CN113466305A - Construction method of ratio adapter sensor based on simultaneous acquisition of double signals of self-enhanced luminescent material and methylene blue - Google Patents
Construction method of ratio adapter sensor based on simultaneous acquisition of double signals of self-enhanced luminescent material and methylene blue Download PDFInfo
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Abstract
The invention belongs to the technical field of biosensing detection, and particularly relates to a construction method of a ratio aptamer sensor based on simultaneous acquisition of double signals of a self-enhanced luminescent material and methylene blue, which is used for sensitively, quickly and accurately detecting Zearalenone (ZEN); specifically, by introducing ZEN aptamer complementary DNA (cDNA) with different sequences, a ratio aptamer sensor obtained by simultaneously obtaining self-enhanced electrochemiluminescence-electrochemistry double signals is constructed, the sensitivity of ZEN detection is regulated, different sequences can be selected according to different requirements, and the sensor has good selectivity and stability; the ratio adapter sensor capable of simultaneously acquiring the self-enhanced electrochemical luminescence peak signal and the electrochemical peak signal developed by the invention can realize quick and high-selectivity sensitive analysis on ZEN detection by adjusting the base pair sequence of cDNA, and obtain better recovery rate (97.3-102.7%).
Description
Technical Field
The invention belongs to the technical field of aptamer sensing detection, and particularly relates to a construction method of a ratio aptamer sensor based on simultaneous acquisition of dual signals of a self-enhanced luminescent material (NRS) and Methylene Blue (MB), which is used for sensitively and accurately detecting zearalenone in corn kernels.
Background
Zearalenone (ZEN) is a toxic metabolite mainly produced by fusarium graminearum, fusarium oxysporum, fusarium niveum and other strains, and is often present in crops such as wheat, corn, sorghum, barley, soybean and the like. The pollution of ZEN seriously affects the quality of agricultural products and the food safety, and further causes huge economic loss and great threat to human health. Excessive ZEN (10-100 mu g kg) in human body-1) Can lead to serious diseases such as teratogenicity, carcinogenesis, neurotoxicity and abortion. Currently, established ZEN detection methods have respective advantages and disadvantages. For example, high performance liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry have good analysis results and high accuracy in practical application, but the used instruments are expensive and complicated in pretreatment, and are not beneficial to large-scale sample screening and internal control detection. Electrochemical, electrochemiluminescence and photoelectrochemical methods are gaining more and more attention in the field of mycotoxin detection due to the advantages of simple and rapid operation, high sensitivity, good selectivity and the like, but the accuracy of such analysis methods still needs to be improved. Therefore, the development of a sensitive, efficient, accurate and easily-popularized ZEN analysis and detection method has important significance for guaranteeing the safety of crops and protecting the health of human beings.
In recent years, a single detection technology is usually adopted for detecting a target object, each method has respective advantages and disadvantages, and if the two methods are combined, the cooperation between the different methods can be realized, and the accuracy of the sensor is improved. The two detection methods are adopted to simultaneously analyze the target object, double signal output can be obtained, the target object can be accurately and sensitively detected, and the method is suitable for monitoring and analyzing the mycotoxin. Two methods developed at present are as follows: electrochemiluminescence-colorimetric double detection, electrochemistry-photoelectrochemical double detection and the like, but the methods cannot realize simultaneous acquisition of signals of the two methods and are time-consuming in detection. And no report is found for detecting zearalenone in corn by combining electrochemical luminescence and electrochemical methods at present.
Disclosure of Invention
Aiming at the problems existing in the single technology, the invention aims to combine the sensing technology of electrochemical luminescence and electrochemical double methods to construct a ratio aptamer sensor which can simultaneously obtain double signals of a self-enhanced luminescent material and methylene blue, and realize ZEN detection with adjustable and controllable sensitivity based on complementary DNA (cDNA) of different sequences.
A construction method of a ratiometric aptamer sensor based on simultaneous acquisition of dual signals of a self-enhanced luminescent material and methylene blue comprises the following steps:
(1) preparation of self-reinforced composite material:
firstly, adding ammonium citrate into ultrapure water, carrying out heating reaction under normal pressure, and in the reaction process, when the color of the solution changes from colorless to yellow, adjusting the pH value to obtain a nitrogen-doped graphene quantum dot solution, which is marked as an NGQDs solution;
then, mixing triton X-100, cyclohexane, 1-hexanol and water to obtain a mixed solution A; adding a terpyridyl ruthenium solution into the mixed solution A, stirring for a period of time, adding tetraethyl orthosilicate and ammonia water to initiate polymerization reaction, adding acetone to precipitate nanoparticles after the reaction is finished, then washing and centrifuging by using ethanol and ultrapure water respectively, drying the centrifuged product to obtain a dried product, and marking the dried product as Ru @ SiO2(preservation in powder form);
subsequently, Ru @ SiO2Dissolving the mixture by ethanol to obtain Ru @ SiO2A solution; then Ru @ SiO2Adding 3-aminopropyltriethoxysilane into the solution, stirring for reaction, washing with ethanol, centrifuging, and drying to obtain powdered amino-functionalized Ru @ SiO2Is denoted as NH2-Ru@SiO2；
Finally, NH is added2-Ru@SiO2Dissolving in ultrapure water to obtain NH2-Ru@SiO2Mixing the solution with NGQDs, stirring, dialyzing to obtain orange yellowThe color solution is marked as NRS solution and stored away from light at room temperature;
(2) sequentially treating a Glassy Carbon Electrode (GCE) with aluminum oxide powder with different particle sizes, then sequentially ultrasonically cleaning in water, ethanol, acetone and water, and airing to obtain a treated glassy carbon electrode;
(3) dripping the NRS solution prepared in the step (1) on the surface of the glassy carbon electrode treated in the step (2), and airing at room temperature to obtain a product marked as NRS/GCE;
(4) modifying the surface of the NRS/GCE sensor prepared in the step (3) with a chitosan solution, and airing; at the moment, a layer of film structure is formed on the surface of the sensor and is marked as CS/NRS/GCE;
(5) designing ZEN aptamer complementary DNA (cDNA) according to the known sequence and base complementary pairing principle of the ZEN aptamer, modifying the surface of the sensor obtained in the step (4) with the ZEN aptamer complementary DNA (cDNA), enabling the cDNA and chitosan to be fixed on the surface of an electrode through electrostatic adsorption, washing the sensor with PBS, and marking the washed sensor as cDNA/CS/NRS/GCE;
(6) modifying Bovine Serum Albumin (BSA) on the surface of the electrode obtained in the step (5) to seal the non-specific binding site on the surface of the chitosan, simultaneously leaching with PBS, and marking the leached sensor as BSA/cDNA/CS/NRS/GCE;
(7) modifying the sensor surface obtained in the step (6) with an aptamer (ZBA) of ZEN, and forming double-stranded DNA by using a hybridization reaction of the aptamer and cDNA; then washing with PBS to remove unbound ZBA, and marking the washed sensor as ZBA/BSA/cDNA/CS/NRS/GCE;
(8) soaking the sensor obtained in the step (7) in a Methylene Blue (MB) solution, wherein the MB is embedded into double-stranded DNA, and washing with ultrapure water to remove the embedded MB to obtain a ratio aptamer sensor based on the NRS and MB self-enhanced electrochemiluminescence-electrochemistry double method, which is marked as MB/ZBA/BSA/cDNA/CS/NRS/GCE;
(9) modifying ZEN standard solutions with different concentrations on the surface of the sensor prepared in the step (8), wherein each ZEN standard solution with one concentration is correspondingly modified with one sensor, and the concentrations and the sensors are in one-to-one correspondence; after a period of incubation, the cells were rinsed with PBS to remove ZBA bound to ZEN and a portion of MB, thereby obtaining a ratiometric aptamer sensor based on simultaneous acquisition of dual signals from the enhanced luminescent material and methylene blue, labeled ZEN/MB/ZBA/BSA/cDNA/CS/NRS/GCE.
Further, in the step (1), the using amount ratio of the ammonium citrate to the ultrapure water is 2 g: 60 mL; the heating reaction temperature is 200 ℃, and the reaction time is 30 min; the pH value is adjusted to 7.0 by using 1M sodium hydroxide solution.
Further, in the step (1), the volume ratio of the triton X-100, the cyclohexane, the 1-hexanol and the water in the mixed solution A is 1.77: 7.5: 1.8: 0.34; the triton X-100, the terpyridyl ruthenium solution, the tetraethyl orthosilicate, the ammonia water and the acetone are 1.77: 0.08: 0.1: 0.06: 20-30; the concentration of the terpyridyl ruthenium solution is 0.1M; stirring for a period of 20-30 min; the polymerization time was 24 h.
Further, in the step (1), the Ru @ SiO2The concentration of the solution was 2mg mL-1(ii) a The Ru @ SiO2The volume ratio of the solution to the 3-aminopropyltriethoxysilane is 1: 0.4; the stirring reaction time is 4 hours; the NH2-Ru@SiO2The concentration of the solution was 1mg mL-1(ii) a The NH2-Ru@SiO2The volume ratio of the solution to the NGQDs is 1: 5; the specific operation during dialysis is as follows: dialyzing for 24h with dialysis bag with molecular weight cutoff of 3500 Da.
Further, in the step (2), the diameter d of the glassy carbon electrode is 3 mm; the grain size of the aluminium oxide powder is 0.3 μm and 0.05 μm in sequence.
Further, in the step (3), the NRS solution is added dropwise in an amount of 6. mu.L.
Further, in the step (4), the concentration of the chitosan solution is 0.5 wt%, and the pH value is 5.0; the amount used at the time of the dropping was 2. mu.L.
Further, in the step (5), the base pair sequence of the ZEN aptamer complementary DNA (cDNA) is designed to comprise the following five base pair sequences, namely cDNA-1, cDNA-2, cDNA-3, cDNA-4 and cDNA-5, and the specific sequences are as follows:
cDNA-1(SEQ.ID.NO.1)：5′-ATA GAT GA-3′
cDNA-2(SEQ.ID.NO.2)：5′-TAC CAT AGA TAG ATG A-3′
cDNA-3(SEQ.ID.NO.3)：5′-TAG TAA TGT ACC ATA GAT AGA TGA-3′
cDNA-4(SEQ.ID.NO.4)：5′-ATT ACA GAT AGT AAT GTA CCA TAG ATA GAT GA-3′
cDNA-5(SEQ.ID.NO.5)：5′-CAT ATC ACA TTA CAG ATA GTA ATG TAC CAT AGA TAG ATG A-3′；
the concentration of the cDNA is 1 μ M, and the amount of the modified sensor surface obtained in step (4) is 3 μ L.
Further, in the step (6), the concentration of the bovine serum albumin is 1 wt%, and the amount of the modified electrode surface obtained in the step (5) is 3 μ L.
Further, in the step (7), the aptamer concentration of the ZEN is 0.6-1.8 mu M, and the surface usage amount of the sensor obtained in the step (6) is 3 mu L; the temperature of the hybridization reaction is 37 ℃, and the hybridization time is 20-60 min.
Further, the concentration of the methylene blue solution in the step (8) is 0.1-20 mu M, and the soaking time is 1 min. Further, in the step (9), the concentration of the ZEN standard solution is 1fg mL-1～10pg mL-1(ii) a The incubation temperature is 37 ℃, and the period of time is 20-70 min; the amount of the ZEN standard solution with different concentrations on the sensor surface is 3 mu L.
The ZEN aptamer complementary dna (cdna) and ZEN aptamer used in the present invention were purchased from bio-engineering (shanghai) gmbh.
The invention also relates to the use of a ratiometric aptamer sensor based on the simultaneous acquisition of dual signals of self-enhanced luminescent material (NRS) and Methylene Blue (MB) for the detection of ZEN, comprising the following steps:
(1) sequentially modifying V1 volumes of ZEN solutions with different concentrations on the surface of the sensor prepared in the step (9); incubating at 37 ℃ for 20-70 min, and then washing the electrode with 0.1M PBS (pH 7.5), wherein a sensor is correspondingly modified by a ZEN solution with one concentration, and the concentrations and the sensors are in one-to-one correspondence;
(2) the sensor prepared above (ZEN/MB/ZBA/BSA/cDNA/CS/NRS/GCE) was used as a working electrode, an Ag/AgCl (saturated KCl) electrode was used as a reference electrode, a platinum wire electrode was used as a counter electrode, and electrochemical and electrochemiluminescence signals were detected and recorded by an electrochemical workstation model CHI832C and an electrochemiluminescence instrument model ECL-MPI EII. All tests were performed in 0.1M PBS (pH 7.5) buffer, with a sweep voltage range of-0.4 to 1.25V, an amplitude of 0.025V, and a frequency of 15 Hz. And detecting electrochemical peak signals and electrochemical luminescence peak signals corresponding to the working electrode by using a Square Wave Voltammetry (SWV) method and an electrochemical luminescence (ECL) method, taking the ratio of the electrochemical luminescence peak signals of NRS and the electrochemical peak signals of MB as a vertical coordinate, taking the log value of the corresponding ZEN concentration as a horizontal coordinate, and establishing corresponding standard curves based on different sequence cDNA for detecting the ZEN concentration in an actual sample.
(3) Detection of ZEN in samples: firstly, obtaining a sample solution, modifying the sample solution with the volume of V2 on the surface of a sensor, and obtaining corresponding electrochemiluminescence intensity and current value through electrochemical test; substituting the ratio of the electrochemiluminescence intensity to the current into the standard curve constructed in the step (2), so as to obtain the concentration of ZEN in the sample; the application of ZEN detection in unknown samples is realized.
The concentration of the ZEN solution in the step (1) is 1fg mL-1～10pg mL-1。
The volumes of V1 and V2 in the above steps are both 3 μ L.
The invention has the beneficial effects that:
(1) the invention provides a self-enhanced electrochemiluminescence-electrochemistry double-method detection ZEN for the first time, so that the sensor has the advantages of low background signal of electrochemiluminescence and high speed and sensitivity of an electrochemistry method.
(2) The invention carries out ratio processing aiming at the double-signal data result obtained simultaneously, and can detect the target object more accurately.
(3) The invention forms double-stranded DNA through cDNA and an aptamer specifically recognizing ZEN, and can simultaneously obtain self-enhanced electrochemiluminescence and electrochemical signals based on NRS and MB when MB is embedded.
(4) The invention realizes the sensitivity regulation and control of ZEN detection by designing a base pair sequence of cDNA; the contrast ratio adapter sensor has no influence basically when other interferents exist, and the signal of the sensor is obviously changed only when ZEN exists, so that the selectivity is good; the signal of the ratio aptamer sensor on the seventh day is obviously reduced by less than 10 percent compared with the initial value by continuously inspecting one week, and the stability is proved to be good.
(5) According to the invention, the constructed ratio adapter sensor with the simultaneous acquisition of double signals is used for detecting the ZEN in the actual sample, so that a better recovery rate (97.3-102.7%) is obtained, and the result is basically consistent with the comparison result of a national standard method.
Drawings
FIG. 1A is a schematic diagram of the construction process of the ratiometric aptamer sensor; b is a linear relation graph for detecting different concentrations of ZEN by using different cDNA chains (cDNA-1 and cDNA-5); c is a sensitivity comparison graph when different cDNA strands (cDNA-1, cDNA-5) were used for the detection of ZEN.
In FIG. 2, A is a graph of aptamer concentration of ZEN as a function of self-enhanced electrochemiluminescence signal and electrochemical signal, respectively; and B is a graph of the hybridization time of the reaction of the cDNA and the ZEN aptamer respectively in relation to a self-enhanced electrochemiluminescence signal and an electrochemical signal.
In FIG. 3, A is the sensitivity regulation of cDNA with different sequences for detecting ZEN; b is the sensitivity comparison of different sequence cDNA.
FIG. 4A is a selectivity plot of a ratiometric aptamer sensor; b is a 7-day stability plot for the ratiometric aptamer sensor.
Detailed Description
The cDNA and ZEN aptamers of different sequences used in the present invention were purchased by bio (shanghai) corporation;
the invention is further described with reference to the following detailed description and the accompanying drawings.
Optimizing conditions:
(1) preparation of self-reinforced composite material:
firstly, adding 2g of ammonium citrate into 60mL of ultrapure water, carrying out heating reaction at 200 ℃ under normal pressure for 30min, wherein the color of the solution is changed from colorless to yellow, and then adding 1M sodium hydroxide solution to adjust the pH value to 7.0 to obtain a nitrogen-doped graphene quantum dot solution, and marking the solution as an NGQDs solution;
then, 1.77mL of triton X-100, 7.5mL of cyclohexane, 1.8mL of 1-hexanol and 340 μ L of water are mixed to obtain a mixed solution A; then adding 80 mu L of 0.1M terpyridyl ruthenium solution into the mixed solution A, and stirring for 20 min; then, 100 mu L of tetraethyl orthosilicate and 60 mu L of ammonia water are added to initiate polymerization reaction, after the reaction is finished for 24 hours, 30mL of acetone is added to precipitate nano particles, then ethanol and ultrapure water are respectively used for washing and centrifuging, the mixture is placed in an oven for drying, and finally the obtained substance is stored in a powder form and is recorded as Ru @ SiO2；
Ru @ SiO2Dissolving with ethanol, and making into solution with mass concentration of 2mg mL-1Ru @ SiO of2The solution was then pipetted 1mL Ru @ SiO2Adding 400 mu L of 3-aminopropyltriethoxysilane into the solution, stirring for reaction for 4h, washing with ethanol, centrifuging, and drying to obtain powdered amino-functionalized Ru @ SiO2Is denoted as NH2-Ru@SiO2；
Finally, NH is added2-Ru@SiO2The powder was dissolved in ultrapure water to give a concentration of 1mg mL-1NH of (2)2-Ru@SiO2Mixing the solution with 5mg mL-1NGQDs are mixed according to the volume ratio of 1:5, stirred and reacted for 12 hours, and dialyzed for 24 hours by a dialysis bag with the molecular weight cutoff of 3500Da to obtain an orange solution which is marked as NRS solution and stored at room temperature in a dark place;
(2) treating a glassy carbon electrode with the diameter of 3mm with 0.3 mu m and 0.05 mu m of aluminum oxide powder in sequence, then ultrasonically cleaning in water, ethanol, acetone and water in sequence, and airing;
(3) dripping 6 mu L of the NRS solution prepared in the step (1) on the surface of the glassy carbon electrode treated in the step (2), and airing at room temperature to obtain a sensor represented as NRS/GCE;
(4) modifying the surface of the NRS/GCE sensor prepared in the step (3) by 2 mu L of chitosan solution (pH 5.0) with the mass concentration of 0.5 wt% and airing, wherein the sensor surface forms a film structure, and the sensor is expressed as CS/NRS/GCE;
(5) modifying the sensor surface obtained in the step (4) with 3 μ L of ZEN aptamer complementary DNA (cDNA) with concentration of 1 μ M, immobilizing the cDNA on the electrode surface through electrostatic adsorption between the cDNA and chitosan, and rinsing the sensor with PBS, wherein the sensor is expressed as cDNA/CS/NRS/GCE;
(6) modifying the surface of the electrode obtained in the step (5) with 3 mu L of Bovine Serum Albumin (BSA) with the mass concentration of 1 wt% to seal the non-specific binding sites on the surface of the chitosan, and simultaneously rinsing with PBS, wherein the sensor is expressed as BSA/cDNA/CS/NRS/GCE;
(7) (a) concentration optimization: modifying the surface of the sensor obtained in the step (6) by 3 mu L of aptamer of ZEN with the concentration of 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 mu M in one-to-one correspondence, forming double-stranded DNA by hybridization reaction of the aptamer and cDNA, forming the double-stranded DNA after the hybridization reaction is carried out for 60min at 37 ℃, and leaching by PBS to remove unbound ZBA to obtain the sensor; from fig. 2(a), it can be seen that, when the aptamer concentration of ZEN in step (7) is increased from 0.6 μ M to 1.4 μ M, the self-enhanced electrochemiluminescence signal difference of NRS and the electrochemical signal of MB are gradually increased, and when the aptamer concentration is continuously increased, the self-enhanced electrochemiluminescence signal difference and the electrochemical signal are gradually flattened, which indicates that the hybridization of the aptamer and cDNA reaches saturation; therefore, the optimal ZEN aptamer concentration was chosen to be 1.6 μ M.
(b) Optimizing hybridization time: modifying the surface of the sensor obtained in the step (6) with 3 μ L of an aptamer of ZEN with the concentration of 1.6 μ M, forming double-stranded DNA by utilizing the hybridization reaction of the aptamer and cDNA, forming the double-stranded DNA after 20, 30, 40, 50 and 60min of the hybridization reaction at 37 ℃, wherein one hybridization reaction time corresponds to one sensor, the hybridization reaction time and the sensor are in one-to-one correspondence, washing is carried out by PBS (phosphate buffer solution) to remove unbound ZBA, and the sensor is represented as ZBA/BSA/cDNA/CS/NRS/GCE;
from FIG. 2(B), it can be seen that, as the hybridization time of aptamer and cDNA in step ZEN is increased, the difference of self-enhanced electrochemical luminescence signal of NRS and the electrochemical signal of MB are gradually increased, and after the hybridization time reaches 40min, the signals are gradually smoothed, which indicates that the hybridization of aptamer and cDNA reaches saturation; therefore, the optimal hybridization time was selected to be 50 min.
Example 1:
the construction method flow of the ratiometric aptamer sensor based on the simultaneous acquisition of the dual signals of the self-enhanced luminescent material (NRS) and the Methylene Blue (MB) is shown in the attached figure 1, and the specific steps are as follows:
(1) preparation of self-reinforced composite material:
firstly, adding 2g of ammonium citrate into 60mL of ultrapure water, carrying out heating reaction at 200 ℃ under normal pressure for 30min, wherein the color of the solution is changed from colorless to yellow, and then adding 1M sodium hydroxide solution to adjust the pH value to 7.0 to obtain a nitrogen-doped graphene quantum dot solution, and marking the solution as an NGQDs solution;
then, 1.77mL of triton X-100, 7.5mL of cyclohexane, 1.8mL of 1-hexanol and 340 μ L of water are mixed to obtain a mixed solution A; then adding 80 mu L of 0.1M terpyridyl ruthenium solution into the mixed solution A, and stirring for 20 min; then, 100 mu L of tetraethyl orthosilicate and 60 mu L of ammonia water are added to initiate polymerization reaction, after the reaction is finished for 24 hours, 30mL of acetone is added to precipitate nano particles, then ethanol and ultrapure water are respectively used for washing and centrifuging, the mixture is placed in an oven for drying, and finally the obtained substance is stored in a powder form and is recorded as Ru @ SiO2；
Ru @ SiO2Dissolving with ethanol, and making into solution with mass concentration of 2mg mL-1Ru @ SiO of2The solution was then pipetted 1mL Ru @ SiO2Adding 400 mu L of 3-aminopropyltriethoxysilane into the solution, stirring for reaction for 4h, washing with ethanol, centrifuging, and drying to obtain powdered amino-functionalized Ru @ SiO2Is denoted as NH2-Ru@SiO2；
Finally, NH is added2-Ru@SiO2The powder was dissolved in ultrapure water to give a concentration of 1mg mL-1NH of (2)2-Ru@SiO2Mixing the solution with 5mg mL-1NGQDs are mixed according to the volume ratio of 1:5, stirred and reacted for 12h, and dialyzed for 24h by a dialysis bag with the molecular weight cutoff of 3500Da to obtain an orange solution which is marked as NRS solutionStoring at room temperature in dark place;
(2) treating a glassy carbon electrode with the diameter of 3mm with 0.3 mu m and 0.05 mu m of aluminum oxide powder in sequence, then ultrasonically cleaning in water, ethanol, acetone and water in sequence, and airing;
(3) dripping 6 mu L of the NRS solution prepared in the step (1) on the surface of the glassy carbon electrode treated in the step (2), and airing at room temperature to obtain a sensor represented as NRS/GCE;
(4) modifying the surface of the NRS/GCE sensor prepared in the step (3) by 2 mu L of chitosan solution (pH 5.0) with the mass concentration of 0.5 wt% and airing, wherein the sensor surface forms a film structure, and the sensor is expressed as CS/NRS/GCE;
(5) modifying the sensor surface obtained in the step (4) with 3 μ L of ZEN aptamer complementary DNA (cDNA) with concentration of 1 μ M, immobilizing the cDNA on the electrode surface through electrostatic adsorption between the cDNA and chitosan, and rinsing the sensor with PBS, wherein the sensor is expressed as cDNA/CS/NRS/GCE;
(6) modifying the surface of the electrode obtained in the step (5) with 3 mu L of Bovine Serum Albumin (BSA) with the mass concentration of 1 wt% to seal the non-specific binding sites on the surface of the chitosan, and simultaneously rinsing with PBS, wherein the sensor is expressed as BSA/cDNA/CS/NRS/GCE;
(7) modifying the surface of the sensor obtained in the step (6) with 3 μ L of an aptamer of ZEN at a concentration of 1.6 μ M, forming double-stranded DNA by hybridization with cDNA, forming double-stranded DNA after hybridization for 50min at 37 ℃, rinsing with PBS to remove unbound ZBA, wherein the sensor is represented by ZBA/BSA/cDNA/CS/NRS/GCE;
(8) soaking the sensor obtained in the step (7) in a Methylene Blue (MB) solution with the concentration of 10 mu M for 1min, wherein MB is embedded into double-stranded DNA, and removing the embedded MB by rinsing with ultrapure water to obtain a ratio aptamer sensor based on the self-enhanced electrochemical luminescence-electrochemical double method of NRS and MB, which is represented by MB/ZBA/BSA/cDNA/CS/NRS/GCE;
(9) the concentration of the sensor surface modification 3 mul prepared in the step (8) is 1fg mL respectively-1,10fg mL-1,100fg mL-1,1pg mL-1,10pg mL-1The ZEN standard solution of a concentration is correspondingly modified with a sensor, and the concentration and the sensor are in one-to-one correspondence; after incubation at 37 ℃ for 60min, ZBA bound to ZEN and a portion of MB were removed by rinsing with PBS to obtain a ratiometric aptamer sensor based on simultaneous acquisition of dual signals from the self-enhancing luminescent material and methylene blue, labeled ZEN/MB/ZBA/BSA/cDNA/CS/NRS/GCE.
In addition, the sensor surface prepared above was modified with 3 μ L of ZEN solutions (1fg mL) of different concentrations-1,10fg mL-1,100fg mL-1,1pg mL-1,10pg mL-1) Correspondingly modifying a sensor by a ZEN solution with a concentration, wherein the concentration and the sensor are in one-to-one correspondence; after incubation at 37 ℃ for 60min at room temperature, the electrodes were washed with 0.1M PBS (pH 7.45); the sensor prepared above (ZEN/MB/ZBA/BSA/cDNA/CS/NRS/GCE) was used as a working electrode, an Ag/AgCl (saturated KCl) electrode was used as a reference electrode, a platinum wire electrode was used as a counter electrode, and electrochemical and electrochemiluminescence signals were detected and recorded by an electrochemical workstation model CHI832C and an electrochemiluminescence instrument model ECL-MPI EII. All tests were carried out in 0.1MPBS (pH 7.5) buffer solution with a sweep voltage range of-0.4 to 1.25V, an amplitude of 0.025V and a frequency of 15 Hz. The method comprises the steps of detecting electrochemical peak signals and electrochemical luminescence peak signals corresponding to a working electrode by using a Square Wave Voltammetry (SWV) method and an electrochemical luminescence (ECL) method, taking the ratio of the self-enhanced electrochemical luminescence peak signal of NRS to the electrochemical peak signal of MB as a vertical coordinate, taking the log value of the corresponding ZEN concentration as a horizontal coordinate, and establishing a corresponding standard curve based on cDNA of different sequences for detecting the ZEN concentration.
In the step (5), ZEN aptamer complementary DNA (cDNA) with different sequences is designed according to the known sequence and the base complementary pairing principle of the ZEN aptamer, and the base pair sequences are as follows:
cDNA-1：5′-ATA GAT GA-3′
cDNA-2：5′-TAC CAT AGA TAG ATG A-3′
cDNA-3：5′-TAG TAA TGT ACC ATA GAT AGA TGA-3′
cDNA-4：5′-ATT ACA GAT AGT AAT GTA CCA TAG ATA GAT GA-3′
cDNA-5：5′-CAT ATC ACA TTA CAG ATA GTA ATG TAC CAT AGA TAG ATG A-3′
in fig. 3, (a) cDNA with different sequences is used for detecting sensitivity control of ZEN, wherein a is cDNA-1, and the standard curve is y ═ 0.145x + 4.595; b is cDNA-2, and the standard curve is y is 0.166x + 4.444; c is cDNA-3, and the standard curve is y is 0.203x + 4.931; d is cDNA-4, and the standard curve is y ═ 0.306x + 6.047; e is cDNA-5, and the standard curve is y ═ 0.420x + 7.609. From FIG. 3(A), it can be seen that as the number of cDNA base pair sequences increases, the slope of the self-enhanced electrochemiluminescence-electrochemical ratio aptamer sensor also gradually increases, indicating that the sensitivity gradually becomes better. FIG. 3 (B) is a graph showing a comparison of sensitivity of cDNAs having different sequences; as can be seen, the sensitivity was best when cDNA-5 was used to detect ZEN.
As is clear from Table 1, the sensitivity of cDNAs having different sequences, R2And its linear regression equation. As a result of comparison, the sensitivity of the cDNA was gradually increased as the number of base pairs was increased, and in the experiment of the present invention, cDNA-5 was selected for the subsequent experiment because it was necessary to obtain the most sensitive sensor. Different cDNAs can be selected for experiments aiming at different substrates, and the adjustment is convenient.
TABLE 1 sensitivity analysis of self-enhanced electrochemiluminescence-electrochemical ratio signals based on different sequence cDNAs
The performance of the self-enhanced electrochemiluminescence-electrochemical ratio aptamer sensor constructed based on cDNA-5 was analyzed.
FIG. 4(A) is the selectivity of the ratiometric aptamer sensor, wherein blank refers to the ratiometric aptamer sensor without ZEN, i.e., the sensor of step (8) in example 1, defined as a blank; ZEN means modification of 3. mu.L to 1ng mL-1The ratio adapter sensor at ZEN, i.e., the sensor obtained by the operation of step (9) in example 1; AFB1, FB1, OTA and Mix as interferents, and are divided intoBased on the sensor obtained in step (8), the difference is that 3. mu.L of 10ng mL is modified on the surface thereof according to the operation of step (9)-1The ratio aptamer sensor obtained by the interferent, wherein AFB1 is aflatoxin B1, FB1 is fumonisin B1, OTA is ochratoxin A, and Mix is a mixed solution of AFB1, FB1 and OTA); ZEN-AFB1, ZEN-FB1, ZEN-OTA and ZEN-Mix as the mixed solution of ZEN and the corresponding interferent, respectively, denoted as solution B, refer to the operation according to step (9) on the basis of obtaining the sensor in step (8), except that 3 μ L10 ng mL of the sensor is modified on the surface-1Aptamer sensor obtained from solution B, wherein the concentration of ZEN in solution B is 1ng mL-1ZEN, concentration of interferent 10ng mL-1. From fig. 4(a), it can be seen that the ratio of self-enhanced electrochemiluminescence peak signal and electrochemical peak signal caused by interferents (AFB1, FB1, OTA and mixed solution of the three) almost agreed with those of the blank, while similar results were shown when ZEN was present, including ZEN and other interferents mixed, indicating that the sensor has good selectivity and is capable of specifically detecting ZEN.
From fig. 4(B), it can be seen that the rate adapter sensor detects ZEN for 7 consecutive days, and 92.7% of the initial value is maintained by day 7, indicating that the sensor has good stability.
Meanwhile, the self-enhanced electrochemical luminescence-electrochemical ratio aptamer sensor constructed based on cDNA-5 is used for analyzing an actual sample, and the steps are as follows:
(1) the sensor surface prepared as described in step (8) in example 1 was modified with 3 μ L of ZEN standard solutions of different concentrations (1fg mL)-1,10fg mL-1,100fg mL-1,1pg mL-1,10pg mL-1) (ii) a Correspondingly modifying a sensor by using a ZEN solution with one concentration, wherein the concentration and the sensor are in one-to-one correspondence; after the modification, the cells were incubated at 37 ℃ for 50min, and then the electrodes were washed with a 0.1MPBS (pH 7.5) solution; obtaining a sensor ZEN/MB/ZBA/BSA/cDNA/CS/NRS/GCE;
(2) the sensor prepared above (ZEN/MB/ZBA/BSA/cDNA/CS/NRS/GCE) was used as a working electrode, an Ag/AgCl (saturated KCl) electrode was used as a reference electrode, a platinum wire electrode was used as a counter electrode, and electrochemical and electrochemiluminescence signals were detected and recorded by an electrochemical workstation model CHI832C and an electrochemiluminescence instrument model ECL-MPI EII. All tests were performed in 0.1M PBS (pH 7.5) buffer, with a sweep voltage range of-0.4 to 1.25V, an amplitude of 0.025V, and a frequency of 15 Hz. An electrochemical peak signal and an electrochemical luminescence peak signal corresponding to the working electrode are detected by using a Square Wave Voltammetry (SWV) method and an electrochemical luminescence (ECL) method, a ratio of the electrochemical luminescence peak signal of NRS to the electrochemical peak signal of MB is used as a vertical coordinate, a corresponding log value of ZEN concentration is used as a horizontal coordinate, and a corresponding standard curve (cDNA-5, wherein y is 0.420x +7.609) based on cDNA-5 is established for detecting the ZEN concentration in an actual sample.
(3) Detection of ZEN in samples: firstly, obtaining a sample solution, modifying 3 mu L of the sample solution on the surface of a sensor, and obtaining corresponding electrochemiluminescence intensity and current value through electrochemical test; substituting the ratio of the electrochemical luminescence intensity to the current into the standard curve constructed in the step (2), so that the concentration of ZEN in the sample can be obtained (as shown in table 2), the ZEN is not detected in a blank non-standard sample, the recovery rate of the ratio aptamer sensor constructed after standard addition is 97.3-102.8%, and the method is basically consistent with a national standard method (HPLC-MS), and the method has reliability and accuracy; the application of ZEN detection in unknown samples is realized.
Table 2 determination of ZEN content in corn flour using the constructed ECL-SWV ratio aptamer sensing method (n-3) and the national standard method
Description of the drawings: the above embodiments are only used to illustrate the present invention and do not limit the technical solutions described in the present invention; thus, while the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted; all such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims.
Sequence listing
<110> university of Jiangsu
<120> construction method of ratio aptamer sensor based on simultaneous acquisition of double signals of self-enhanced luminescent material and methylene blue
<160> 5
<170> SIPOSequenceListing 1.0
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<212> DNA
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Claims (10)
1. A construction method of a ratiometric aptamer sensor based on simultaneous acquisition of dual signals of a self-enhanced luminescent material and methylene blue is characterized by comprising the following steps:
(1) preparation of self-reinforced composite material:
firstly, adding ammonium citrate into ultrapure water, carrying out heating reaction under normal pressure, and in the reaction process, when the color of the solution changes from colorless to yellow, adjusting the pH value to obtain a nitrogen-doped graphene quantum dot solution, which is marked as an NGQDs solution;
then, mixing triton X-100, cyclohexane, 1-hexanol and water to obtain a mixed solution A; adding a terpyridyl ruthenium solution into the mixed solution A, stirring for a period of time, adding tetraethyl orthosilicate and ammonia water to initiate polymerization reaction, adding acetone to precipitate nanoparticles after the reaction is finished, then washing and centrifuging by using ethanol and ultrapure water respectively, drying the centrifuged product to obtain a dried product, and marking the dried product as Ru @ SiO2；
Subsequently, Ru @ SiO2Dissolving the mixture by ethanol to obtain Ru @ SiO2A solution; then Ru @ SiO2Adding 3-aminopropyltriethoxysilane into the solution, stirring for reaction, washing with ethanol, centrifuging, and drying to obtain powdered amino-functionalized Ru @ SiO2Is denoted as NH2-Ru@SiO2；
Finally, NH is added2-Ru@SiO2Dissolving in ultrapure water to obtain NH2-Ru@SiO2Mixing the solution with NGQDs, stirring, reacting, dialyzing to obtain orange solution, marking as NRS solution, and storing at room temperature in dark place;
(2) sequentially treating the glassy carbon electrode with aluminum oxide powder with different particle sizes, and then sequentially ultrasonically cleaning and airing in water, ethanol, acetone and water to obtain a treated glassy carbon electrode;
(3) dripping the NRS solution prepared in the step (1) on the surface of the glassy carbon electrode treated in the step (2), and airing at room temperature to obtain a product marked as NRS/GCE;
(4) modifying the surface of the NRS/GCE sensor prepared in the step (3) with a chitosan solution, and airing; at the moment, a layer of film structure is formed on the surface of the sensor and is marked as CS/NRS/GCE;
(5) designing ZEN aptamer complementary DNA according to the known sequence and base complementary pairing principle of the ZEN aptamer, marking as cDNA, modifying the cDNA on the surface of the sensor obtained in the step (4), fixing the cDNA on the surface of an electrode under the electrostatic adsorption effect between the cDNA and chitosan, washing the sensor by PBS, and marking the washed sensor as cDNA/CS/NRS/GCE;
(6) modifying Bovine Serum Albumin (BSA) on the surface of the electrode obtained in the step (5) to seal the non-specific binding site on the surface of the chitosan, simultaneously leaching with PBS, and marking the leached sensor as BSA/cDNA/CS/NRS/GCE;
(7) modifying the sensor surface obtained in the step (6) with an aptamer (ZBA) of ZEN, and forming double-stranded DNA by using a hybridization reaction of the aptamer and cDNA; then, washing with PBS, and marking the washed sensor as ZBA/BSA/cDNA/CS/NRS/GCE;
(8) soaking the sensor obtained in the step (7) in Methylene Blue (MB) solution, and leaching the sensor with ultrapure water after soaking to obtain a ratio aptamer sensor based on the NRS and MB self-enhanced electrochemiluminescence-electrochemistry double method, wherein the ratio aptamer sensor is marked as MB/ZBA/BSA/cDNA/CS/NRS/GCE;
(9) modifying ZEN standard solutions with different concentrations on the surface of the sensor prepared in the step (8), wherein each ZEN standard solution with one concentration is correspondingly modified with one sensor, and the concentrations and the sensors are in one-to-one correspondence; after a period of incubation, the aptamer sensor, labelled ZEN/MB/ZBA/BSA/cDNA/CS/NRS/GCE, based on the simultaneous acquisition of the dual signals of the self-enhancing luminescent material and methylene blue, was rinsed in PBS.
2. The method for constructing the ratiometric aptamer sensor based on the simultaneous acquisition of the dual signals of the self-enhanced luminescent material and the methylene blue according to claim 1, wherein in the step (1), the dosage ratio of the ammonium citrate to the ultrapure water is 2 g: 60 mL; the heating reaction temperature is 200 ℃, and the reaction time is 30 min; the pH value is adjusted to 7.0 by using 1M sodium hydroxide solution;
the volume ratio of triton X-100, cyclohexane, 1-hexanol and water in the mixed solution A is 1.77: 7.5: 1.8: 0.34; the triton X-100, the terpyridyl ruthenium solution, the tetraethyl orthosilicate, the ammonia water and the acetone are 1.77: 0.08: 0.1: 0.06: 20-30; the concentration of the terpyridyl ruthenium solution is 0.1M; stirring for a period of 20-30 min; the time of the polymerization reaction is 24 hours;
the Ru @ SiO2The concentration of the solution was 2mg mL-1(ii) a The Ru @ SiO2The volume ratio of the solution to the 3-aminopropyltriethoxysilane is 1: 0.4; the stirring reaction time is 4 hours; the NH2-Ru@SiO2The concentration of the solution was 1mg mL-1(ii) a The NH2-Ru@SiO2The volume ratio of the solution to the NGQDs is 1: 5; the specific operation during dialysis is as follows: dialyzing for 24h with dialysis bag with molecular weight cutoff of 3500 Da.
3. The method for constructing the ratiometric aptamer sensor based on the simultaneous acquisition of the dual signals of the self-enhanced luminescent material and the methylene blue, according to claim 1, wherein in the step (2), the diameter of the glassy carbon electrode is d ═ 3 mm; the grain diameter of the aluminum oxide powder is 0.3 μm and 0.05 μm in sequence; in the step (3), the dosage of the NRS solution is 6 mu L when the NRS solution is added dropwise.
4. The method for constructing a ratiometric aptamer sensor based on simultaneous acquisition of dual signals of a self-enhanced luminescent material and methylene blue according to claim 1, wherein in the step (4), the concentration of the chitosan solution is 0.5 wt%, and the pH is 5.0; the amount used at the time of the dropping was 2. mu.L.
5. The method for constructing the ratiometric aptamer sensor based on the simultaneous acquisition of the dual signals of the self-enhanced luminescent material and the methylene blue as claimed in claim 1, wherein in the step (5), the base pair sequences of the complementary DNA of the ZEN aptamer are designed to comprise the following five types, namely cDNA-1, cDNA-2, cDNA-3, cDNA-4 and cDNA-5, and the specific sequences are as follows:
cDNA-1：5′-ATA GAT GA-3′
cDNA-2：5′-TAC CAT AGA TAG ATG A-3′
cDNA-3：5′-TAG TAA TGT ACC ATA GAT AGA TGA-3′
cDNA-4：5′-ATT ACA GAT AGT AAT GTA CCA TAG ATA GAT GA-3′
cDNA-5：5′-CAT ATC ACA TTA CAG ATA GTA ATG TAC CAT AGA TAG ATG A-3′；
the concentration of the cDNA is 1 μ M, and the amount of the modified sensor surface obtained in step (4) is 3 μ L.
6. The method for constructing the ratiometric aptamer sensor based on the simultaneous acquisition of dual signals of the self-enhanced luminescent material and methylene blue of claim 1, wherein in the step (6), the concentration of the bovine serum albumin is 1 wt%, and the amount of the modified electrode surface obtained in the step (5) is 3 μ L.
7. The method for constructing the ratiometric aptamer sensor based on the simultaneous acquisition of dual signals of the self-enhanced luminescent material and methylene blue, according to claim 1, wherein in the step (7), the aptamer concentration of the ZEN is 0.6-1.8 μ M, and the amount of the modified sensor surface obtained in the step (6) is 3 μ L; the temperature of the hybridization reaction is 37 ℃, and the hybridization time is 20-60 min.
8. The method for constructing the ratiometric aptamer sensor based on simultaneous acquisition of dual signals of the self-enhanced luminescent material and methylene blue according to claim 1, wherein the concentration of the methylene blue solution in the step (8) is 0.1-20 μ M, and the soaking time is 1 min.
9. The method for constructing the ratiometric aptamer sensor based on the simultaneous acquisition of dual signals of the self-enhanced luminescent material and the methylene blue, according to claim 1, wherein in the step (9), the ZEN standard solution has a concentration of 1fg mL-1～10pg mL-1(ii) a The incubation temperature is 37 ℃, and the period of time is 20-70 min; the amount of the ZEN standard solution with different concentrations on the sensor surface is 3 mu L.
10. Use of a ratiometric aptamer sensor prepared according to the method of any one of claims 1 to 9 for the detection of ZEN.
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