CN113466305B - Construction method of ratio aptamer sensor based on simultaneous acquisition of self-enhanced luminescent material and methylene blue dual signals - Google Patents
Construction method of ratio aptamer sensor based on simultaneous acquisition of self-enhanced luminescent material and methylene blue dual signals 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 a self-enhanced luminescent material and a methylene blue double signal, which is used for sensitively, rapidly and accurately detecting Zearalenone (ZEN); specifically, by introducing ZEN aptamer complementary DNA (cDNA) with different sequences, a ratio aptamer sensor obtained by self-enhancing electrochemiluminescence-electrochemistry double signals simultaneously is constructed, the sensitivity control on detecting ZEN is realized, different sequences can be selected according to different requirements, and the sensor has good selectivity and stability; the ratio aptamer sensor for simultaneously obtaining the self-enhanced electrochemiluminescence peak signal and the electrochemiluminescence peak signal, which is developed by the invention, can realize rapid and high-selectivity sensitive analysis on ZEN detection by adjusting the base pair sequence of cDNA, and obtain a 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 double 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 nivale and other strains, and is commonly present in crops such as wheat, corn, sorghum, barley, soybeans and the like. The contamination of ZEN severely affects the quality of agricultural products and food safety, and further poses a great economic loss and a significant threat to human health. ZEN (10-100. Mu.g kg) -1 ) Serious diseases such as teratogenesis, carcinogenesis, neurotoxicity and abortion can result. Currently, the established ZEN detection methods have advantages and disadvantages. For example, high performance liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry have better analysis results and high accuracy in practical application, but the use of instruments is expensive and the pretreatment is complex, which is unfavorable for large-scale sample screening and internal control detection. Electrochemical, electrochemiluminescence and photoelectrochemical methods are getting more and more attention in the mycotoxin detection field due to the advantages of simple and rapid operation, high sensitivity, good selectivity and the like, but the accuracy of the analysis method is still to be improved. Therefore, developing a kind of sensitive, high-efficient, accurate and easy to popularize 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 generally adopted for detecting a target object, each method has respective advantages and disadvantages, and if the two methods are combined, the cooperation among different methods can be realized, so that the accuracy of the sensor is improved. The two detection methods are adopted to analyze the target object at the same time, so that double-signal output can be obtained, accurate and sensitive detection of the target object is realized, and the method is suitable for monitoring and analysis of mycotoxin. The two methods developed at present are as follows: electrochemiluminescence-colorimetric double detection, electrochemiluminescence-photoelectrochemical double detection and the like, but the methods cannot achieve the simultaneous acquisition of signals of the two methods, and are time-consuming in detection. And the electrochemical luminescence-electrochemical method combination is not reported at present for detecting zearalenone in corns.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention aims to construct a ratio aptamer sensor capable of simultaneously acquiring double signals of self-enhanced luminescent materials and methylene blue by combining the electrochemical luminescence and electrochemical double-method sensing technology, and realizes the ZEN detection with adjustable sensitivity based on complementary DNA (cDNA) with different sequences.
A construction method of a ratio 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, and performing heating reaction under normal pressure, wherein in the reaction process, when the color of the solution is changed from colorless to yellow, the pH value is regulated to obtain a nitrogen doped graphene quantum dot solution, and the nitrogen doped graphene quantum dot solution is recorded as an NGQDs solution;
then, by mixing the tritonIntroducing X-100, cyclohexane, 1-hexanol and water to obtain a mixed solution A; then adding terpyridine 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 nano particles after the reaction is finished, washing and centrifuging with ethanol and ultrapure water respectively, drying the centrifuged product to obtain a dried product, namely Ru@SiOSiO 2 (stored in powder form);
subsequently Ru@SiO 2 Dissolving with ethanol to obtain Ru@SiO 2 A solution; then at Ru@SiO 2 Adding 3-aminopropyl triethoxy silane into the solution, stirring for reaction, washing with ethanol, centrifuging, and oven drying to obtain powdered amino-functionalized Ru@SiO 2 Is marked as NH 2 -Ru@SiO 2 ；
Finally, NH 2 -Ru@SiO 2 Dissolving in ultrapure water to obtain NH 2 -Ru@SiO 2 Mixing the solution with NGQDs, dialyzing after stirring reaction, and storing the obtained orange-yellow solution, which is designated as NRS solution, at room temperature and in a dark place;
(2) Sequentially treating a Glassy Carbon Electrode (GCE) with aluminum oxide powder with different particle sizes, and 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, wherein the obtained product is marked as NRS/GCE;
(4) Modifying the chitosan solution on the surface of the NRS/GCE sensor prepared in the step (3), and airing; at this time, a film structure is formed on the surface of the sensor, and is marked as CS/NRS/GCE;
(5) Designing a ZEN aptamer complementary DNA (cDNA) according to the known sequence and base complementary pairing principle of the ZEN aptamer, modifying the ZEN aptamer complementary DNA (cDNA) on the surface of the sensor obtained in the step (4), fixing the cDNA and chitosan on the surface of an electrode through electrostatic adsorption, leaching the sensor by using PBS, and marking the leached sensor as cDNA/CS/NRS/GCE;
(6) Modifying Bovine Serum Albumin (BSA) on the surface of the electrode obtained in the step (5) to block non-specific binding sites on the surface of chitosan, and eluting with PBS, wherein the eluted sensor is named BSA/cDNA/CS/NRS/GCE;
(7) Modifying the aptamer (ZBA) of the ZEN on the surface of the sensor obtained in the step (6), and forming double-stranded DNA by utilizing hybridization reaction of the aptamer and cDNA; elution was then performed with PBS to remove unbound ZBA, and the eluted sensor was designated ZBA/BSA/cDNA/CS/NRS/GCE;
(8) Immersing the sensor obtained in the step (7) in a Methylene Blue (MB) solution, wherein the MB is embedded into double-stranded DNA, eluting the double-stranded DNA with ultrapure water to remove the embedded MB, and obtaining the self-enhanced electrochemiluminescence-electrochemiluminescence dual-method ratio aptamer sensor based on NRS and MB, wherein the ratio aptamer sensor is marked as MB/ZBA/BSA/cDNA/CS/NRS/GCE;
(9) Modifying the surface of the sensor prepared in the step (8) with ZEN standard solutions with different concentrations, wherein one concentration of ZEN solution is correspondingly modified with one sensor, and the concentrations and the sensors are in one-to-one correspondence; after a period of incubation, the mixture was rinsed with PBS to remove ZBA and part of MB bound to ZEN, thereby obtaining a dual signal simultaneous acquisition ratio aptamer sensor based on self-enhancing luminescent material and methylene blue, labeled ZEN/MB/ZBA/BSA/cDNA/CS/NRS/GCE.
Further, in the step (1), the dosage ratio of the ammonium citrate to the ultrapure water is 2g:60mL; the temperature of the heating reaction is 200 ℃, and the reaction time is 30min; 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, terpyridyl ruthenium solution, tetraethyl orthosilicate, ammonia water and acetone 1.77:0.08:0.1:0.06: 20-30 parts; the concentration of the terpyridyl ruthenium solution is 0.1M; stirring for 20-30 min; the polymerization time was 24 hours.
Further, in the step (1), the Ru@SiO 2 The concentration of the solution was 2mg mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The Ru@SiO 2 Solution and 3-aminopropyl triethoxyThe volume ratio of the base silane is 1:0.4; the stirring reaction time is 4 hours; the NH is 2 -Ru@SiO 2 The concentration of the solution was 1mg mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The NH is 2 -Ru@SiO 2 The volume ratio of the solution to NGQDs is 1:5; the specific operation during dialysis is as follows: dialysis was performed for 24h using dialysis bags with a molecular weight cut-off of 3500 Da.
Further, in the step (2), the diameter of the glassy carbon electrode is d=3 mm; the particle size of the aluminum oxide powder used was 0.3 μm and 0.05 μm in this order.
Further, in the step (3), the amount of the NRS solution to be added is 6. Mu.L.
Further, in the step (4), the concentration of the chitosan solution is 0.5wt% and the pH is 5.0; the amount used at the time of the dropping was 2. Mu.L.
Further, in the step (5), the designed base pair sequence of the ZEN aptamer complementary DNA (cDNA) comprises the following five types of sequences respectively designated as 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 was 1. Mu.M, and the amount of the modified sensor surface obtained in the step (4) was 3. Mu.L.
Further, in the step (6), the concentration of the bovine serum albumin is 1wt%, and the electrode surface amount obtained in the step (5) is modified to be 3. Mu.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 modified to be 3 mu L; the hybridization reaction temperature is 37 ℃ and the hybridization time is 20-60 min.
Further, in step (8)The concentration of the methylene blue solution is 0.1-20 mu M, and the soaking time is 1min. Further, in the step (9), the ZEN standard solution concentration is 1fg mL -1 ～10pg mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The temperature of the incubation is 37 ℃ and the period of time is 20-70 min; the amount of the ZEN standard solution modification with different concentrations on the surface of the sensor is 3 mu L.
Both the ZEN aptamer complementary DNA (cDNA) and ZEN aptamer used in the present invention were purchased from the division of bioengineering (Shanghai) co.
The invention also relates to a use of the ratio aptamer sensor for detecting ZEN based on simultaneous acquisition of double signals of self-enhanced luminescent material (NRS) and Methylene Blue (MB), comprising the following steps:
(1) Sequentially modifying the volumes of the ZEN solutions with different concentrations of V1 on the surface of the sensor prepared in the step (9); after incubation for 20-70 min at 37 ℃, the electrode is cleaned by 0.1M PBS (pH=7.5) solution, a sensor is correspondingly modified by ZEN solution with one concentration, and the concentration and the sensor 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 scan voltage ranging from-0.4 to 1.25V, an amplitude of 0.025V, and a frequency of 15Hz. Detecting corresponding electrochemical peak signals and electrochemical luminescence peak signals of a working electrode by using a Square Wave Voltammetry (SWV) and an electrochemical luminescence method (ECL), taking the ratio of the electrochemical luminescence peak signals of NRS to the electrochemical peak signals of MB as an ordinate, taking the log value of the corresponding ZEN concentration as an abscissa, and establishing corresponding standard curves based on cDNA with different sequences for detecting the ZEN concentration in an actual sample.
(3) Detection of ZEN in sample: firstly, obtaining a sample liquid, modifying the sample liquid 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), and obtaining the concentration of ZEN in the sample; the application of ZEN detection in unknown samples is realized.
The concentration of the ZEN solution in step (1) was 1fg mL -1 ～10pg mL -1 。
The volumes of V1 and V2 in the steps are 3 mu L.
The beneficial effects of the invention are as follows:
(1) The invention provides a self-enhanced electrochemiluminescence-electrochemistry method for detecting ZEN for the first time, so that the sensor has the advantages of low background signal of electrochemiluminescence and rapidness and sensitivity of an electrochemistry method.
(2) The invention carries out ratio processing on the dual-signal data result obtained at the same time, and can detect the target object more accurately.
(3) According to the invention, cDNA and an aptamer specifically recognizing ZEN form double-stranded DNA, and when MB is embedded, self-enhanced electrochemiluminescence and electrochemical signals based on NRS and MB can be obtained simultaneously.
(4) The invention realizes the sensitivity regulation and control of detecting ZEN by designing the base pair sequence of cDNA; the contrast ratio aptamer sensor is basically not influenced when other interferents exist, and the signal of the sensor only changes obviously when ZEN exists, so that the selectivity is good; the signal of the ratio aptamer sensor on the seventh day can be obviously found to be reduced by less than 10% compared with the initial value after continuously examining for one week, and the stability is good.
(5) The ZEN in the actual sample is detected through the constructed ratio aptamer sensor with double signals simultaneously acquired, so that a good recovery rate (97.3-102.7%) is obtained, and the comparison result is basically consistent with that of the national standard method.
Drawings
FIG. 1A is a schematic diagram of the construction process of a ratio aptamer sensor; b is a linear plot of the use of different cDNA strands (cDNA-1, cDNA-5) for detection of different concentrations ZEN; c is a comparison of sensitivity using different cDNA strands (cDNA-1, cDNA-5) for detection of ZEN.
FIG. 2A is a graph of aptamer concentration of ZEN versus self-enhanced electrochemical luminescence signal and electrochemical signal, respectively; b is a graph of hybridization time of cDNA and ZEN aptamer reactions versus self-enhanced electrochemiluminescence signal, respectively.
FIG. 3A shows the sensitivity control of cDNA of different sequences for detecting ZEN; b is the sensitivity contrast of cDNA with different sequences.
FIG. 4A is a selective plot of a ratio aptamer sensor; b is a 7 day stability profile of the ratio aptamer sensor.
Detailed Description
The cDNA and ZEN aptamers of different sequences used in the invention are purchased from the company of the biological industry (Shanghai);
the invention will be further described with reference to specific examples and figures of the specification.
Condition optimization:
(1) Preparation of self-reinforced composite material:
firstly, adding 2g of ammonium citrate into 60mL of ultrapure water, heating at the normal pressure of 200 ℃ for reaction, changing the color of the solution from colorless to yellow in the process of reaction for 30min, 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 nitrogen-doped graphene quantum dot solution as an NGQDs solution;
then, 1.77mL of triton X-100, 7.5mL of cyclohexane, 1.8mL of 1-hexanol and 340. Mu.L of water were mixed to obtain a mixed solution A; then 80. Mu.L of 0.1M ruthenium terpyridyl solution was added to the mixed solution A and stirred for 20min; subsequently, 100. Mu.L of tetraethyl orthosilicate and 60. Mu.L of ammonia water were added to initiate polymerization, after the reaction was completed for 24 hours, 30mL of acetone was added to precipitate nanoparticles, which were then washed and centrifuged with ethanol and ultrapure water, respectively, and dried in an oven, and finally the obtained material was stored in the form of powder, denoted Ru@SiO 2 ；
Ru@SiO 2 Dissolving with ethanol to give a mass concentration of 2mg mL -1 Ru@SiO 2 The solution was then removed with a pipette gun to 1mL Ru@SiO 2 Adding 400 mu L of 3-aminopropyl triethoxysilane into the solution, stirring and reacting for 4 hours, washing and centrifuging with ethanol, and drying to obtain powdery amino-functionalized Ru@SiO 2 Is marked as NH 2 -Ru@SiO 2 ；
Finally, NH 2 -Ru@SiO 2 Dissolving the powder in ultrapure water to obtain a concentration of 1mg mL -1 NH of (C) 2 -Ru@SiO 2 The solution was mixed with a concentration of 5mg mL -1 Mixing NGQDs according to the volume ratio of 1:5, stirring and reacting for 12 hours, dialyzing for 24 hours by using a dialysis bag with the molecular weight cutoff of 3500Da, and obtaining an orange-yellow solution which is marked as NRS solution and storing at room temperature in a dark place;
(2) Sequentially treating a glassy carbon electrode with the diameter of 3mm with aluminum oxide powder with the diameter of 0.3 mu m and aluminum oxide powder with the diameter of 0.05 mu m, and then sequentially ultrasonically cleaning and airing in water, ethanol, acetone and water;
(3) Dropwise adding 6 mu L of the NRS solution prepared in the step (1) to the surface of the glassy carbon electrode treated in the step (2), and airing at room temperature, wherein the obtained sensor is expressed as NRS/GCE;
(4) Modifying 2 mu L of chitosan solution (pH=5.0) with mass concentration of 0.5wt% on the surface of the NRS/GCE sensor prepared in the step (3) and airing, wherein the surface of the sensor forms a film structure, and the sensor is expressed as CS/NRS/GCE;
(5) Modifying 3 mu L of ZEN aptamer complementary DNA (cDNA) with the concentration of 1 mu M on the surface of the sensor obtained in the step (4), fixing the ZEN aptamer complementary DNA (cDNA) on the surface of an electrode through electrostatic adsorption between the cDNA and chitosan, and leaching the sensor by using PBS, wherein the sensor is expressed as cDNA/CS/NRS/GCE;
(6) Modifying 3 mu L of Bovine Serum Albumin (BSA) with the mass concentration of 1wt% on the surface of the electrode obtained in the step (5) to block non-specific binding sites on the surface of chitosan, and eluting 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) with 3 mu L of ZEN aptamer with concentration of 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 mu M in a one-to-one correspondence manner, forming double-stranded DNA by utilizing hybridization reaction of the aptamer and cDNA, forming double-stranded DNA after hybridization reaction for 60min at 37 ℃, eluting by using PBS to remove unbound ZBA, and obtaining the sensor; from FIG. 2 (A), it can be seen that when the concentration of the aptamer of ZEN in step (7) increases from 0.6. Mu.M to 1.4. Mu.M, the self-enhanced electrochemical luminescence signal difference of NRS and the electrochemical signal of MB are both gradually increasing, and when the concentration of the aptamer continues to increase, the self-enhanced electrochemical luminescence signal difference and the electrochemical signal tend to be flat, indicating that hybridization of the aptamer to cDNA is saturated; thus, the optimal ZEN aptamer concentration was chosen to be 1.6 μm.
(b) Hybridization time optimization: modifying 3 mu L of the ZEN aptamer with the concentration of 1.6 mu M on the surface of the sensor obtained in the step (6), forming double-stranded DNA by utilizing hybridization reaction of the ZEN aptamer and cDNA, forming double-stranded DNA after hybridization reaction for 20, 30, 40, 50 and 60 minutes at 37 ℃, wherein one hybridization reaction time is in one-to-one correspondence with one sensor, leaching with PBS 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 the aptamer and cDNA of ZEN increases, the self-enhanced electrochemical luminescence signal difference of NRS and the electrochemical signal of MB are gradually increased, and when the hybridization time reaches 40min, the signals are gradually gentle, indicating that the hybridization of the aptamer and cDNA is saturated; therefore, the optimal hybridization time was chosen to be 50min.
Example 1:
the construction method flow of the ratio aptamer sensor based on simultaneous acquisition of double signals of self-enhanced luminescent material (NRS) and Methylene Blue (MB) is shown in the accompanying 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, heating at the normal pressure of 200 ℃ for reaction, changing the color of the solution from colorless to yellow in the process of reaction for 30min, 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 nitrogen-doped graphene quantum dot solution as an NGQDs solution;
then, 1.77mL of triton X-100, 7.5mL of cyclohexane, 1.8mL of 1-hexanol and 340. Mu.L of water were mixed to obtain a mixed solution A; then 80. Mu.L of 0.1M ruthenium terpyridyl solution was added to the mixed solution A and stirred for 20min; subsequently, 100. Mu.L of tetraethyl orthosilicate and 60. Mu.L of aqueous ammonia were added to initiate the polymerization, and after the reaction was completed for 24 hoursPrecipitating nanoparticles with 30mL of acetone, washing with ethanol and ultrapure water, centrifuging, oven drying, and storing the obtained material in powder form, denoted Ru@SiO 2 ；
Ru@SiO 2 Dissolving with ethanol to give a mass concentration of 2mg mL -1 Ru@SiO 2 The solution was then removed with a pipette gun to 1mL Ru@SiO 2 Adding 400 mu L of 3-aminopropyl triethoxysilane into the solution, stirring and reacting for 4 hours, washing and centrifuging with ethanol, and drying to obtain powdery amino-functionalized Ru@SiO 2 Is marked as NH 2 -Ru@SiO 2 ；
Finally, NH 2 -Ru@SiO 2 Dissolving the powder in ultrapure water to obtain a concentration of 1mg mL -1 NH of (C) 2 -Ru@SiO 2 The solution was mixed with a concentration of 5mg mL -1 Mixing NGQDs according to the volume ratio of 1:5, stirring and reacting for 12 hours, dialyzing for 24 hours by using a dialysis bag with the molecular weight cutoff of 3500Da, and obtaining an orange-yellow solution which is marked as NRS solution and storing at room temperature in a dark place;
(2) Sequentially treating a glassy carbon electrode with the diameter of 3mm with aluminum oxide powder with the diameter of 0.3 mu m and aluminum oxide powder with the diameter of 0.05 mu m, and then sequentially ultrasonically cleaning and airing in water, ethanol, acetone and water;
(3) Dropwise adding 6 mu L of the NRS solution prepared in the step (1) to the surface of the glassy carbon electrode treated in the step (2), and airing at room temperature, wherein the obtained sensor is expressed as NRS/GCE;
(4) Modifying 2 mu L of chitosan solution (pH=5.0) with mass concentration of 0.5wt% on the surface of the NRS/GCE sensor prepared in the step (3) and airing, wherein the surface of the sensor forms a film structure, and the sensor is expressed as CS/NRS/GCE;
(5) Modifying 3 mu L of ZEN aptamer complementary DNA (cDNA) with the concentration of 1 mu M on the surface of the sensor obtained in the step (4), fixing the ZEN aptamer complementary DNA (cDNA) on the surface of an electrode through electrostatic adsorption between the cDNA and chitosan, and leaching the sensor by using PBS, wherein the sensor is expressed as cDNA/CS/NRS/GCE;
(6) Modifying 3 mu L of Bovine Serum Albumin (BSA) with the mass concentration of 1wt% on the surface of the electrode obtained in the step (5) to block non-specific binding sites on the surface of chitosan, and eluting with PBS, wherein the sensor is expressed as BSA/cDNA/CS/NRS/GCE;
(7) Modifying 3 mu L of the ZEN aptamer with the concentration of 1.6 mu M on the surface of the sensor obtained in the step (6), forming double-stranded DNA by utilizing the hybridization reaction of the ZEN aptamer and cDNA, forming double-stranded DNA after the hybridization reaction for 50min at 37 ℃, leaching by using PBS to remove unbound ZBA, wherein the sensor is represented by ZBA/BSA/cDNA/CS/NRS/GCE;
(8) Immersing 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 in double-stranded DNA, eluting the sensor with ultrapure water to remove the embedded MB, and obtaining the self-enhanced electrochemiluminescence-electrochemiluminescence dual-method ratio aptamer sensor based on NRS and MB, wherein the ratio aptamer sensor is expressed by MB/ZBA/BSA/cDNA/CS/NRS/GCE;
(9) The concentration of 3 mu L of the surface modification of the sensor prepared in the step (8) is 1fg mL respectively -1 ,10fg mL -1 ,100fg mL -1 ,1pg mL -1 ,10pg mL -1 A sensor is correspondingly modified by the ZEN standard solution with one concentration, and the concentration and the sensor are in one-to-one correspondence; after incubation at 37 ℃ for 60min, the PBS was rinsed to remove ZBA and part of the MB bound to ZEN, thereby obtaining a dual signal simultaneous acquisition ratio aptamer sensor based on self-enhancing luminescent material and methylene blue, labeled ZEN/MB/ZBA/BSA/cDNA/CS/NRS/GCE.
In addition, 3. Mu.L of ZEN solutions (1 fg mL -1 ,10fg mL -1 ,100fg mL -1 ,1pg mL -1 ,10pg mL -1 ) A sensor is correspondingly modified by a ZEN solution with one concentration, and the concentration and the sensor are in one-to-one correspondence; after incubation for 60min at 37 ℃ at room temperature, the electrodes were washed with 0.1M PBS (ph=7.45) solution; 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 buffered at 0.1MPBS (ph=7.5)The scanning is carried out in liquid, the scanning voltage range is-0.4-1.25V, the amplitude is 0.025V, and the frequency is 15Hz. Detecting corresponding electrochemical peak signals and electrochemical luminescence peak signals of a working electrode by using a Square Wave Voltammetry (SWV) and an electrochemical luminescence method (ECL), taking the ratio of the self-enhanced electrochemical luminescence peak signals of NRS to the electrochemical peak signals of MB as an ordinate, taking the log value of the corresponding ZEN concentration as an abscissa, and establishing corresponding standard curves based on cDNA with 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 of the ZEN aptamer and the base complementary pairing principle, 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) is the sensitivity control of cDNA of different sequences for detection ZEN, wherein a is cDNA-1 and the standard curve is y=0.145 x+4.595; b is cDNA-2, standard curve y=0.166x+4.444; c is cDNA-3, standard curve y=0.203x+4.931; d is cDNA-4, and the standard curve is y=0.3068x+6.047; e is cDNA-5, standard curve 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-enhancing electrochemical luminescence-electrochemical ratio aptamer sensor also gradually increases, indicating that its sensitivity gradually becomes better. FIG. 3 (B) shows the sensitivity comparison of cDNAs of different sequences; as can be seen, the sensitivity was best when using cDNA-5 to detect ZEN.
From Table 1, it is clear that the sensitivity, R, of cDNAs of different sequences 2 And linear regression equations thereof. By comparison, it was found that as the number of base pairs increases, the corresponding sensitivity increases gradually, and in the experiment of the present invention, c was selected because the sensor with the highest sensitivity was obtainedDNA-5 was used for the later experiments. 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 a self-enhancing electrochemiluminescence-electrochemical ratio aptamer sensor constructed based on cDNA-5 was analyzed.
FIG. 4 (A) is the selectivity of a ratio aptamer sensor, wherein blank refers to the ratio aptamer sensor without ZEN, i.e., the sensor of step (8) in example 1, defined as a blank; ZEN means that the concentration of 3. Mu.L was modified to 1ng mL -1 A ratio aptamer sensor at ZEN, i.e. a sensor obtained by the operation of step (9) in example 1; AFB1, FB1, OTA, mix as interferents, refer to the operation according to step (9) on the basis of the sensor obtained in step (8), respectively, except that 3 μL 10ng mL was modified on its surface -1 A ratio aptamer sensor obtained by an interferent, wherein AFB1 is aflatoxin B1, FB1 is fumonisin B1, OTA is ochratoxin A, mix is a mixed solution of AFB1, FB1 and OTA); ZEN-AFB1, ZEN-FB1, ZEN-OTA and ZEN-Mix respectively as mixed solutions of ZEN and the corresponding interferents, denoted as solution B, respectively, refer to the operation according to step (9) on the basis of the sensor obtained in step (8), except that 3. Mu.L of 10ng mL was modified on its surface -1 Solution B resulted in a ratio aptamer sensor, wherein the concentration of ZEN in solution B was 1ng mL -1 ZEN, concentration of interferents at 10ng mL -1 . From fig. 4 (a), it can be seen that the ratio of self-enhanced electrochemiluminescence peak signal to electrochemiluminescence peak signal caused by the interferents (AFB 1, FB1, OTA and their mixed solution) is almost consistent with that of the blank, and when ZEN exists, the mixture of ZEN and other interferents is included, which shows similar results, indicating that the sensor has good selectivity and can specifically detect ZEN.
From fig. 4 (B), it can be seen that the ratio aptamer sensor was able to maintain 92.7% of its initial value by day 7 after detection of ZEN for 7 consecutive days, indicating that the sensor has good stability.
Meanwhile, the actual sample is analyzed by using a self-enhanced electrochemiluminescence-electrochemiluminescence ratio aptamer sensor constructed based on cDNA-5, and the steps are as follows:
(1) In example 1, 3. Mu.L of ZEN standard solutions (1 fg mL -1 ,10fg mL -1 ,100fg mL -1 ,1pg mL -1 ,10pg mL -1 ) The method comprises the steps of carrying out a first treatment on the surface of the A sensor is correspondingly modified by a ZEN solution with one concentration, and the concentration and the sensor are in one-to-one correspondence; after finishing the modification, incubating for 50min at 37 ℃, and then cleaning the electrode by using 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 scan voltage ranging from-0.4 to 1.25V, an amplitude of 0.025V, and a frequency of 15Hz. Detecting corresponding electrochemical peak signals and electrochemical luminescence peak signals of a working electrode by using Square Wave Voltammetry (SWV) and electrochemical luminescence method (ECL), taking the ratio of the electrochemical luminescence peak signals of NRS to the electrochemical peak signals of MB as an ordinate, taking the log value of the corresponding ZEN concentration as an abscissa, and establishing a corresponding standard curve (cDNA-5, standard curve is y=0.420x+7.609) based on cDNA-5 for detecting the ZEN concentration in an actual sample.
(3) Detection of ZEN in sample: firstly, obtaining a sample liquid, modifying 3 mu L of the sample liquid on the surface of a sensor, and obtaining corresponding electrochemiluminescence intensity and current value through electrochemical test; substituting the ratio of the electrochemiluminescence intensity and the current into the standard curve constructed in the step (2), obtaining the concentration of ZEN in the sample (shown in table 2), and detecting no ZEN in the blank non-standard sample, wherein the recovery rate of the ratio aptamer sensor constructed by the standard after the standard is 97.3-102.8%, which is basically consistent with that of a national standard method (HPLC-MS), so that the method has reliability and accuracy; the application of ZEN detection in unknown samples is realized.
Table 2 measurement of ZEN content in corn flour using the constructed ECL-SWV ratio aptamer sensing method (n=3) and national standard method
Description: the above embodiments are only for illustrating the present invention and not for limiting the technical solution described in the present invention; thus, while the invention has been described in detail with reference to the various embodiments described above, it will be understood by those skilled in the art that the invention may be modified or equivalents; all technical solutions and modifications thereof that do not depart from the spirit and scope of the present invention are intended to be included in the scope of the appended claims.
Sequence listing
<110> university of Jiangsu
<120> method for constructing a ratio aptamer sensor based on simultaneous acquisition of two signals of a self-enhanced luminescent material and methylene blue
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Claims (10)
1. The construction method of the ratio aptamer sensor based on simultaneous acquisition of double signals of the 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, and performing heating reaction under normal pressure, wherein in the reaction process, when the color of the solution is changed from colorless to yellow, the pH value is regulated to obtain a nitrogen doped graphene quantum dot solution, and the nitrogen doped graphene quantum dot solution is recorded as an NGQDs solution;
then, mixing triton X-100, cyclohexane, 1-hexanol and water to obtain a mixed solution A; then adding terpyridine 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 nano particles after the reaction is finished, washing and centrifuging with ethanol and ultrapure water respectively, drying the centrifuged product to obtain a dried product, namely Ru@SiOSiO 2 ；
Subsequently Ru@SiO 2 Dissolving the mixture by using ethanol,obtaining Ru@SiO 2 A solution; then at Ru@SiO 2 Adding 3-aminopropyl triethoxy silane into the solution, stirring for reaction, washing with ethanol, centrifuging, and oven drying to obtain powdered amino-functionalized Ru@SiO 2 Is marked as NH 2 -Ru@SiO 2 ；
Finally, NH 2 -Ru@SiO 2 Dissolving in ultrapure water to obtain NH 2 -Ru@SiO 2 Mixing the solution with NGQDs, dialyzing after stirring reaction, and storing the obtained orange-yellow solution, which is designated as NRS solution, at room temperature and in a 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, wherein the obtained product is marked as NRS/GCE;
(4) Modifying the chitosan solution on the surface of the NRS/GCE sensor prepared in the step (3), and airing; at this time, a 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 of the ZEN aptamer and the base complementary pairing principle, marking the ZEN aptamer complementary DNA as cDNA, wherein the concentration of the cDNA is 1 mu M, modifying the cDNA on the surface of the sensor obtained in the step (4), fixing the cDNA on the surface of an electrode by electrostatic adsorption between the cDNA and chitosan, leaching the sensor by using PBS, and marking the leached sensor as cDNA/CS/NRS/GCE;
the designed base pair sequences of the ZEN aptamer complementary DNA comprise the following five types of cDNA-1, cDNA-2, cDNA-3, cDNA-4 and cDNA-5, respectively, 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'; (6) Modifying Bovine Serum Albumin (BSA) on the surface of the electrode obtained in the step (5) to block non-specific binding sites on the surface of chitosan, and eluting with PBS, wherein the eluted sensor is named BSA/cDNA/CS/NRS/GCE;
(7) Modifying the aptamer (ZBA) of the ZEN on the surface of the sensor obtained in the step (6), and forming double-stranded DNA by utilizing hybridization reaction of the aptamer and cDNA; eluting with PBS, and marking the eluted sensor as ZBA/BSA/cDNA/CS/NRS/GCE;
(8) Soaking the sensor obtained in the step (7) in Methylene Blue (MB) solution, wherein the concentration of the methylene blue solution is 0.1-20 mu M; leaching with ultrapure water after soaking to obtain a self-enhanced electrochemical luminescence-electrochemical dual-method ratio aptamer sensor based on NRS and MB, wherein the ratio aptamer sensor is marked as MB/ZBA/BSA/cDNA/CS/NRS/GCE;
(9) Modifying the surface of the sensor prepared in the step (8) with ZEN standard solutions with different concentrations, wherein one concentration of ZEN solution is correspondingly modified with one sensor, and the concentrations and the sensors are in one-to-one correspondence; after a period of incubation, the ratio aptamer sensor based on simultaneous acquisition of the two signals of self-enhancing luminescent material and methylene blue, labeled ZEN/MB/ZBA/BSA/cDNA/CS/NRS/GCE, was obtained by rinsing with PBS.
2. The method for constructing a ratio-adaptor sensor based on simultaneous acquisition of two signals of self-enhanced luminescent material and methylene blue according to claim 1, wherein in the step (1), the usage ratio of ammonium citrate to ultrapure water is 2g:60mL; the temperature of the heating reaction is 200 ℃, and the reaction time is 30min; the pH value is adjusted to 7.0 by using a sodium hydroxide solution of 1M;
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, terpyridyl ruthenium solution, tetraethyl orthosilicate, ammonia water and acetone 1.77:0.08:0.1:0.06: 20-30 parts; the concentration of the terpyridyl ruthenium solution is 0.1M; stirring for 20-30 min; the polymerization reaction time is 24 hours;
the Ru@SiO 2 The concentration of the solution was 2mg mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The Ru@SiO 2 The volume ratio of the solution to the 3-aminopropyl triethoxysilane is 1:0.4; the stirring reaction time is 4 hours; the NH is 2 -Ru@SiO 2 The concentration of the solution was 1mg mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The NH is 2 -Ru@SiO 2 The volume ratio of the solution to NGQDs is 1:5; the specific operation during dialysis is as follows: the 24. 24h is dialyzed using dialysis bags having a molecular weight cut-off of 3500 Da.
3. The method for constructing a ratio-adaptor sensor based on simultaneous acquisition of dual signals of self-enhanced luminescent material and methylene blue according to claim 1, wherein in step (2), the diameter of the glassy carbon electrode is d=3 mm; the particle size of the aluminum oxide powder used is 0.3 μm and 0.05 μm in sequence; in the step (3), the amount of the NRS solution to be added is 6. Mu.L.
4. The method for constructing a ratio-adaptor sensor based on simultaneous acquisition of two 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 0.5wt%, and the pH is 5.0; the amount used at the time of the dropping was 2. Mu.L.
5. The method for constructing a ratio-adaptor sensor based on simultaneous acquisition of two signals of self-enhanced luminescent material and methylene blue according to claim 1, wherein in the step (5), the cDNA modification is performed in such a manner that the amount of the surface of the sensor obtained in the step (4) is 3. Mu.L.
6. The method for constructing a ratio-adaptive body sensor based on simultaneous acquisition of two signals of a self-enhanced luminescent material and methylene blue according to claim 1, wherein in the step (6), the concentration of bovine serum albumin is 1wt%, and the electrode surface amount obtained in the step (5) is modified to be 3 μl.
7. The method for constructing a ratio aptamer sensor based on simultaneous acquisition of two signals of a self-enhanced luminescent material and methylene blue according to claim 1, wherein in the step (7), the aptamer concentration of ZEN is 0.6-1.8 μm, and the amount of the modified sensor surface obtained in the step (6) is 3 μl; the hybridization reaction temperature is 37 ℃, and the hybridization time is 20-60 min.
8. The method for constructing a ratio-adaptive body sensor based on simultaneous acquisition of two signals of a self-enhanced luminescent material and methylene blue according to claim 1, wherein the soaking time in the step (8) is 1min.
9. The method for constructing a ratio-adaptive body sensor based on simultaneous acquisition of two signals of a self-enhanced luminescent material and methylene blue according to claim 1, wherein in the step (9), the ZEN standard solution concentration is 1fg mL -1 ~10 pg mL -1 The method comprises the steps of carrying out a first treatment on the surface of the The temperature of the incubation is 37 ℃ and the period of time is 20-70 min; the amount of the ZEN standard solution modification with different concentrations on the surface of the sensor is 3 mu L.
10. Use of a ratio aptamer sensor prepared according to the method of any one of claims 1 to 9 for detecting ZEN.
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