CN113092556B - Preparation method and application of electrochemical sensor for detecting transgenic soybeans through double signal output based on gene editing technology - Google Patents
Preparation method and application of electrochemical sensor for detecting transgenic soybeans through double signal output based on gene editing technology Download PDFInfo
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Abstract
The invention discloses a preparation method and application of an electrochemical sensor for detecting transgenic soybeans by double signal output based on a gene editing technology, which is characterized by comprising a magnetic material Fe3O4The step of @ AuNPs synthesis; signal unit Fe3O4@AuNPs&DNA‑Fc/[Ru(bpy)2]2+A step of synthesis; uniformly mixing a Cas12a enzyme solution and a crRNA solution, incubating at room temperature, adding a t-DNA solution and a binding buffer solution, uniformly mixing, incubating at room temperature, adding a signal unit solution, after cutting, absorbing a magnetic material part by adopting a magnetic separation method, and re-dispersing the magnetic material part into 20 mu L of water; and finally, dripping part of the magnetic material on the surface of the treated magnetic glassy carbon electrode to obtain the electrochemical sensor, wherein the transgenic soybean can be detected by adopting a rapid scanning voltammetry technology and an electrochemical luminescence method, and the electrochemical sensor has the advantages of high sensitivity, high selectivity and simplicity and rapidness in operation.
Description
Technical Field
The invention relates to a detection method of transgenic soybeans, in particular to a preparation method and application of an electrochemical sensor for detecting transgenic soybeans by double signal output based on a gene editing technology.
Background
As an emerging biotechnology, the transgenic technology is a new biotechnology, which is characterized in that a desired target gene is added into genetic materials of other organisms by utilizing genetic engineering so as to improve the original characters of the organisms or endow the organisms with new excellent characters. In recent years, the application and development of transgenic technology in the agricultural field are very rapid, and the transgenic technology becomes the focus of agricultural science and technology competition of various countries in the world. At present, transgenic soybeans as important grain crops occupy 57 percent (5860 ten thousand hectares) of the total planting area of the transgenic crops in the world. Meanwhile, the safety problem of transgenic food is also concerned, so that the development of a detection method which is high in sensitivity, simple and easy to operate is necessary.
CRISPR/Cas is an RNA-mediated, heritable, adaptive immune system widely found in bacteria and archaea. Is developed into a novel gene editing technology and is successfully applied to the fields of agriculture, medicine, biology and the like. CRISPR/Cas12a belongs to the second major class, type V systems. After Cas12a specifically binds to target DNA under crRNA guidance, the property of non-specifically cleaving single-stranded DNA is activated, and can be applied to the development of a sensing detection system.
Electrochemiluminescence (ECL) has been widely used in the fields of bioanalysis such as immunoassay and nucleic acid hybridization analysis as a highly sensitive analysis method. The method combines the advantages of high chemiluminescence sensitivity, wide linear range and simple instrument, and the advantages of good reproducibility and easy control of the electrochemical method. Fast sweep voltammetry (FSCV) is a sub-second resolution electrochemical method. The detection sensitivity is improved along with the increase of the scanning speed, and the method is suitable for detecting trace analytes. At present, no report related to the detection of transgenic soybean by an electrochemical biosensor based on a CRISPR/Cas12 system exists at home and abroad, and no report related to the detection of transgenic products by ECL and FSCV double signal output exists.
Disclosure of Invention
The invention aims to provide a preparation method and application of an electrochemical sensor for detecting transgenic soybeans by double signal output based on a gene editing technology, which has high sensitivity, high selectivity, simple and quick operation.
The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method of an electrochemical sensor for detecting transgenic soybeans based on double signal output of a gene editing technology comprises the following steps:
(1) magnetic material Fe3O4Synthesis of @ AuNPs
(2) Signal unit (Fe)3O4@AuNPs&DNA-Fc/[Ru(bpy)2]2+) Synthesis of
180-220 μ L Fe3O4@ AuNPs were transferred to a flask and then 15-20. mu.L of 3-5 mmol/L ruthenium complex [ Ru (bpy) ]was added2]2+And 150-200 mu L of 90-100 mu g/mL DNA-Fc (ferrocene), adjusting the pH value to 7.5, incubating for 4-6 hours at 35 ℃, adding 160-200 mu L of 2 wt% bovine serum albumin solution to block non-specific binding sites, removing free DNA-Fc by a magnetic cleaning method, and re-dispersing into 200 mu L water to obtain a signal unit Fe3O4@AuNPs&DNA-Fc/[Ru(bpy)2]2+；
(3) CRISPR/Cas12 non-specific cleavage process
Mixing 2-5 mu L of 1-3 pmol/L Cas12a enzyme (one type in a CRISPR/Cas gene editing system and having the function of non-specifically cutting DNA single strands) solution and 2-5 mu L of 5-10 pmol/L crRNA solution uniformly, incubating at room temperature for 5-10 minutes, adding 2-5 mu L of 10 mu mol/L t-DNA solution and 10-20 mu L of 10 multiplied binding buffer solution, mixing uniformly, incubating at room temperature for 10-15 minutes, and then adding 5-10 mu L of signal unit Fe3O4@AuNPs&DNA-Fc/[Ru(bpy)2]2+Incubating the solution at 37 ℃ for 45-90 minutes (after base complementary pairing of the crRNA for guiding Cas12a enzyme and the target t-DNA in the system, the function of nonspecifically cutting single-stranded DNA by the Cas12a enzyme is activated, and the modification of the single-stranded DNA on Fe3O4Cutting DNA-Fc on the surface of the @ AuNPs to enable Fc to be dissociated in a solution), after the cutting is finished, absorbing a magnetic material part by adopting a magnetic separation method, and re-dispersing the magnetic material part into 20 mu L of water for subsequent experiments;
(4) construction of electrochemical biosensor
Al for magnetic glassy carbon electrode with diameter of 2mm2O3Polishing the mixture into a mirror surface, then ultrasonically washing the mirror surface by using absolute ethyl alcohol and water for 2 to 5 minutes in sequence, and blowing the mirror surface by using nitrogen for later use after the mirror surface is completely washed by water; dripping the part of the magnetic material obtained in the step (3) of 5-8 mu L on the surface of the treated magnetic glassy carbon electrode, and tightly combining the magnetic material and the electrode through magnetic force action to obtain the magnetic material based on the gene editing technologyAn electrochemical sensor for detecting transgenic soybean with double signal output.
Preferably, the magnetic material Fe described in step (1)3O4The specific steps for synthesis of @ AuNPs are as follows:
(1) 2.5-3.0 g FeCl3·6H2O、7.5~8.0 g NH4Ac and 0.5-1.0 g of sodium citrate are dissolved in 120-150 mL of ethylene glycol, stirred vigorously for 1 hour at 140-170 ℃ under the protection of nitrogen, then transferred into a stainless steel autoclave with a polytetrafluoroethylene lining, heated for 14-16 hours at 200 ℃, then naturally cooled to room temperature to obtain magnetic beads, and the magnetic beads are washed with ethanol and ultrapure water for multiple times and then dispersed in 50mL of ethanol to obtain the ferroferric oxide nano particles Fe3O4NPs；
(2) 40-50 mL of Fe3O4NPs and 0.4-0.6 mL of 3-Aminopropyltriethoxysilane (APTES) are mixed, ultrasonic treatment is carried out for 0.5 hour, stirring is carried out for 5-8 hours at room temperature, and magnetic separation, cleaning and re-dispersion are carried out in 50mL of water, so that aminated Fe is obtained3O4NPs；
(3) 0.6-1.0 mL of 0.1-0.3 mol/L NaOH solution and 0.5-0.7 mL of 80 wt% tetrakis (hydroxymethyl) phosphonium chloride (THPC) solution are added to 27mL of water, and after stirring for 5 minutes, 0.9-1.3 mL of 1wt% HAuCl is added4·4H2Stirring the O solution for 10-20 minutes to obtain an Au seed solution;
(4) subjecting 400-500 mu L of aminated Fe to ultrasonic treatment3O4NPs are mixed with 0.8-1.2 mL of gold seed solution, and the mixture is stirred for 0.5-1 hour at room temperature and then dispersed in 500 mu L of ultrapure water to obtain Fe3O4@ Au seed; mixing 90-100 μ L Fe3O4Adding @ Au seeds into 2-4 mL of gold growth solution, then adding 110-130 mu L of 0.35 wt% polyvinylpyrrolidone (PVP) aqueous solution and 13-16 mu L of formaldehyde solution, and stirring for 35-45 minutes to obtain the magnetic material Fe3O4@ AuNPs; the formula of the gold growth solution is as follows: 5.0-5.5 mu mol/L HAuCl4·4H2O and 1.0 to 1.2. mu. mol/L K2CO3The mixed solution of (1).
Preference is given toThe t-DNA sequence described in the step (3) is 5' -GCT AAG CAC ATG CATTTT AAC GAA TTA ATT CGG GGG ATCT-3', the sequence of the crRNA is 5' -UAA UUU CUA CUA AGU GUA GAUACG AAU UAA UUC GGG GGA UC3' and the DNA-Fc sequence is 5' -Fc-TTATT-C6-SH-3' (the underlined part in the t-DNA sequence is the recognition site PAM of the crRNA, the underlined part of the crRNA is the sequence complementary to the base of the t-DNA, and the rest is a fixed sequence).
Preferably, the formulation of 10 × binding buffer described in step (3) is as follows: 20mmol/L Tris-HCl (pH = 7.5), 100mmol/L KCl, 5mmol/L MgCl21mmol/L Dithiothreitol (DTT), 5wt% glycerol.
The method for detecting the transgenic soybean by the electrochemical sensor prepared by the method comprises the following steps:
(1) detecting the transgenic soybean by a rapid scanning voltammetry technology: the electrochemical sensor prepared according to any one of claims 1 to 4 is used as a working electrode, a platinum electrode is used as an auxiliary electrode, a PBS buffer solution (pH = 7.4) is placed, a Fast Scanning Cyclic Voltammetry (FSCV) is adopted, the potential range is-0.5-1.3V, and the potential scanning speed is 200-1000V/s; measuring the corresponding oxidation peak current under the condition of t-DNA with different concentrations, establishing a quantitative relation between the concentration of the t-DNA and the peak current, and measuring the concentration of the t-DNA in an unknown sample according to the quantitative relation;
(2) detecting the transgenic soybean by an electrochemical luminescence method: an electrochemical sensor prepared according to any one of claims 1 to 4 is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode, 0.1mol/L PBS solution with pH =8.0 and 20-30 mmol/L tripropylamine is placed in the electrochemical sensor, a chronoamperometry is used as an excitation signal, the voltage range is 0-1.5V, the pulse width is 0.25 seconds, the pulse period is 30 seconds, the high voltage value of a photomultiplier of a weak luminometer is 800V, and the Fc pair in a signal unit can perform electrochemiluminescence [ Ru (bpy) ]2]2+Has inhibitory effect, so that non-specific function of Cas12a is activated and DNA-Fc is cleaved to [ Ru (bpy) ]with increasing t-DNA concentration2]2+Decrease in inhibition and increase in electrochemiluminescence intensity, and determination of corresponding electrochemistry under different concentrations of t-DNAAnd (3) establishing a quantitative relation between the concentration of the t-DNA and the electrochemical luminescence intensity, thereby measuring the concentration of the t-DNA in the unknown sample.
The invention principle is as follows: the invention constructs an electrochemical biosensor for detecting transgenic soybean SHZD32-1 by using ECL and FSCV signals by using the non-specific cutting principle of CRISPR/Cas12 a. Firstly, AuNPs are coated on aminated Fe by utilizing the electrostatic adsorption effect of gold-ammonia bond3O4NPs surface, followed by gold-amino bond [ Ru (bpy)2]2+Assembled on the AuNPs surface. In addition, DNA-Fc (5 ' -Fc-TTA TT-C6-SH-3 ') is a DNA single chain with 3' -end modified Sulfhydryl (SH), and can be subjected to self-assembly modification and fixed on the surfaces of AuNPs based on Au-S bonding. Upon complementary pairing of crRNA mediating Cas12a enzyme with t-DNA, the cleavage function of Cas12a is activated. The single-stranded DNA-Fc immobilized on the AuNPs surface was cleaved, and the detection signal of FSCV corresponding to Fc as an electrochemical signal marker was reduced, but its pair [ Ru (bpy)2]2+The inhibition of ECL decreased, with a consequent increase in ECL detection signal. Thus, both signal offsets can be used to establish a quantitative relationship with t-DNA concentration and to detect t-DNA concentration in unknown samples.
Compared with the prior art, the invention has the advantages that:
(1) the selectivity is high: realizing specific recognition of t-DNA through a CRISPR/Cas gene editing system;
(2) the sensitivity is high: ECL and FSCV, both very sensitive detection methods;
(3) the dual signal detection avoids the occurrence of false positives or false negatives: the ECL method and the FSCV method are adopted for simultaneous detection, and the two signals are mutually verified in terms of their lengths, so that false positive or false negative of the detection result can be avoided;
(4) the operation is simple and does not need complex instruments: cas12a has the action temperature of 37 ℃, does not need complex temperature changing conditions, and therefore has low requirements on instruments and is easy to operate.
Drawings
FIG. 1 is a flow chart of electrochemical biosensor based on CRISPR/Cas12 system for detecting transgenic soybean SHZD 32-1;
FIG. 2 is a graph showing the linear relationship between the electrochemical signal intensity and the t-DNA concentration measured by the FSCV method;
FIG. 3 is a graph showing a linear relationship between an electrochemiluminescence intensity and a t-DNA concentration measured by the ECL method;
FIG. 4 shows the FSCV method for blank (blank) and 107fmol/L transgenic maize NK603, 107fmol/L transgenic maize BT63, 107fmol/L transgenic soybean GTS40-3-2, 107fmol/L non-transgenic Soybean, 107A current signal graph measured by fmol/L transgenic soybean SHZD 32-1;
FIG. 5 shows the ECL method for blank (blank) and blank (10) respectively7fmol/L transgenic maize NK603, 107fmol/L transgenic maize BT63, 107fmol/L transgenic soybean GTS40-3-2, 107fmol/L non-transgenic Soybean, 107An electrochemiluminescence signal graph measured by fmol/L transgenic soybean SHZD 32-1.
Detailed Description
The invention is described in further detail below with reference to the accompanying examples.
Detailed description of the preferred embodiment
Example 1
A preparation method of an electrochemical sensor for detecting transgenic soybeans based on double signal output of a gene editing technology is shown in figure 1 and comprises the following steps:
1. magnetic material Fe3O4Synthesis of @ AuNPs
(1) 2.8 g FeCl3·6H2O、7.8 g NH4Ac and 0.8g of sodium citrate are dissolved in 135mL of ethylene glycol, stirred vigorously for 1 hour at 155 ℃ under the protection of nitrogen, then transferred into a stainless steel autoclave with a polytetrafluoroethylene lining, heated for 15 hours at 200 ℃, naturally cooled to room temperature to obtain magnetic beads, the magnetic beads are washed for multiple times by ethanol and ultrapure water and then dispersed in 50mL of ethanol to obtain the ferroferric oxide nano particles Fe3O4NPs；
(2) 45 mL of Fe3O4NPs were mixed with 0.5 mL 3-Aminopropyltriethoxysilane (APTES) and incubatedAfter 0.5 hour of sound, stirring for 7 hours at room temperature, performing magnetic separation, cleaning, and re-dispersing in 50mL of water to obtain aminated Fe3O4NPs；
(3) 0.8 mL of a 0.2 mol/L NaOH solution and 0.6mL of a 80 wt% Tetrakis Hydroxymethyl Phosphonium Chloride (THPC) solution were added to 27mL of water, and after stirring for 5 minutes, 1.1 mL of 1wt% HAuCl was added4·4H2Stirring the solution O for 15 minutes to obtain an Au seed solution;
(4) 450 μ L of aminated Fe under sonication3O4NPs are mixed with 1 mL of gold seed solution, and the mixture is dispersed in 500 mu L of ultrapure water after being stirred for 0.8 hour at room temperature to obtain Fe3O4@ Au seed; 95 μ L of Fe3O4@ Au seed is added into 3mL of gold growth solution, then 120 mu L of 0.35 wt% polyvinylpyrrolidone (PVP) aqueous solution and 14 mu L of formaldehyde solution are added and stirred for 40 minutes to obtain the magnetic material Fe3O4@ AuNPs; the formula of the gold growth solution is as follows: 5.2. mu. mol/L HAuCl4·4H2O and 1.1. mu. mol/L K2CO3The mixed solution of (1).
2. Signal unit (Fe)3O4@AuNPs&DNA-Fc/[Ru(bpy)2]2+) Synthesis of
200 μ L of Fe3O4@ AuNPs were transferred to a flask, and 18. mu.L of 4 mmol/L ruthenium complex [ Ru (bpy)2]2+And 180 mu L of 95 mu g/mL DNA-Fc (ferrocene), adjusting the pH value to 7.5, incubating at 35 ℃ for 5 hours, adding 180 mu L of 2 wt% bovine serum albumin solution to block non-specific binding sites, removing free DNA-Fc by magnetic cleaning, and re-dispersing in 200 mu L water to obtain a signal unit Fe3O4@AuNPs&DNA-Fc/[Ru(bpy)2]2+。
3. CRISPR/Cas12 non-specific cleavage process
Mixing 3 μ L of Cas12a enzyme (one type of CRISPR/Cas gene editing system, with non-specific DNA single strand cleavage function) solution 2 pmol/L and 3 μ L of crRNA solution 8 pmol/L, incubating at room temperatureAfter 8 minutes, 3. mu.L of a 10. mu. mol/L t-DNA solution and 15. mu.L of 10 Xbinding buffer were added and mixed, incubated at room temperature for 12 minutes, followed by addition of 8. mu.L of the signal element Fe3O4@AuNPs&DNA-Fc/[Ru(bpy)2]2+Solution, incubation for 70 minutes at 37 ℃ (after base complementary pairing of the crRNA of the Cas12a guiding enzyme and the target t-DNA in the system, the function of nonspecifically cutting single-stranded DNA by the Cas12a enzyme is activated, and the modification is carried out on Fe3O4Cutting DNA-Fc on the surface of the @ AuNPs to enable Fc to be dissociated in a solution), after the cutting is finished, absorbing a magnetic material part by adopting a magnetic separation method, and re-dispersing the magnetic material part into 20 mu L of water for subsequent experiments; wherein the t-DNA sequence is 5' -GCT AAG CAC ATG CATTTT AAC GAA TTA ATT CGG GGG ATCT-3', crRNA sequence 5' -UAA UUU CUA CUA AGU GUA GAUACG AAU UAA UUC GGG GGA UC3' and the DNA-Fc sequence is 5' -Fc-TTATT-C6-SH-3' (the underlined part in the t-DNA sequence is the recognition site PAM of the crRNA, the underlined part of the crRNA is the sequence complementary to the base of the t-DNA, and the rest is a fixed sequence).
4. Construction of electrochemical biosensor
Al for magnetic glassy carbon electrode with diameter of 2mm2O3Polishing the mixture into a mirror surface, then ultrasonically washing the mirror surface by using absolute ethyl alcohol and water for 2 to 5 minutes in sequence, and blowing the mirror surface by using nitrogen for later use after the mirror surface is completely washed by water; and (4) taking the part of the magnetic material obtained in the step (3) of 7 mu L, dripping the part of the magnetic material on the surface of the treated magnetic glassy carbon electrode, and tightly combining the magnetic material and the electrode through magnetic force action to obtain the electrochemical sensor for detecting the transgenic soybean through double signal output based on the gene editing technology.
Example 2
The difference from the above example 1 is that:
(1) 2.5g FeCl3·6H2O、7.5 g NH4Ac and 0.5 g sodium citrate were dissolved in 150mL ethylene glycol, vigorously stirred at 140 deg.C under nitrogen for 1 hour, transferred to a stainless steel autoclave lined with Teflon, heated at 200 deg.C for 14 hours, and allowed to standCooling to room temperature to obtain magnetic beads, washing the magnetic beads with ethanol and ultrapure water for multiple times, and dispersing in 50mL of ethanol to obtain the ferroferric oxide nanoparticles Fe3O4NPs；
(2) 40 mL of Fe3O4NPs and 0.4 mL of 3-Aminopropyltriethoxysilane (APTES) are mixed, ultrasonic treatment is carried out for 0.5 hour, the mixture is stirred for 5 hours at room temperature, and then the mixture is subjected to magnetic separation, cleaning and re-dispersing in 50mL of water to obtain aminated Fe3O4NPs；
(3) 0.6mL of a 0.3mol/L NaOH solution and 0.5 mL of a 80 wt% tetrakis (hydroxymethyl) phosphonium chloride (THPC) solution were added to 27mL of water, and after stirring for 5 minutes, 0.9 mL of 1wt% HAuCl was added4·4H2Stirring the solution O for 10 minutes to obtain an Au seed solution;
(4) 400 μ L of aminated Fe under sonication3O4NPs are mixed with 0.8 mL of gold seed solution, and the mixture is dispersed in 500 mu L of ultrapure water after being stirred for 0.5 hour at room temperature to obtain Fe3O4@ Au seed; mixing 90 μ L Fe3O4Adding the @ Au seeds into 2-4 mL of gold growth solution, then adding 110 mu L of 0.35 wt% polyvinylpyrrolidone (PVP) aqueous solution and 13 mu L of formaldehyde solution, and stirring for 35 minutes to obtain the magnetic material Fe3O4@ AuNPs; the formula of the gold growth solution is as follows: 5.0. mu. mol/L HAuCl4·4H2O and 1.0. mu. mol/L K2CO3The mixed solution of (1).
Step 3CRISPR/Cas12 non-specific cleavage: will be provided withmu.L of 3pmol/L Cas12a enzyme solution and 2. mu.L of 10pmol/L crRNA solution were mixed, incubated at room temperature for 5 minutes, 2. mu.L of 10. mu. mol/L t-DNA solution and 10. mu.L of 10 Xbinding buffer were added, mixed, incubated at room temperature for 10 minutes, and then 5. mu.L of signal element Fe was added3O4@AuNPs&DNA-Fc/[Ru(bpy)2]2+Solution, after incubation for 45 minutes at 37 ℃;
Example 3
The difference from the above example 1 is that:
(1) 3.0g FeCl3·6H2O、8.0 g NH4Ac and 1.0g of sodium citrate are dissolved in 150mL of ethylene glycol, stirred vigorously for 1 hour at 170 ℃ under the protection of nitrogen, then transferred into a stainless steel autoclave with a polytetrafluoroethylene lining, heated for 16 hours at 200 ℃, naturally cooled to room temperature to obtain magnetic beads, and the magnetic beads are washed for multiple times by ethanol and ultrapure water and then dispersed in 50mL of ethanol to obtain the ferroferric oxide nano particles Fe3O4NPs；
(2) 50mL of Fe3O4NPs and 0.6mL of 3-Aminopropyltriethoxysilane (APTES) are mixed, ultrasonic treatment is carried out for 0.5 hour, stirring is carried out for 8 hours at room temperature, and magnetic separation, cleaning and re-dispersion are carried out in 50mL of water, thus obtaining aminated Fe3O4NPs；
(3) 1.0mL of a 0.1mol/L NaOH solution and 0.7mL of a 80 wt% Tetrakis Hydroxymethyl Phosphonium Chloride (THPC) solution were added to 27mL of water, and after stirring for 5 minutes, 1.3mL of 1wt% HAuCl was added4·4H2Stirring the solution O for 20 minutes to obtain an Au seed solution;
(4) 500. mu.L of aminated Fe under sonication3O4NPs and 1.2mL goldMixing the seed solutions, stirring the mixture at room temperature for 1 hr, and dispersing in 500 μ L ultrapure water to obtain Fe3O4@ Au seed; 100 mu L of Fe3O4@ Au seed is added into 4mL of gold growth solution, then 130 mu L of 0.35 wt% polyvinylpyrrolidone (PVP) aqueous solution and 16 mu L of formaldehyde solution are added, and stirring is carried out for 45 minutes, thus obtaining the magnetic material Fe3O4@ AuNPs; the formula of the gold growth solution is as follows: 5.5. mu. mol/L HAuCl4·4H2O and 1.2. mu. mol/L K2CO3The mixed solution of (1).
Step 3CRISPR/Cas12 non-specific cleavage: mu.L of 1 pmol/L Cas12a enzyme solution and 5. mu.L of 5 pmol/L crRNA solution were mixed, incubated at room temperature for 10 minutes, 5. mu.L of 10. mu. mol/L t-DNA solution and 20. mu.L of 10 Xbinding buffer were added, mixed, incubated at room temperature for 15 minutes, and then 10. mu.L of signal element Fe was added3O4@AuNPs&DNA-Fc/[Ru(bpy)2]2+The solution was incubated at 37 ℃ for 90 minutes;
Detailed description of the invention
Method for double-signal detection of transgenic soybean by using electrochemical sensor prepared in specific example 1
1. Detecting the transgenic soybean SHZD32-1 by a rapid sweep voltammetry technology: the method comprises the following steps of taking a magnetic glassy carbon electrode as a working electrode and a platinum electrode as an auxiliary electrode, putting a PBS (phosphate buffer solution) (pH = 7.4), and adopting a Fast Scanning Cyclic Voltammetry (FSCV), wherein the potential range is-0.5-1.3V, and the potential scanning speed is 200-1000V/s; measuring the corresponding oxidation peak current under the condition of t-DNA with different concentrations, establishing a quantitative relation between the concentration of the t-DNA and the peak current, and measuring the concentration of the t-DNA in an unknown sample according to the quantitative relation; as shown in FIG. 2, the electrochemical signal intensities of different concentrations of t-DNA: (y) t-DNA concentration: (x) A logarithmic linear relationship, linear equation ofy=-0.0434 xlogx+0.437, correlation coefficient R =0.995, linear relation is good, can be used for the detection of transgenic soybean SHZD32-1 in unknown samples.
2. Detecting the transgenic soybean SHZD32-1 by an electrochemiluminescence method: using a magnetic glassy carbon electrode as a working electrode, a platinum electrode as an auxiliary electrode, Ag/AgCl as a reference electrode, putting 0.1mol/L of PBS (phosphate buffer solution) with pH =8.0 and 20-30 mmol/L of tripropylamine into the PBS, adopting a chronoamperometry as an excitation signal, wherein the voltage range is 0-1.5V, the pulse width is 0.25 s, the pulse period is 30 s, the high voltage value of a photomultiplier of a weak luminometer is 800V, and the Fc pair in a signal unit can carry out electrochemiluminescence [ Ru (bpy) ]2]2+Has inhibitory effect, so that non-specific function of Cas12a is activated and DNA-Fc is cleaved to [ Ru (bpy) ]with increasing t-DNA concentration2]2+The inhibition of (a) is reduced and the electrochemiluminescence intensity is increased, thereby determining the concentration of t-DNA in the unknown sample. As shown in FIG. 3, the electrochemiluminescence intensity for t-DNA of different concentrations ((C))y) t-DNA concentration: (x) A logarithmic linear relationship, linear equation ofy=869.1 x logx1306.9, the correlation coefficient R =0.996, the linear relation is good, can be used for detecting the transgenic soybean SHZD32-1 in unknown samples.
Detailed description of the preferred embodiment
As shown in FIG. 4, the method is applied to blank (blank) and 107fmol/L transgenic maize NK603, 107fmol/L transgenic maize BT63, 107fmol/L transgenic soybean GTS40-3-2, 107fmol/L non-transgenic Soybean, 107A current signal graph measured by fmol/L transgenic soybean SHZD 32-1. As can be seen from the figure, the FSCV detection method has good selectivity on the detection of the transgenic soybean SHZD 32-1;
as shown in FIG. 5, the method is applied to blank (blank) and 107fmol/L transgenic maize NK603, 107fmol/L transgenic maize BT63, 107fmol/L transgenic soybean GTS40-3-2, 107fmol/L non-transgenic Soybean, 107An electrochemiluminescence signal graph measured by fmol/L transgenic soybean SHZD 32-1. As can be seen from the figure, the ECL detection method has good selectivity on the detection of the transgenic soybean SHZD 32-1;
in conclusion, the detection results of the two methods are verified mutually, so that false positive possibly caused by a single detection method is avoided, and the detection result of the electrochemical sensor is reliable.
The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Those skilled in the art should also realize that changes, modifications, additions and substitutions can be made without departing from the true spirit and scope of the invention.
<110> Ningbo university
<120> preparation method and application of electrochemical sensor for detecting transgenic soybean based on gene editing technology and double signal output
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 40
<212> DNA
<213> t-DNA（5'-GCTAAGCACATGCATTTTAACGAATTAATTCGGGGG ATCT-3'）
<400> 1
<210> 2
<211> 41
<212> DNA
<213> crRNA（5'-UAAUUUCUACUAAGUGUAGAUACGAAUUAAUUCGGG GGA UC-3'）
<400> 2
<210> 3
<211> 5
<212> DNA
<213> DNA-Fc（5'-Fc-TTATT-C6-SH-3'）
<400> 3
Claims (5)
1. A preparation method of an electrochemical sensor for detecting transgenic soybeans based on double signal output of a gene editing technology is characterized by comprising the following steps:
(1) magnetic material Fe3O4Synthesis of @ AuNPs
(2) Signal unit synthesis
180-220 μ L Fe3O4@ AuNPs were transferred to a flask and then 15-20. mu.L of 3-5 mmol/L ruthenium complex [ Ru (bpy) ]was added2]2+And 150-200 mu L of 90-100 mu g/mL DNA-Fc, adjusting the pH value to 7.5, incubating at 35 ℃ for 4-6 hours, adding 160-200 mu L of 2 wt% bovine serum albumin solution to block non-specific binding sites, removing free DNA-Fc by a magnetic cleaning method, and re-dispersing into 200 mu L water to obtain a signal unit Fe3O4@AuNPs&DNA-Fc/[Ru(bpy)2]2+；
(3) CRISPR/Cas12 non-specific cleavage process
Mixing 2-5 mu L of 1-3 pmol/L Cas12a enzyme solution and 2-5 mu L of 5-10 pmol/L crRNA solution uniformly, incubating at room temperature for 5-10 minutes, adding 2-5 mu L of 10 mu mol/L t-DNA solution and 10-20 mu L of 10 multiplied binding buffer solution, mixing uniformly, incubating at room temperature for 10-15 minutes, and then adding 5-10 mu L of signal unit Fe3O4@AuNPs&DNA-Fc/[Ru(bpy)2]2+Incubating the solution at 37 ℃ for 45-90 minutes, absorbing the magnetic material part by adopting a magnetic separation method, and re-dispersing the magnetic material part into 20 mu L of water to obtain a magnetic material part solution;
(4) construction of electrochemical biosensor
Al for magnetic glassy carbon electrode with diameter of 2mm2O3Polishing the mixture into a mirror surface, then ultrasonically washing the mirror surface by using absolute ethyl alcohol and water for 2 to 5 minutes in sequence, and blowing the mirror surface by using nitrogen for later use after the mirror surface is completely washed by water; taking 5-8 mu L of the product obtained in the step (3)And (3) dripping partial solution of the magnetic material on the surface of the treated magnetic glassy carbon electrode to obtain the electrochemical sensor for detecting the transgenic soybean by double signal output based on the gene editing technology.
2. The method for preparing an electrochemical sensor for detecting transgenic soybean based on double signal output of gene editing technology as claimed in claim 1, wherein the magnetic material Fe in step (1)3O4The specific steps for synthesis of @ AuNPs are as follows:
(1) 2.5-3.0 g FeCl3·6H2O、7.5～8.0g NH4Ac and 0.5-1.0 g of sodium citrate are dissolved in 120-150 mL of ethylene glycol, stirred vigorously for 1 hour at 140-170 ℃ under the protection of nitrogen, then transferred into a stainless steel autoclave with a polytetrafluoroethylene lining, heated for 14-16 hours at 200 ℃, then naturally cooled to room temperature to obtain magnetic beads, and the magnetic beads are washed with ethanol and ultrapure water for multiple times and then dispersed in 50mL of ethanol to obtain the ferroferric oxide nano particles Fe3O4NPs；
(2) 40-50 mL of Fe3O4NPs and 0.4-0.6 mL of 3-aminopropyltriethoxysilane are mixed, subjected to ultrasonic treatment for 0.5 hour, stirred at room temperature for 5-8 hours, subjected to magnetic separation, washed and re-dispersed in 50mL of water to obtain aminated Fe3O4NPs；
(3) 0.6 to 1.0mL of 0.1 to 0.3mol/L NaOH solution and 0.5 to 0.7mL of 80 wt% tetrakis hydroxymethyl phosphonium chloride solution were added to 27mL of water, and after stirring for 5 minutes, 0.9 to 1.3mL of 1wt% HAuCl was added4·4H2Stirring the O solution for 10-20 minutes to obtain an Au seed solution;
(4) subjecting 400-500 mu L of aminated Fe to ultrasonic treatment3O4NPs are mixed with 0.8-1.2 mL of gold seed solution, and the mixture is stirred for 0.5-1 hour at room temperature and then dispersed in 500 mu L of ultrapure water to obtain Fe3O4@ Au seed; mixing 90-100 μ L Fe3O4@ Au seed is added into 2-4 mL gold growth solution, then added with 110-130 μ L0.35 wt% polyvinylpyrrolidone water solution and 13-16 μ L formaldehyde solution, and stirred for 35 @Obtaining the magnetic material Fe after 45 minutes3O4@ AuNPs; the formula of the gold growth solution is as follows: 5.0-5.5 mu mol/L HAuCl4·4H2O and 1.0 to 1.2. mu. mol/L K2CO3The mixed solution of (1).
3. The method for preparing an electrochemical sensor for detecting transgenic soybean with double signal output based on gene editing technology as claimed in claim 1, wherein the t-DNA sequence in step (3) is 5'-GCT AAG CAC ATGCATTTTAACGAATTAATTCGGGGGATCT-3', and the crRNA sequence is 5'-UAA UUU CUACUAAGUGUAGAUACGAAUUAAUUCGGGGGAUC-3', DNA-Fc sequence 5 '-Fc-TTATT-C6-SH-3'.
4. The method for preparing an electrochemical sensor for detecting transgenic soybean based on double signal output of gene editing technology as claimed in claim 1, wherein the formulation of 10 x binding buffer in step (3) is as follows: 20mmol/L Tris-HCl, 100mmol/L KCl, 5mmol/L MgCl21mmol/L dithiothreitol and 5wt% glycerol.
5. A method for double-signal detection of transgenic soybean by the electrochemical sensor prepared by the preparation method of any one of claims 1 to 4, which is characterized by comprising the following steps:
(1) detecting the transgenic soybean by a rapid scanning voltammetry technology: the prepared electrochemical sensor is used as a working electrode, a platinum electrode is used as an auxiliary electrode, a PBS (phosphate buffer solution) with the pH value of 7.4 is placed in the electrochemical sensor, a rapid scanning cyclic voltammetry method is adopted, the potential range is-0.5-1.3V, and the potential scanning speed is 200-1000V/s; measuring the corresponding oxidation peak current of the transgenic soybean under the condition of t-DNA with different concentrations, establishing a quantitative relation between the concentration of the t-DNA and the peak current, and measuring the concentration of the t-DNA in an unknown sample according to the quantitative relation;
(2) detecting the transgenic soybean by an electrochemical luminescence method: the prepared electrochemical sensor is used as a working electrode, a platinum electrode is used as an auxiliary electrode, Ag/AgCl is used as a reference electrode, 0.1mol/L PBS solution with the pH value of 8.0 and 20-30 mmol/L tripropylamine is put into the electrochemical sensor, a chronoamperometry method is used as an excitation signal, the voltage range is 0-1.5V, the pulse width is 0.25 second, the pulse period is 30 seconds, the high voltage value of a photomultiplier of a weak luminometer is 800V, the corresponding electrochemical luminescence intensity under the condition of transgenic soybean t-DNA with different concentrations is measured, the quantitative relation between the concentration of the t-DNA and the electrochemical luminescence intensity is established, and the concentration of the t-DNA in an unknown sample is measured according to the quantitative relation.
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