, ECL signal is reduced, and different single base mutants are detected by a signal difference method.
Preparation of cadmium sulfide (CdS) 50mL of 0.01M CdCl was reacted under nitrogen 2 ·5/2H 2 The O solution was added to a 100mL three-necked flask, 250. Mu.L of MPA was added thereto, and the mixture was stirred with heating, and the pH was adjusted to 11 with saturated NaOH. Slowly add 5.5mL of 0.1M Na 2 S, refluxing for 4 hours at 110 ℃. And stopping heating, adding a proper amount of isopropanol after the solution is naturally cooled, centrifuging for 10min at the rotating speed of 10000r/min by using a centrifuge, settling particles at the bottom of a centrifuge tube, and discarding supernatant. And transferring the purified CdS sample to a centrifuge tube, and placing the centrifuge tube in a refrigerator at 4 ℃ for standby. The purified particles can be used for dynamic light scattering analysis and TEM characterization.
Preparation of nanogold (AuNPs): taking 99mL of pure water and 1mL of HAuCl 4 After mixing, the mixture was put into a 250mL three-necked flask, stirred and heated until the mixture was sufficiently refluxed, 3.5mL of 38.8mmol/L sodium citrate was added, the mixture was heated and stirred for 15 minutes, the solution was gradually changed from pale yellow to reddish wine, and the reaction was terminated. The cooled nanogold sample was transferred to a centrifuge tube, and 1 XPBS (0.1M NaCl,5mM MgCl) 2 ) Centrifuging at 14000r/min for 15min, settling the particles at the bottom of the centrifuge tube, discarding supernatant, centrifuging, cleaning for 2 times, and dispersing with pure water. Stored in a-4 ℃ refrigerator.
Assembling and electrode modification of a Y-shaped structure:
(1) Sequentially polishing the glassy carbon electrode by using alumina powder with different particle sizes, sequentially performing ultrasonic treatment in ethanol and pure water to remove surface residues, and scanning a cyclic voltammogram by using 0.1M KCl (0.05M potassium ferricyanide) as electrolyte.
(2) And (2) modifying Probe DNA (Probe DNA, hairpin structure) with two ends marked with amino and sulfhydryl groups on the electrode treated in the step (1), and standing at 4 ℃ for 16 hours, so that the Probe DNA is fixed on the surface of the glassy carbon electrode by utilizing Au-S bonds.
(3) Activating cadmium sulfide by adopting EDC/NHS mixed solution, and modifying the cadmium sulfide to the electrode treated in the step (2) for amidation reaction, wherein the method specifically comprises the following steps: 20 μL 0.2M NHS,0.04M EDC was added dropwise to the electrode to activate 1mL CdS, and the reaction was carried out at room temperature for 30min. 20uL of activated CdS was added dropwise, at 4℃overnight. ECL quench signals were tested.
(4) And (3) modifying (37 ℃ C., 60 min) target strand and auxiliary DNA (Help DNA,1 mu M) on the electrode treated in the step (3) to form a Y-type structure, and testing ECL recovery signals.
(5) After mixing Cas9 nuclease and sgRNA, preincubation was performed in reaction buffer (10 μl of 1 xbuffer buffer containing 50nM Cas9 nuclease and 50nM sgRNA was preincubated at 25 ℃ for 10 min) at a final concentration of 50nM, 10 μl of the mixture was reacted at 37 ℃ for 15min, RNP complex assembly was performed, and then added dropwise to the electrode treated in step (4) for 20min at 37 ℃ to test ECL cleavage signal.
ECL detection liquid is 0.05M K 2 S 2 0 8 1 XTE buffer solution of (A). Electrochemical measurement: all electrochemical tests were signal read out by the Chenhua CHI660E electrochemical workstation. A three-electrode system is adopted: the glassy carbon electrode is used as a working electrode, the platinum wire electrode is used as a counter electrode, and the Ag/AgCl electrode is used as a reference electrode. ECL signals were detected from-1.25V to 0V potential with photomultiplier tube (PMT) set at-480V.
The prepared AuNPs aqueous solution, cdS aqueous solution and mixed reaction (obtained in the step (3) in this example) were dripped on a copper mesh to observe morphology, and high power transmission electron microscope pictures are shown as a, b and c in fig. 2.
As shown in FIG. 3a, the assembled Y-shaped gel electrophoresis pattern shows different DNA sequences from left to right (wherein M is 20bp DNA marker,1 is target DNA,2 is Help DNA,3 is Probe DNA,4 is target DNA/Help DNA,5 is target DNA/Probe DNA,6 is Probe DNA/Help DNA,7 is target DNA/Probe DNA/Help DNA, and 8 is target DNA/Probe DNA/Help DNA/RNP). By comparing the sizes of the DNA bands, it can be seen that the Y-type structure was assembled gradually. Fig. 3b is a gel electrophoresis diagram of an embodiment of the present invention validating CRISPR/Cas9 digestion Y-type structures.
FIG. 4 shows that after optimization, na + At a concentration of 0.1M, mg 2+ At a concentration of 5mM, the buffer solution was PBS and the experimental conditions were optimized at a concentration of 1. Mu.M of the hellp strand.
The method of this example was used to detect target DNA at different concentrations at different stages, and as shown in FIG. 5, it can be seen from FIG. 5a that the target DNA concentration and ECL signal all have a logarithmic linear relationship in the concentration range of 0.01 to 500 nM. In the concentration range of 0.01-500 nM, the linear equation is
y=0.0285 log c (nmol/L) +0.0867, correlation coefficient R 2 A detection limit of 0.9994 and a signal to noise ratio of 3 was 8pM. FIG. 5b is a graph showing the calibration curve of the logarithm of the concentration of different target DNA in the second phase and ECL signal, y= -0.02027log C (nmol/L) +0.09164, R 2 0.99544, wherein the correlation work curve of concentration of the genotype with perfect match of sgRNA and ECL signal was obtained with a total amount of immobilized nucleic acids (nucleic acids of two genotypes, one wild type and the other single base mutant) of 1.1. Mu.M.
To further investigate the discrimination ability between mutant DNA and normal wtDNA, mixed DNA samples were prepared as test subjects at different molar ratios of quenching signal (a), 0.1%:99.9% (b), 1%:99% (c), 10%:90% (d), 50%:50% (e), 90%:10% f), 99%:1% (g), 99.9%:0.1% (h) with a total concentration of 100 nM. As shown in fig. 6, it can be seen from fig. 6a that ECL signal in the first stage does not change with increasing concentration of mutant DNA in the mixed DNA sample, and that ECL signal in the second stage dynamically decreases with increasing concentration of mutant DNA in fig. 6 b.
This example also tested ECL responses of electrochemical biosensor systems using H.Pylori, shigella, S.Aureus, salmonella and E.coli strains of different genus, to evaluate the specificity of the method. As shown in FIG. 7, H.Pylori elicits a strong ECL response, while Shigella, S.Aureus, salmonella and E.coli were below the minimum limit of detection of the target bacteria, indicating that the method can specifically differentiate helicobacter pylori from pathogenic bacteria of different genus, and the electrochemical detection technique has high specificity for helicobacter pylori.
Pretreatment of ITO glass and preparation of PDMS film: the ITO glass is sequentially treated by ultrasonic treatment for 15min by acetone, ethanol and pure water, and then dried by nitrogen for standby. Uniformly mixing a liquid PDMS matrix and a curing agent according to the mass ratio of 10:1, uniformly stirring the mixture by using a glass rod, and removing bubbles. PDMS was cast on ITO glass, cured at 60 ℃ for 3 hours to form a PDMS film, which was cut into the desired shape, as shown in fig. 8. Each electrode unit integrated on the ITO chip can complete analysis and visual detection of the sample.
The nucleic acid sequences used in this example are shown in the following table:
Figure BDA0004001256690000101
Figure BDA0004001256690000111
wherein the underline is a mismatched base.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. An electrochemical sensor comprising a sensing electrode, an auxiliary DNA, an sgRNA, and a Cas9 nuclease;
the sensing electrode is composed of a conductive matrix, gold nanoparticles, probe DNA and nano CdS, wherein the probe DNA is of a hairpin structure, the two ends of the probe DNA are respectively connected with the gold nanoparticles and the nano CdS, and the conductive matrix is connected with the gold nanoparticles;
the probe DNA, the auxiliary DNA and the target DNA can be hybridized with each other to form a Y-shaped structure;
the sgrnas and the Cas9 nucleases are capable of forming Cas9/sgRNA complexes, which Cas9/sgRNA complexes are capable of disrupting the Y-structure, allowing separation of nano CdS from the Y-structure.
2. The electrochemical sensor according to claim 1, wherein one end of the probe DNA is modified with a thiol group, and the gold nanoparticle is attached to the probe DNA through an Au-S bond.
3. The electrochemical sensor according to claim 1, wherein the probe DNA is modified at one end with an amine group, and the nano CdS is connected to the probe DNA through an amide bond.
4. The electrochemical sensor of claim 1, wherein the conductive substrate is a glassy carbon electrode or conductive glass.
5. The electrochemical sensor according to claim 1, wherein the probe DNA is shown in SEQ ID No. 2;
or, the auxiliary DNA is shown as SEQ ID NO. 3;
or, the target DNA is shown as SEQ ID NO.1, 4 and/or 5;
alternatively, the sgRNA is as shown in SEQ ID NO.11, 12 and/or 13.
6. An electrochemical detection method of a single base mutant of multiple helicobacter pylori, characterized by providing an electrochemical sensor according to any one of claims 1 to 5; the method comprises the following steps:
adding auxiliary DNA and target DNA to be detected into a sensing electrode for incubation, and detecting ECL signals;
pre-incubating the sgrnas and Cas9 nucleases to obtain Cas9/sgRNA complexes;
adding the obtained Cas9/sgRNA complex to the incubated sensing electrode, continuing incubation, and detecting ECL signals;
detecting target DNA to be detected through ECL signal difference;
the target DNA is helicobacter pylori single base mutant.
7. The electrochemical detection method of multiple helicobacter pylori single base mutants according to claim 6, characterized in that probe DNA and gold nanoparticles are added to the surface of a conductive substrate, and the probe DNA is immobilized on the surface of the conductive substrate by Au-S bond.
8. The electrochemical detection method for single base mutants of multiple helicobacter pylori according to claim 7, characterized in that cadmium sulfide is activated by using EDC/NHS mixed solution, and then the activated cadmium sulfide is amidated with probe DNA immobilized on the surface of the conductive substrate.
9. An electrochemical detection kit for helicobacter pylori single base mutants, which is characterized by comprising the electrochemical sensor and a buffer solution according to any one of claims 1-5.
10. The kit for electrochemical detection of single base mutants of helicobacter pylori according to claim 9, characterized in that the buffer is a PBS buffer.
CN202211618727.7A 2022-12-15 2022-12-15 Electrochemical staged detection method for single base mutant of multiple helicobacter pylori Pending CN116203094A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117030820A (en) * 2023-09-28 2023-11-10 艾康生物技术(杭州)有限公司 Measuring method of biosensor

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117030820A (en) * 2023-09-28 2023-11-10 艾康生物技术(杭州)有限公司 Measuring method of biosensor
CN117030820B (en) * 2023-09-28 2024-01-09 艾康生物技术(杭州)有限公司 Measuring method of biosensor

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