CN117665073A - Electrochemical small molecule biosensor based on nano enzyme signal amplification, microfluidic chip device, and preparation method and application thereof - Google Patents
Electrochemical small molecule biosensor based on nano enzyme signal amplification, microfluidic chip device, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an electrochemical small molecule biosensor based on nano enzyme signal amplification, a microfluidic chip device, a preparation method and application thereof, and successfully constructs an electrochemical method for rapidly detecting small molecules based on CRISPR/Cas12a and Pd@PCN-222 signal amplification, and amplifies electrochemical signals by coupling SH-ssDNA and Pd@PCN-222 and combining the trans-cleavage of Cas12 a; when target DNA fragments exist in the DNA to be detected in the CRISPR/Cas12a system, ca s12a protein can be activated under the guidance of crRNA, nonspecific Pd@PCN-222-SH-ssDNA fixed on the surface of a cutting electrode is cut, and electrochemical signals from the Pd@PCN-222-SH-ssDNA are reduced.
Description
Technical Field
The invention belongs to the technical field of electrochemical analysis and detection, and particularly relates to an electrochemical small-molecule biosensor based on nano enzyme signal amplification, a microfluidic chip device, and a preparation method and application thereof.
Background
The CRISPR/Cas system (aggregated short palindromic repeats/CRISPR related nucleases) is a new technology developed in recent years, and has wide application in the fields of molecular diagnosis, genome editing and the like, and even has wide application in the fields of food safety and environment due to the unique cis-cleavage and trans-cleavage capability.
However, most of the assays based on CRISPR/Cas systems currently require amplification of targets or additional introduction of signal amplification strategies such as hybridization chain reactions, catalytic hairpin DNA self-assembly reactions, entropy driven catalytic reactions, etc., while these signal amplification techniques can increase the sensitivity of CRISPR-based detection methods, their required reaction time, complex primer design and cumbersome procedure limit the flexibility and speed of CRISPR detection. In particular, detection of non-nucleic acid targets, such as toxins, proteins, pathogens, etc., requires processing of the non-nucleic acid signal into a nucleic acid signal by recognition switching elements for subsequent detection.
Therefore, in order to ensure food safety, such as meeting the requirement of rapid field detection, it is necessary to develop an amplification-free detection platform.
Disclosure of Invention
This section is intended to outline some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section as well as in the description summary and in the title of the application, to avoid obscuring the purpose of this section, the description summary and the title of the invention, which should not be used to limit the scope of the invention.
The present invention has been made in view of the above and/or problems occurring in the prior art.
Therefore, the invention aims to overcome the defects in the prior art and provide an electrochemical small molecule biosensor based on nano enzyme signal amplification.
In order to solve the technical problems, the invention provides the following technical scheme: the sensor comprises a gold electrode 100 and a functional layer 200 modified on the surface of the reaction end of the gold electrode 100;
wherein the functional layer 200 is a non-specific single-stranded ssDNA labeled with 6-mercaptoethanol and pd@pcn-222 nanoenzyme, the nucleotide sequence of which is capable of being cleaved by the activated Cas12a protein.
As a preferable scheme of the electrochemical small molecule biosensor based on nano enzyme signal amplification, the invention comprises the following steps: the length of the nucleotide sequence of the single-stranded DNA is 10-30 nt.
As a preferable scheme of the electrochemical small molecule biosensor based on nano enzyme signal amplification, the invention comprises the following steps: the nucleotide sequence of the single-stranded DNA comprises a nucleotide sequence shown as SEQ ID No. 1.
It is still another object of the present invention to provide a method for manufacturing an electrochemical small molecule biosensor.
In order to solve the technical problems, the invention provides the following technical scheme: comprising the steps of (a) a step of,
dropwise adding a single-stranded ssDNA solution to the surface of the pretreated gold electrode 100, and blocking a non-specific binding site by using a 6-mercaptoethanol solution after drying to obtain a ssDNA modified gold electrode 100;
and (3) dropwise adding the Pd@PCN-222 nano enzyme solution onto the single-stranded ssDNA modified gold electrode 100, performing condensation reaction, and combining to form a functional layer 200 to obtain the electrochemical small molecule biosensor.
As a preferable scheme of the preparation method of the electrochemical small molecule biosensor, the invention comprises the following steps: the concentration of the single-stranded ssDNA solution is 1-5 mu M, the concentration of the 6-mercaptoethanol solution is 2-3mM, and the concentration of the Pd@PCN-222 nano enzyme solution is 2-6 mg/ml.
As a preferable scheme of the preparation method of the electrochemical small molecule biosensor, the invention comprises the following steps: the proportion of the single-stranded ssDNA solution, the 6-mercaptoethanol solution and the Pd@PCN-222 nano enzyme solution is 3:2:4.
as a preferable scheme of the preparation method of the electrochemical small molecule biosensor, the invention comprises the following steps: the drying temperature of the drying is 20-40 ℃ and the drying time is 8-15 h.
It is still another object of the present invention to provide an electrochemical small molecule biosensor for detecting ochratoxin a.
In order to solve the technical problems, the invention provides the following technical scheme: comprising the steps of (a) a step of,
the application is that ochratoxin A is detected by a biosensor, wherein the method for detecting the ochratoxin A comprises the following steps of,
mixing and heating an aptamer chain with a nucleotide sequence shown as SEQ ID No.2 and a complementary chain with a nucleotide sequence shown as SEQ ID No.3 according to a molar ratio of 0.75:1-1.75:1 to form double-stranded DNA;
incubating the double-stranded DNA and a target detection object containing ochratoxin A together to obtain a reaction solution;
mixing the reaction solution with CRISPR/Cas12a system solution with the concentration of 10-130 nM to obtain a solution to be tested;
adding the liquid to be detected on the surface of the gold electrode 100 of the biosensor, cutting for 6-60 min in a reverse mode, immersing the gold electrode 100 into a buffer solution after cutting, performing differential square wave voltammetry analysis, and calculating the change of a current signal to realize detection analysis of ochratoxin A;
the CRISPR/Cas12a system comprises crRNA with a nucleotide sequence shown as SEQ ID No.4, cas12a protein, a ribonuclease inhibitor and a buffer II, wherein the volume ratio is 0.5-1.5: 0.5 to 1.5:0.5 to 1.5:2.
it is still another object of the present invention to provide a microfluidic chip device for detecting ochratoxin a, which is composed of a PMMA chip 300 and a microfluidic intelligent platform 400, wherein the PMMA chip 300 has a plurality of test cells a composed of a reaction zone a-1, a detection zone a-2 and a waste zone a-3 which are sequentially communicated;
the reaction zone A-1 is composed of a first reaction chamber A-101 for containing a sample solution, a second reaction chamber A-102 for containing a gold electrode 100 modified with a functional layer 200 and a third reaction chamber A-103 for containing a CRISPR-Cas12a system solution, wherein the first reaction chamber A-101, the second reaction chamber A-102 and the third reaction chamber A-103 are sequentially communicated, and the third reaction chamber A-103 is communicated with the detection zone A-2, and the reaction chambers are all provided with sample injection holes a.
It is a further object of the present invention to provide a microfluidic chip device for detecting ochratoxin a, comprising,
the sample solution of the object to be detected and the double-stranded DNA solution are added into the first reaction chamber A-101 of the chip reaction zone A-1 through the sample injection hole a, and the CRISPR-Cas12a system solution is added into the third reaction chamber A-103;
placing the PMMA chip 300 loaded with the solution into a microfluidic intelligent platform 400;
electrochemical detection is carried out by a chronoamperometric method, the gold electrode 100 modified with the functional layer 200 is inserted into the detection area A-2 before detection, and detection analysis of ochratoxin A is realized by calculating the change of a current signal.
The invention has the beneficial effects that:
(1) The preparation method of the electrochemical sensor has the advantages of simple process, convenient and fast operation, safety, low cost, no pollution and high manufacturing efficiency, and the Pd@PCN-222 serving as a functional nano material has peroxidase activity, so that the preparation steps of a working electrode can be reduced, and the detection sensitivity of the electrochemical sensor can be improved.
(2) When the electrochemical sensor provided by the invention detects ochratoxin A, the detection of the ochratoxin A in water and plant-derived foods can be realized, and the electrochemical sensor has the advantages of high stability, long service life, wide detection range, low detection limit, strong anti-interference capability, capability of replacing natural enzymes and other signal amplification technologies, wide application range, high application value and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the description of the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a schematic structural diagram of an electrochemical small molecule biosensor of the present invention;
FIG. 2 is a graph showing the evaluation result of the activity of peroxidase-like substance Pd@PCN-222 of the present invention;
FIG. 3 is a graph showing the current change results of example 4 of the present invention at various stages of electrochemical biosensor construction using EIS and CV measurements;
FIG. 4 is a diagram of SWV response signals for detecting the feasibility of OTA according to embodiment 5 of the present invention;
FIG. 5 is a graph showing comparison of signal intensities of detection OTA under different SH-ssDNA conditions in preparing an electrochemical sensor;
FIG. 6 is a graph of sensitivity analysis of the electrochemical biosensor prepared according to the present invention;
FIG. 7 is a schematic diagram of a microfluidic chip device according to embodiment 10 of the present invention;
FIG. 8 is a schematic view of a PMMA chip according to embodiment 10 of the present invention;
FIG. 9 is a schematic diagram of a test unit according to embodiment 10 of the present invention;
fig. 10 is a schematic diagram of the detection principle of the microfluidic chip device according to embodiment 10 of the present invention.
Detailed Description
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
Further, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic can be included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The raw materials used in the invention are as follows:
tris (hydroxymethyl) aminomethane (Tris) was purchased from ala Ding Huaxue reagent company (Shanghai, china);
ochratoxin a standard was purchased from behcet biotechnology limited, south tokyo;
gold electrode, electrochemical workstation from Shanghai Chen Hua instruments Co., ltd;
other materials and reagents are conventional commercial products.
In the examples below, room temperature refers to 25.+ -. 5 ℃ and overnight to 8-12h without specific explanation.
The Pd@PCN-222 nano-enzyme used in the invention is synthesized by referring to the following method:
70mg ZrCl 4 50mg of Fe-TCPP and 3.0mg of benzoic acid are dissolved in 15mL of N, N-diethyl formamide solution, the mixture is subjected to ultrasonic dispersion after being mixed, the obtained mixed solution is heated at 110 ℃ for 48 hours, then cooled and centrifuged, and the obtained product is repeatedly washed by N, N-diethyl formamide, ultrapure water and ethanol and dried in vacuum for 12 hours to obtain PCN-222;
50mg of PCN-222, 100mg of polyvinylpyrrolidone, 50mg of vitamin C and 50mg of citric acid are dissolved in 8mL of ultrapure water, the mixture is subjected to ultrasonic dispersion after mixing, the obtained mixed solution is transferred into a three-necked flask, and the mixture is refluxed in an oil bath at 120 ℃ and kept for 5min, and simultaneously the mixture is subjected to ultrasonic dispersion5mL of K with concentration of 1mg mL-1 2 PdCl 4 Slowly dripping the solution into the three-necked flask within 30min, continuously heating for 3h, cooling to room temperature, centrifuging, washing the obtained product with ultrapure water, acetone and ethanol in sequence until the product is colorless, and vacuum drying to obtain Pd@PCN-222 nano-enzyme.
The sequences used in the present invention correspond to the following:
SEQ ID No.1：5’-SH-TTTTTTTTAAAAAAAAAAAAAAATTTTTTT-NH 2 -3’；
SEQ ID No.2：5’-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCG GACA-3’；
SEQ ID No.3：5’-CCGATGCTCCCTTTACGCC-3’；
SEQ ID No.4：5’-UAAUUUCUACUAAGUGUAGAUGGCGUAAAGGG AGCAUCGG-3’；
example 1
Referring to fig. 1, the present embodiment provides a method for preparing an electrochemical small molecule biosensor based on nano-enzyme signal amplification, the sensor realizes signal amplification based on pd@pcn-222 and Cas12a, and a structural gold electrode 100 is formed from bottom to top, and a reaction end of the gold electrode 100 is modified with a pd@pcn-222-SH-ssDNA functional layer 200, specifically:
1) Dripping ssDNA solution with the nucleotide sequence shown as SEQ ID No.1 and the concentration of 3 mu M and the length of 30nt on the surface of a treated gold electrode, incubating at 37 ℃ for 2 hours to enable the-SH end of the ssDNA to be covalently bound with the gold electrode, and covering the electrode with 5 mu L of MCH (2 mM) solution for 30 minutes to seal non-specific binding sites on the surface of the electrode so as to enable the SH-ssDNA to stand upright;
2) And (3) dropwise adding 10 mu L of Pd@PCN-222 nano enzyme solution with the concentration of 4 mu M onto the ssDNA modified gold electrode, combining to form a signal probe (Pd@PCN-222-SH-ssDNA) according to the condensation reaction between amino and carboxyl, namely the functional layer 200, and airing at room temperature for one night to obtain the electrochemical biosensor based on nano enzyme signal amplification.
Example 2
The embodiment provides an application method of the small molecular biosensor prepared in the embodiment 1 in detecting ochratoxin A, which specifically comprises the following steps:
1) Preparing a DNA mixed solution of target detection molecules:
the aptamer strand with the nucleotide sequence shown in SEQ ID No.2 and the complementary strand with the nucleotide sequence shown in SEQ ID No.3 are heated at 95 ℃ for 5min to form double-stranded DNA, 10 mu L of double-stranded DNA is incubated with a target detection object solution containing ochratoxin A for 0.5h at 37 ℃ to obtain a DNA mixed solution.
2) Preparing detection reaction liquid:
mixing 1 mu L of the DNA mixed solution obtained in the step 1) with 2 mu L of 10 XBuffer, 1 mu L of ribonuclease inhibitor, 1 mu L of crRNA with a nucleotide sequence shown as SEQ ID No.4, 1 mu L of Cas12a protein and 14 mu L of ultrapure water to obtain a CRISPR/Cas12a reaction system, and reacting the CRISPR/Cas12a reaction system at 37 ℃ for 20min to obtain a reaction solution.
3) And (3) detection:
and (3) dripping the reaction liquid obtained in the step (2) on the surface of the electrochemical biosensor obtained in the step (1), respectively reacting for 60 minutes at the temperature of 37 ℃, and detecting the electrochemical signal of the electrochemical biosensor after the reaction is finished to obtain a detection result.
Example 3
Referring to FIG. 2, this example was used to evaluate the peroxidase-like activity of Pd@PCN-222 used in example 1, and the peroxidase substrate TMB was selected as a chromogenic substrate, specifically:
in the presence of 20mM H 2 O 2 In the test solution (2), the gold electrode before and after the modification Pd@PCN-222 was subjected to Cyclic Voltammetry (CVs), and the results are shown in FIG. 2, it can be seen that with H 2 O 2 A significant color change was observed with the introduction of (C), indicating that Pd@PCN-222 can be found in H 2 O 2 Is involved in catalyzing TMB oxidation in TMB-H 2 O 2 In the system, pd@PCN-222 showed a strong adsorption peak at 652nm, which can be attributed to the single electron charge transfer of the oxTMB.
Example 4
Referring to fig. 3, the feasibility of detecting small molecules by using the method of example 2 is evaluated in the present embodiment, and specifically, the method is as follows:
the current changes at various stages of electrochemical biosensor construction were measured using EIS and CV, the results are shown in fig. 3, wherein:
curve a corresponds to a gold electrode without any modification;
curve b corresponds to modifying ssDNA only on the gold electrode surface;
curve c corresponds to assembling MCH after modifying ssDNA on the gold electrode surface;
curve d corresponds to assembling MCH and introducing Pd@PCN-222 after modifying ssDNA on the surface of the gold electrode;
curve e corresponds to the assembly of MCH after modification of ssDNA on the gold electrode surface and introduction of pd@pcn-222 and Cas12a trans-cleavage reaction solution.
FIG. 3 shows the CV curve of a modified electrode, with bare gold electrodes exhibiting typical [ Fe (CN) 6 ] 3- / 4- The oxidation-reduction peak (curve a) shows that the gold electrode interface has effective electron transmission capability, and an SH-ssDN A probe is added on the gold electrode to form a DNA monolayer on the surface of the electrode, so that the electron medium is prevented from diffusing to the surface of AuE, and the oxidation current peak is prevented from being obviously reduced (curve b); the peak of the oxidation current is further reduced (curve c) with MCH assembly;
due to the conductivity of pd@pcn-222, a significant increase in peak current can be observed when pd@pcn-222 is added to the electrode surface (curve d), after the Cas12a trans-lysis reaction solution is added to the gold electrode, the pd@pcn-222-SH-ssDNA probe is lysed, the pd@pcn-222 falls off the gold electrode surface, increasing the peak current (curve e), illustrating the feasibility of the invention for achieving signal amplification based on pd@pcn-222 and Cas12 a.
Example 5
The embodiment provides a specific application of the small molecular biosensor prepared in the embodiment 1 in detecting ochratoxin A, which is specifically as follows:
1) Preparing a DNA mixed solution of target detection molecules:
the aptamer chain with the nucleotide sequence shown in SEQ ID No.2 and the complementary chain with the nucleotide sequence shown in SEQ ID No.3 are heated at 95 ℃ for 5min to form double-stranded DNA, 10 mu L of double-stranded DNA is incubated with target detection object solutions containing ochratoxin A with different concentrations for 0.5h at 37 ℃ to obtain a DNA mixture.
2) Preparing detection reaction liquid:
mixing 1 mu L of the DNA mixed solution obtained in the step 1) with 2 mu L of 10 XBuffer, 1 mu L of ribonuclease inhibitor, 1 mu L of crRNA with a nucleotide sequence shown as SEQ ID No.4, 1 mu L of Cas12a protein and 14 mu L of ultrapure water to obtain a CRISPR/Cas12a reaction system, and reacting the CRISPR/Cas12a reaction system at 37 ℃ for 20min to obtain a reaction solution.
3) And (3) detection:
after incubating the electrodes at 37℃for 0.5H, the reaction solution was immersed in 0.1M Tris-HCl buffer (pH 7.4, nitrogen saturated) and contained 20mM H 2 O 2 Differential Square Wave Voltammetry (SWV) analysis was performed. Under the condition of 0.05V/s, the SWV is used for recording the conditions that the MCH/SH-ssDNA-Pd@PCn-222/AuE system and the MCH/SH-ssDNA-Pd@PCn-222/AuE are incubated together before and after the Pd@PCn-222 catalyzes H in the voltage range of 0.1 to-0.6V 2 O 2 Reducing the change in the generated current signal.
As a result, as shown in fig. 4, when only MCH/pd@pcn-222-SH-ssDNA/AuE (curve a) was incubated with Cas12a/crRNA (curve b), no significant change in peak current was observed, indicating that trans-cleavage activity of Cas12a was not activated in the absence of the target OTA;
however, in the presence of OTA, after simultaneous addition of Cas12a/crRNA, the current signal drops significantly (curve c), a change indicating that only when OTA, cas12a/crRNA, co-occur, the aptamer/acna duplex recognizes OTA, releases cDNA to activate Cas12a in combination with crRNA, thereby cleaving the electrode surface signaling probe pd@pcn-222-SH-ssDNA.
The above results clearly demonstrate the feasibility of the developed sensing system based on the CRISPR/Cas12a system integrated pd@pcn-222 to detect ochratoxin a.
Example 6
Referring to fig. 5, the present example was used to evaluate the optimal conditions for detecting SH-ssDNA in an electrochemical sensor prepared by ochratoxin a (OTA) in the biosensor prepared in example 1, specifically:
the SH-ssDNA concentrations in example 1 were adjusted to 1, 3 and 5. Mu.M, and the lengths were adjusted to 10, 20 and 30nt, respectively, and the other process parameters were the same as in example 1, and the signal intensity of ochratoxin A (OTA) was measured by a sensor prepared by detecting SH-ssDNA at different concentrations, and the results are shown in FIG. 5.
As shown in FIG. 5A, the ΔI (%) value increases rapidly from 1 to 3. Mu.M and then reaches a plateau from 3 to 5. Mu.M, indicating that the amount of SH-ssDNA modified at the electrode surface tends to saturate. Thus, SH-ssDNA at a concentration of 3. Mu.M was selected for subsequent experiments.
As shown in FIG. 5B, it was found that ΔI (%) increased with an increase in SH-ssDNA length, reached a maximum at 30nt, and did not significantly increase after ΔI (%) exceeded 30nt. Thus, SH-ssDNA of 30nt in length was selected for the experiment.
Example 7
This example was used to determine the sensitivity and limit of detection of ochratoxin a (OTA) using the biosensor prepared in example 1, and as shown in fig. 6A and 6B, the peak current signal detected was reduced in proportion to the increase in the concentration of the target OTA, with the detection linearity ranging from 0.005ng/m to 50ng/mL.
From fig. 6C and 6D, a good linear relationship between Δi (%) and the logarithm of OTA concentration was observed, with a corresponding regression equation of Δi=8.63 log ota+70.83 (R 2 =0.997), the limit of detection was 1.21pg/mL.
Example 9
This example was used to evaluate the biosensor prepared in example 1 for detection of recovery of ochratoxin a (OTA), and specifically:
the electrochemical biosensor was used for target detection in an actual sample by a standard addition method (measurement method is described in example 2), and recovery rate experiments were performed, and the measurement results are shown in table 1:
TABLE 1 results of verification of recovery of ochratoxin A in actual samples
As can be seen from the table, the recovery rate of the sensor provided by the invention to the standard corn samples with ochratoxin A concentration of 0.05, 5 and 50ng/mL is 98.8% -102.2%, the RSD value is 2.0% -3.82%, the standard recovery rate measurement result of the standard edible oil sample is 96.8% -103.4%, and the RSD value is 2.35% -4.52%. The detection limit of the electrochemical biosensor can be converted into 0.024 mug/kg, which is lower than the detection limit of ochratoxin A in national standards by using high performance liquid chromatography, and the electrochemical biosensor has the potential of being applied to detection and analysis of actual samples.
Example 10
Referring to fig. 7 to 10, the present embodiment uses the principle that the electrochemical biosensor forms the change of the electrochemical signal, and finally integrates on a microfluidic chip, and establishes a portable microfluidic chip device for small molecule detection, which is used for rapid, sensitive, specific and multichannel quantitative detection of small molecule substances, specifically:
referring to fig. 7, the microfluidic chip device is composed of a PMMA chip 300 and a microfluidic intelligent platform 400;
referring to fig. 8, a PMMA chip 300 has a plurality of test cells a;
referring to fig. 9, a test unit a is composed of a reaction area a-1, a detection area a-2 and a waste liquid area a-3 which are sequentially communicated, the reaction area a-1 is composed of a first reaction chamber a-101 containing a sample solution, a second reaction chamber a-102 containing a gold electrode 100 modified with a functional layer 200, and a third reaction chamber a-103 containing a CRISPR/Cas12a system solution, the first reaction chamber a-101, the second reaction chamber a-102, the third reaction chamber a-103 are sequentially communicated, and the third reaction chamber a-103 is communicated with the detection area a-2;
the first reaction chamber A-101, the second reaction chamber A-102 and the third reaction chamber A-103 have the capacities of 50 mu L, 10 mu L and 50 mu L respectively;
the micro-injection molding technology is adopted to manufacture the CD PMMA chip, and the whole micro-fluidic chip can realize constant temperature control, rotation speed control, time control and steering control.
Referring to fig. 10, the detection principle of the microfluidic chip device is as follows:
the sample solution of the object to be detected and the double-stranded DNA solution are added into the first reaction chamber A-101 of the chip reaction zone A-1 through the sample injection hole a, and the CRISPR-Cas12a system solution is added into the third reaction chamber A-103;
placing the PMMA chip 300 loaded with the solution into a microfluidic intelligent platform 400;
electrochemical detection is carried out by a chronoamperometric method, the gold electrode 100 modified with the functional layer 200 is inserted into the second reaction chamber A-102 before detection, and detection and analysis of ochratoxin A are realized by calculating the change of a current signal.
In conclusion, the invention successfully constructs an electrochemical method for rapidly detecting small molecules based on CRISPR/Cas12a and Pd@PCN-222 signal amplification, and the electrochemical signal is amplified by coupling SH-ssDNA and Pd@PCN-222 to combine with the trans-cleavage of Cas12 a; when a target DNA fragment exists in the DNA to be detected in the CRISPR/Cas12a system, the Cas12a protein can be activated under the guidance of crRNA, the nonspecific Pd@PCN-222-SH-ssDNA fixed on the surface of the cutting electrode is cut, and the electrochemical signal from the Pd@PCN-222-SH-ssDNA is reduced.
The trans-cleavage activity of CRISPR-Cas12a was introduced into the electrochemical biosensor and the traditional ferrocene electrochemical signal was replaced with pd@pcn-222. Under the optimized condition, the portable electrochemical biosensor provided by the invention can be used for detecting the target object with low concentration, and further shows that the electrochemical biosensor can realize rapid and simple high-sensitivity detection.
Meanwhile, the electrochemical biosensor forms the change of electrochemical signals, and is finally integrated on a microfluidic chip to establish a portable biosensor platform for small molecule detection, so as to be used for rapidly, sensitively, specifically and quantitatively detecting small molecule substances in multiple channels.
It should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered in the scope of the claims of the present invention.
Claims (10)
1. An electrochemical small molecule biosensor based on nano enzyme signal amplification is characterized in that: the sensor comprises a gold electrode (100) and a functional layer (200) modified on the surface of the reaction end of the gold electrode (100);
wherein the functional layer (200) is a non-specific single-stranded ssDNA labeled with 6-mercaptoethanol and pd@pcn-222 nanoenzyme, the nucleotide sequence of which is capable of being cleaved by an activated Cas12a protein.
2. The nano-enzyme signal amplification-based electrochemical small molecule biosensor of claim 1, wherein: the length of the nucleotide sequence of the single-stranded DNA is 10-30 nt.
3. The nano-enzyme signal amplification-based electrochemical small molecule biosensor of any one of claims 1 or 2, wherein: the nucleotide sequence of the single-stranded DNA comprises a nucleotide sequence shown as SEQ ID No. 1.
4. A method for preparing an electrochemical small molecule biosensor according to any one of claims 1 to 3, characterized in that: comprising the steps of (a) a step of,
dropwise adding a single-stranded ssDNA solution to the surface of the pretreated gold electrode (100), and blocking a non-specific binding site by using a 6-mercaptoethanol solution after drying to obtain a ssDNA modified gold electrode (100);
and (3) dropwise adding the Pd@PCN-222 nano enzyme solution onto a single-stranded ssDNA modified gold electrode (100) to perform condensation reaction, and combining to form a functional layer (200) to obtain the electrochemical small molecule biosensor.
5. The method for preparing the electrochemical small molecule biosensor based on nano enzyme signal amplification according to claim 4, which is characterized in that: the concentration of the single-stranded ssDNA solution is 1-5 mu M, the concentration of the 6-mercaptoethanol solution is 2-3mM, and the concentration of the Pd@PCN-222 nano enzyme solution is 2-6 mg/ml.
6. The method for preparing the electrochemical small molecule biosensor according to claim 5, wherein: the proportion of the single-stranded ssDNA solution, the 6-mercaptoethanol solution and the Pd@PCN-222 nano enzyme solution is 3:2:4.
7. the method for preparing the electrochemical small molecule biosensor according to claim 4, wherein: the drying temperature of the drying is 20-40 ℃ and the drying time is 8-15 h.
8. Use of an electrochemical small molecule biosensor according to any one of claims 1-3 for detecting ochratoxin a, characterized in that: the application is that ochratoxin A is detected by a biosensor, wherein the method for detecting the ochratoxin A comprises the following steps of,
mixing and heating an aptamer chain with a nucleotide sequence shown as SEQ ID No.2 and a complementary chain with a nucleotide sequence shown as SEQ ID No.3 according to a molar ratio of 0.75:1-1.75:1 to form double-stranded DNA;
incubating the double-stranded DNA and a target detection object containing ochratoxin A together to obtain a reaction solution;
mixing the reaction solution with CRISPR/Cas12a system solution with the concentration of 10-130 nM to obtain a solution to be tested;
adding the liquid to be measured on the surface of the gold electrode (100) of the biosensor, cutting for 6-60 min in a reverse mode, and immersing the gold electrode (100) into the liquid containing H after cutting 2 O 2 In the buffer solution of (2), differential square wave voltammetry analysis is carried out, and detection analysis of ochratoxin A is realized by calculating the change of a current signal;
the CRISPR/Cas12a system comprises crRNA with a nucleotide sequence shown as SEQ ID No.4, cas12a protein, a ribonuclease inhibitor and a buffer II, wherein the volume ratio is 0.5-1.5: 0.5 to 1.5:0.5 to 1.5:2.
9. a microfluidic chip device applying the method for detecting ochratoxin a of claim 8, wherein: the device for detecting the micro-fluidic chip comprises a PMMA chip (300) and a micro-fluidic intelligent platform (400), wherein the PMMA chip (300) is provided with a plurality of test units (A), and the test units (A) comprise a reaction area (A-1), a detection area (A-2) and a waste liquid area (A-3) which are sequentially communicated;
the reaction zone (A-1) is composed of a first reaction chamber (A-101) for containing a sample solution, a second reaction chamber (A-102) for containing a gold electrode (100) modified with a functional layer (200) and a third reaction chamber (A-103) for containing a CRISPR-Cas12a system solution, the first reaction chamber (A-101), the second reaction chamber (A-102) and the third reaction chamber (A-103) are sequentially communicated, the third reaction chamber (A-103) is communicated with the detection zone (A-2), and the reaction chambers are all provided with sample injection holes (a).
10. The use of the microfluidic chip device according to claim 9 for detecting small molecule ochratoxin a, wherein:
the sample solution of the object to be detected and the double-stranded DNA solution are added into a first reaction chamber (A-101) of a chip reaction zone (A-1) through a sample injection hole (a), and the CRISPR-Cas12a system solution is added into a third reaction chamber (A-103);
placing the PMMA chip (300) loaded with the solution into a microfluidic intelligent platform (400);
electrochemical detection is carried out by a chronoamperometric method, the gold electrode (100) modified with the functional layer (200) according to claim 1 is inserted into the detection area (A-2) before detection, and detection analysis of ochratoxin A is realized by calculating the change of a current signal.
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