CN113219163B - Colorimetric sensor for detecting mycotoxin in food, detection system and application - Google Patents
Colorimetric sensor for detecting mycotoxin in food, detection system and application Download PDFInfo
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Abstract
The invention relates to the technical field of food toxin detection, in particular to a colorimetric sensor for detecting mycotoxin in food, a detection system and application. The colorimetric sensor comprises an aptamer sequence and a nano material for modifying HS-DNA, wherein the HS-DNA sequence is a sequence which is complementary with the aptamer sequence and has a mismatched base in the middle. The colorimetric sensor has the advantages of no enzyme, low cost, simple preparation, rapid field visualization and high sensitivity, and can realize rapid field visualization detection of mycotoxin in liquid food.
Description
Technical Field
The invention relates to the technical field of food toxin detection, in particular to a colorimetric sensor for detecting mycotoxin in food, a detection system and application.
Background
Ochratoxin a (ota) is a toxic secondary metabolite derived primarily from aspergillus and penicillium. In our daily lives, it is widely found in various foods and beverages, such as wheat, wine and fruit juices. It is considered to have nephrotoxicity, hepatotoxicity, neurotoxicity, teratogenicity, carcinogenicity and immunotoxicity in humans. Therefore, international agency for research on cancer (IARC) has categorized ochratoxin a as a class 2B human carcinogen in 1993. In addition, OTA has strong chemical stability, and once entering the human body and accumulating, the OTA can seriously affect the human health.
Currently, chromatographic methods, such as High Performance Liquid Chromatography (HPLC), Liquid Chromatography (LC), fluorescence detection assisted by Mass Spectrometry (MS), Gas Chromatography (GC) combined with mass spectrometry, capillary electrophoresis, Thin Layer Chromatography (TLC) and other large-scale instrumental detection technologies, are commonly used by first-line food detection institutions such as customs, because they generally have the advantages of high sensitivity, high selectivity and high reliability. However, they are time consuming for the analytical detection process of ochratoxin a in foods and require expensive research equipment and laboratories, trained instrument operators and large amounts of non-reusable toxic reagents harmful to the environment and human body. The traditional enzyme-linked immunosorbent assay is well known for its high specificity detection performance, and is widely used for detecting ochratoxin A in food at present, the method relies on an accurate antibody with high affinity and specificity recognition for ochratoxin A, the preparation cost of the antibody is high, the storage condition requirement is strict, and the wide application of the method is limited. Recently, in order to improve the sensitivity of enzyme-linked immunosorbent assay for detecting ochratoxin A, researchers have combined the method with different reading techniques such as fluorescence, colorimetry and chemiluminescence, but still have problems such as high cost, long reaction time and cumbersome operation.
FIG. 1 is a schematic diagram of an Exo III-assisted amplification fluorometric assay for OTA detection, showing a schematic diagram of OTA detection. In the absence of OTA, OTA aptamers hybridized to cDNA and Exo iii was unable to cleave FAM-labeled single-stranded SP at the 5' end. After the final addition of AuNPs, the nanoparticles can strongly adsorb FAM-labeled single-chain SPs on the surface by van der waals forces between the FAM-labeled SP chains and the bases of the AuNPs. Whereas AuNP is a fluorescence quencher, adsorption of FAM-labeled SP to the surface of AuNP results in FRET and quenching by AuNP. In the presence of OTA, the aptamer recognizes and binds to the target, resulting in the formation of double stranded DNA between the cDNA and SP. Exo III then digests the double stranded DNA from the 3' blunt end of the SP, releasing the fluorophore and releasing the cDNA. The released cDNA is then hybridized with another SP to initiate a new cleavage reaction. Through this cyclic hybridisation-hydrolysis process, OTA molecules can trigger the cleavage of large numbers of SPs. Upon addition of AuNP, the fluorophore cannot be adsorbed and quenched, greatly enhancing fluorescence. The method has complex steps in an experimental scheme, needs DNA amplification, uses ExoIII enzyme, has high requirements on amplification experimental instruments and experimental operators, and increases the detection cost; the time is consumed, the sample to be detected needs to be brought back to a laboratory, and the aim of on-site rapid detection is difficult to meet.
In recent years, the colorimetric sensor detection strategy based on the nano material is expected to correct the defects as a quick, simple, accurate, portable and cheap tool, and is applied to the field detection of ochratoxin A in food. Aptamers are nucleotide sequences obtained by screening using the exponential enrichment (SELEX) technique, which have high affinity and selectivity for a specific target, and which can be chemically synthesized and modified at relatively low cost, and more importantly, which are easy to store for a long time and are chemically stable, compared to recognition molecules currently used for recognizing targets. Since the report of ochratoxin A aptamer, researchers developed various aptamer-based methods for detecting ochratoxin A in foods, such as electrochemical detection methods, test strip detection methods, and the like. For example, the OTA Aptamer and AuNPs are simply adsorbed by the former (Yin X., Wang S., Liu X., et al., Aptamer-based colorimetric biosensing of ochromicin A in a characterized white grain with sample using unmodified gold nanoparticles, 2017,33:659-664.), and the detection of the OTA is realized as low as 49nM by the regulation and control of sodium chloride; others (Tian F, Zhou J, Jiano B, et al, A Nanozyme-based assay for amplified detection of ochratoxin A. nanoscale,2019,11(19): 9547-; joule et al (He Y., Tian F., Zhou J., et al., colorimetric aptamer for ochratoxin A detection based on enzyme-induced gold nanoparticles aggregation. journal of hazardous materials,2020,388:121758.) combine the cascade reaction of aptamer and nanoenzyme, develop a sensor for detecting OTA with higher sensitivity, with a detection limit of 0.069 nM; others (Tian F., Zhou J., Fu R., et al, Multicolor colorimetric detection of ochratoxin A vitamin structure-switching aptamer and enzyme-induced catalysis of gold nanoparticles, 2020,320:126607.) bound the aptamer to the gold nanorods, and developed a flexible method for Multicolor colorimetric detection of OTA with a detection limit of 9 nM. These colorimetric detection methods have many advantages such as visual identification and high sensitivity, but have the disadvantage of long detection reaction time, which is not favorable for rapid detection of ochratoxin A in food on site.
A colorimetric assay for OTA is detailed in figure 2. In the presence of OTA, aptamers on magnetic beads (DNA-ALP-MBs) immobilized with DNA alkaline phosphatase (ALP) were triggered, the structure was converted to G-quadruplexes, and ALP-labeled complementary DNA (cDNA-ALP) was released. After magnetic separation, ALP catalyzes the dephosphorylation reaction of ascorbic acid 2-phosphate (AAP) to Ascorbic Acid (AA), which reduces Ag+Thereby forming an Ag nano-shell on the surface of the AuNRs, and forming the Au/Ag core-shell nanorod (AuNRs @ Ag). The blue-shift of the longitudinal LSPR peak of the AuNRs produces a perceptible color change. However, in the absence of OTA, ALP-mediated AuNRs deposition was hindered because cDNA-ALP was not released from DNA-ALP-MBs. In this case, the color of the solution and the longitudinal LSPR peak change were negligible. The OTA concentration directly affects the ALP concentration, which directly depends on the silver nanostructure deposition on the AuNRs. Thus, the proposed visual analysis method can semi-quantitatively determine OTA concentration since it is based on color and longitudinal LSPR peak variation. But has the following disadvantages: 1. incubating the mixed solution of DNA-ALP-MB and OTA of various concentrations at 25 deg.C for 30 min; after magnetic separation, mixing with AAP and incubating at 37 deg.C for 30 min; then, AuNRs solution and AgNO were added3The mixture of (1) and DEA buffer, incubated at 25 ℃ for 10 min; the reaction time is long, and the aim of rapid visual detection on site is difficult to meet. 2. Various nanometer materials such as DNA alkaline phosphatase, magnetic beads and gold nanorods are used, the preparation process is complex, and the detection cost is increased.
Maeda et al (Sato K., Hosokawa K., Maeda M. Rapid aggregation of gold nanoparticles induced by non-cross-linking DNA hybridization. journal of the American Chemical Society,2003,125:8102-8103.) invented a new method for inducing the aggregation of AuNPs (FIG. 3) which modifies a DNA HS-DNA having a thiol group at the 5' end on the surface of AuNPs, and when a certain concentration of com-DNA and sodium chloride solution are present in the solution, the color change of the colloidal gold solution occurs because when the DNA on the surface of AuNPs and the com-DNA are completely complementary to each other, the repulsive electrostatic force between them is greatly reduced, and at the same time, a cavity is formed by the double-stranded end, a hydrophobic force can be generated to cause the aggregation of the AuNPs, which is expressed as a change of the color of the solution from red to purple. The aggregation form is called non-crosslinking aggregation, and has the advantages that AuNPs are easy to synthesize, the cost is low relative to other materials, the AuNPs are easy to modify different probe molecules on the surface, the prepared deoxyribonucleic acid functionalized AuNPs are stable in chemical property, the aggregation process is rapid, and color change can be seen by naked eyes. The specificity of the DNA-AuNPs to com-DNA and the rapid visual reaction process are fully utilized by scientific researchers, and the DNA-AuNPs are applied to the aspects of rapid visual detection of heavy metal ions, single nucleotide polymorphism and the like, are further expanded into a liquor system with complex components, and obtain favorable results. However, the distance of the AuNPs after agglomeration is close enough, and the ends can form a fully complementary double-stranded DNA structure to generate color change, so that the length of the surface modified DNA is required, the application of the surface modified DNA is limited, and the surface modified DNA is difficult to combine with an aptamer with a longer sequence and simultaneously exerts the capabilities of quick visualization of non-crosslinked agglomeration and high-specificity recognition of the aptamer.
Disclosure of Invention
The invention provides a colorimetric sensor for detecting mycotoxin in food, a detection system and application, has the advantages of no enzyme, low cost, simple preparation, rapid field visualization and high sensitivity, can realize rapid field visualization detection of OTA in liquid food, and solves the problems in the prior art. One of the technical schemes adopted by the invention is as follows:
a colorimetric sensor for mycotoxin detection in food, comprising an aptamer sequence and also comprising a nano material for modifying HS-DNA; the HS-DNA sequence is a sequence which is complementary with the aptamer sequence and has mismatched bases in the middle.
Furthermore, the binding force of the HS-DNA and the aptamer sequence is smaller than that of the mycotoxin to be detected and the aptamer so as to promote the non-crosslinking agglomeration of the HS-DNA modified nano material.
Further, the mycotoxin is ochratoxinA (OTA); the HS-DNA sequence is as follows: HS- (CH)2)6-TGT CCG ATG CTC TTT CCA CAC CCG ATC。
Further, the aptamer sequence is 5 '-3' -GAT CGG GTG TGG GTG GCG TAA AGG GAG CATC GGA CA.
Further, the nano material is nano gold particles (AuNPs), nano gold rods or nano gold sheets; AuNPs are preferred.
Further, the AuNPs have a diameter of 15-50 nm; preferably 20 nm.
Further, the Tm value of the HS-DNA sequence is less than 35 ℃.
Further, the colorimetric sensor for mycotoxin detection in food comprises a nano material solution for modifying HS-DNA, a nucleic acid aptamer solution, a PB solution, a magnesium chloride solution and a sodium chloride solution; the nano material solution for modifying the HS-DNA is an HS-DNA-AuNPs solution.
Further, the preparation process of the HS-DNA-AuNPs solution is as follows:
(1) preparing AuNPs with the diameter of 15-50nm into a solution for later use;
(2) preparing a reduced HS-DNA solution for later use;
(3) mixing the reduced HS-DNA solution and the AuNPs solution according to a certain mass ratio, and incubating for more than 6 hours at room temperature; gradually adding sodium chloride solution to make the final concentration of the solution be 50 mM; standing at room temperature for aging; repeating the steps, treating the mixture by using a sodium chloride solution, gradually dropwise adding, sequentially increasing the salt concentration of the reaction solution to 300mM finally, and incubating overnight; centrifuging, removing the unconnected DNA and NaCl in the solution, and re-dispersing in water to obtain the final product.
Further, the AuNPs solution in the step (1) is HAuCl with the mass concentration of 1%4And 1% sodium citrate. The preparation process comprises the following steps:
clean stirring magnetons and 95mL of ultrapure water were added to a three-neck round-bottom flask, and the apparatus was set up. Note that the lower inlet and the upper outlet are provided; the positive and negative electrodes of the electrode; note that a temperature control program is set. 1mL of 1% HAuCl was added4Simultaneously heating to slightly boil; 4mL of 1% sodium citrate are then added rapidly, stirring is continued andkeeping slight boiling, changing the solution color into blue immediately, continuing heating and stirring, changing the solution color from blue to purple, finally changing into red, continuing heating and stirring for 10min after the solution color is stable and does not change any more, continuing stirring, cooling the solution to room temperature, transferring the solution into a brown bottle (or transferring the solution into a centrifuge tube, wrapping the solution with tin foil paper for shading), and finally storing at 4 ℃; and (5) obtaining the product.
Further, the final concentration of the AuNPs solution in the step (1) is 0.2 mg/mL.
Further, the reduced HS-DNA solution prepared in step (2) is prepared as follows:
a, mixing TCEP and HS-DNA according to a certain molar ratio, and incubating at room temperature to obtain a mixed solution;
b, performing the following operations on the ice box: will CH3Adding COONa into the mixed solution containing TCEP DNA, adding glacial ethanol, incubating at-80 deg.C for a certain time, centrifuging at 10 deg.C, removing supernatant, adding glacial ethanol, vortex, centrifuging at 10 deg.C, removing most of supernatant, and standing the residual liquid at room temperature to volatilize ethanol; adding water, re-dissolving, and mixing.
Further, TCEP: HS-DNA is 100:1 molar ratio.
Further, step (3) HS-DNA solution: AuNPs solution at 1000:1 molar ratio.
Further, the molar concentration of the sodium chloride solution in the step (3) is 5M.
Further, the centrifugation treatment in the step (3) comprises a first centrifugation at 12000rpm for 30min at 25 ℃, a supernatant is discarded and 1mL of water is added for redissolving, and a second centrifugation at 12000rpm for 30min at 25 ℃, a supernatant is discarded and water is added for redissolving.
The second technical scheme adopted by the invention is as follows:
provides a detection system adopting the colorimetric sensor, and the final concentrations of the prepared HS-DNA modified nano material solution, the nucleic acid aptamer solution, the PB solution, the magnesium chloride solution and the sodium chloride solution are respectively 5nM, 0.35 MuM, 10mM, 1mM and 650 mM.
Further, the detection system is prepared according to the following operation steps:
sequentially adding 2.5 mu L of PB solution, 2.5 mu L of magnesium chloride solution and 1.75 mu L of apt solution into an EP tube, adding water to supplement the solution to a final volume of 31.175 mu L, and uniformly mixing by vortex; after reacting for 5min, adding 2.5 mu L of OTA solution with different concentrations, and mixing uniformly by vortex; continuously reacting for 5min, adding 13.3 mu L of HS-DNA-AuNPs solution and 6.5 mu L of sodium chloride solution, mixing uniformly by vortex, immediately adding a liquid transfer gun into a cuvette, reacting for 5min, measuring spectral data by using an ultraviolet spectrophotometer, and processing the spectral data by using origin; in the reaction system, the HS-DNA-AuNPs solution has the final concentration of 5nm, the PB has the final concentration of 10mM, the magnesium chloride has the final concentration of 1mM, the apt has the final concentration of 0.35 mu M, the sodium chloride has the final concentration of 650mM, and the OTA has the final concentration to be detected.
According to the detection system obtained by optimization, the binding acting force of the HS-DNA and the aptamer sequence is smaller than the binding acting force of the mycotoxin to be detected and the aptamer, so that the non-crosslinking aggregation of the HS-DNA modified nano material is promoted.
The third technical scheme adopted by the invention is as follows:
the colorimetric sensor is applied to detection of food mycotoxin, wherein the mycotoxin is ochratoxin A, aflatoxin, fumonisin or vomitoxin. The aptamer sequence of the corresponding toxin is designed as above, so that the binding action force of the aptamer and the corresponding toxin is greater than that of the aptamer and the corresponding toxin, the HS-DNA modified nano material is promoted to be better agglomerated under the condition of adding other detection reagents in the system, and quick and visible sensitive detection is obtained according to color change.
The invention has the beneficial effects that:
the invention takes the non-crosslinking agglomeration of HS-DNA-AuNPs as a detection tool, and fully exerts the advantage that the color of the kit is changed from red to purple quickly; using an aptamer of OTA as com-DNA, and designing a complementary nucleotide sequence corresponding to the com-DNA as HS-DNA; the OTA aptamer used in the invention is a known 36nt sequence with high specificity, the sequence completely complementary with the OTA aptamer is modified on the surface of AuNPs through sulfydryl, the length of double-stranded DNA formed by hybridization is too long, and the color change after agglomeration is influenced, so that the invention designs a limited number of middle mismatching bases on HS-DNA, makes the two ends of the double-stranded DNA combined with the aptamer complementary, and then optimizes the reaction conditions to make the combination acting force of the OTA to be detected and the aptamer larger than the hydrogen bond acting force of complementary pairing of DNA bases, thereby realizing the rapid colorimetric detection of the OTA within 5 min.
By designing the HS-DNA with 27nt and complementary with two ends of an aptamer sequence, wherein two ends of the HS-DNA are respectively provided with a complementary region with 12nt, and the base at the middle position is mismatched, the design ensures that the tail end of the HS-DNA can form double-stranded DNA with completely complementary base, so that hydrophobic acting force is generated, and non-crosslinking agglomeration is caused; secondly, the length of the formed double-stranded DNA is moderate, the distance of the formed double-stranded DNA is close enough after the AuNPs are agglomerated, color change is caused by surface plasma coupling, thirdly, mismatched bases are few, the acting force of hydrogen bonds is weak, and the recognition of OTA by an aptamer is facilitated. The HS-DNA avoids the influence of sequence length on aptamer combination, ensures that the distance of gold nanoparticles is close enough after agglomeration, has obvious color change, and remarkably exerts the capabilities of quick visualization of non-crosslinked agglomeration and high-specificity identification of aptamers.
The HS-DNA designed by the invention can ensure that the OTA aptamer specifically recognizes OTA, the combined acting force is greater than the pairing of the aptamer sequence and the HS-DNA, and the HS-DNA-AuNPs and the OTA aptamer have strong anti-interference capability in non-crosslinking agglomeration reaction, so that the aim of rapidly and visually detecting OTA in liquid food within 5min on site can be fulfilled, and the sensitivity of detection reaction is greatly improved. In addition, the HS-DNA-AuNPs have low preparation cost, simple process and stable chemical property, can be normally used after being stored for more than half a year at 4 ℃, and reduces the cost and the technical requirement in the practical application process.
Drawings
FIG. 1 is a schematic representation of a prior art Exo III-assisted amplification fluorometric assay for OTA detection;
FIG. 2 is a diagram of the working principle of prior art OTA multi-colorimetric detection based on aptamer structural switching and enzyme-induced AuNRs deposition;
FIG. 3 is a schematic diagram of non-crosslinked aggregates of DNA-AuNPs;
FIG. 4 is a specific sequence design schematic for detecting OTA according to the present invention;
FIG. 5a is a schematic diagram of non-crosslinked aggregates detected by the HS-DNA-AuNPs colorimetric sensor of the present invention; FIG. 5b is extinction spectrum (5min) of HS-DNA-AuNPs detection OTA, and the upper inset in FIG. 5b is the change of solution color within 5 min; the final concentration of HS-DNA-AuNPs is 5nM, the final concentration of PB is 10mM, the final concentration of sodium chloride is 250mM, the final concentration of apt is 1. mu.M, and the final concentration of magnesium chloride is 1 mM;
FIG. 6a is a transmission electron microscope image of AuNPs used in the present invention; FIG. 6b is extinction spectra of unmodified HS-DNA and modified HS-DNA of the nano-surface;
FIG. 7 shows the sensitivity of the HS-DNA-AuNPs colorimetric sensor of the present invention for detecting OTA; FIG. 7a is a color change of a solution; FIG. 7b is an extinction spectrum of HS-DNA-AuNPs; FIG. 7c is a fitted curve of maximum absorbance versus OTA concentration; the reaction time is 5 min; the final concentration of HS-DNA-AuNPs is 5nM, the final concentration of PB is 10mM, the final concentration of magnesium chloride is 1mM, the final concentration of apt is 0.35. mu.M, and the final concentration of sodium chloride is 650 mM;
FIG. 8 shows the specificity of the HS-DNA-AuNPs colorimetric sensor of the present invention for detecting OTA; FIG. 8a is an extinction spectrum of HS-DNA-AuNPs detecting different toxins; FIG. 8b is a bar graph of the maximum absorbance difference with the upper inset showing the solution color change; the reaction time is 5 min; the final concentration of HS-DNA-AuNPs is 5nM, the final concentration of PB is 10mM, the final concentration of magnesium chloride is 1mM, the final concentration of apt is 0.35. mu.M, the final concentration of sodium chloride is 650mM, and the final concentration of toxin is 620 nM.
Detailed Description
In order to clearly explain the technical features of the present invention, the following detailed description of the present invention is provided with reference to the accompanying drawings.
The OTA aptamer sequence and the HS-DNA sequence used in the experiment of the invention are synthesized by biological engineering (Shanghai) corporation, and the specific sequence information is shown in Table 1.
Ochratoxin A, ochratoxin B, aflatoxin B1, aflatoxin B2 and deoxynivalenol are purchased from Qingdao Pop bioengineering Co.
Other chemical reagents are all Chinese medicine chemical reagents and can be used without further purification.
TABLE 1 nucleotide sequence
The colorimetric sensor for detecting mycotoxin in food is a colorimetric sensor for OTA detection in food, and comprises an OTA aptamer sequence and AuNPs for modifying an HS-DNA sequence.
The diameter of AuNPs used in the invention is 20nm, and the specific synthesis method is as follows:
solution preparation: 1% HAuCl41% of sodium citrate. 0.1g was diluted to 10mL (1:100 ratio). Sodium citrate, when being prepared, is weighed to be between 0.05g and 0.07g and diluted to be between 5mL and 7mL according to the proportion of 1: 100.
The method comprises the following operation steps: clean stirring magnetons and 95mL of ultrapure water were added to a three-neck round-bottom flask, and the apparatus was set up. Note that: the lower part is arranged in and out of the upper part; the positive and negative electrodes of the electrode; note that a temperature control program is set. 1mL of 1% HAuCl was added4Simultaneously heating to slightly boil; then, 4mL of 1% sodium citrate is rapidly added, stirring is continuously carried out, slight boiling is kept, the solution is changed into blue immediately, heating and stirring are continuously carried out, the solution is changed into purple from blue, and finally changed into red, after the solution is stable and is not changed, heating and stirring are continuously carried out for 10min, stirring is continuously carried out, the solution is cooled to the room temperature, the solution is transferred into a brown bottle (or transferred into a centrifugal tube, and the solution is wrapped by tin foil paper in a dark place), and finally the solution is stored at 4 ℃.
The DNA sequence of The invention is designed by simulation with The UNAFold Web Server, HS-DNA is mainly simulated, and The HS-DNA is prevented from forming an intramolecular ring structure in a solution, so that The HS-DNA sequence with a relatively low Tm value is selected for experiments.
Thirdly, the reduction of the HS-DNA of the invention is as follows:
3.1 reduction of HS-DNA solution preparation:
0.5M TCEP: prepare 100 μ L of water, weigh 0.0125g +100 μ L of water, and store at-20 deg.C in the dark. Molecular weight: 250.19, respectively; tris (2-carboxythroughout) phosphine.
3M CH3COONa: 1mL of water (3M in this case) was weighed as 0.2461g +1mL, and 9mL of water (diluted 10 times) was added.
And (3) glacial ethanol: placing the absolute ethyl alcohol into a refrigerator at the temperature of 20 ℃ below zero. CH (CH)3COONa was previously placed in a 4 ℃ freezer.
3.2 concrete operation: 1mL system
mu.L of TCEP (0.5M) was mixed with 50. mu.L of HS-DNA (the company provides a theoretical concentration of 100. mu.M) and incubated at room temperature for 6h (overnight is preferred). Remarks TCEP: HS-DNA is 100:1 regardless of sequence length. Note that: the following steps are all performed on an ice box. The whole process was carried out on ice. Ethanol precipitation: mu.L of 3M CH3COONa was added to the above mixed solution of DNA containing TCEP, followed by addition of 200. mu.L of glacial ethanol, further incubation at-80 ℃ for 1h, centrifugation at 12000rpm at 10 ℃ for 35min, and removal of the supernatant (150. mu.L removed, 106. mu.L remaining). Then, 500. mu.L of glacial ethanol was added, vortexed, and centrifuged at 12000rpm at 10 ℃ for 35min to remove most of the supernatant, and the remaining liquid was left at room temperature to evaporate off the ethanol (over 5 h). Add 50. mu.L of water (water can be added depending on the initial DNA volume) to redissolve and mix. The DNA concentration diluted with 1. mu.L of the sample + 9. mu.L of water was taken out, the concentration after reduction was measured and the recovery rate was calculated. The remaining sample was used for modification.
Fourthly, the HS-DNA-AuNPs of the invention are prepared as follows
The specific operation is as follows:
to 1mL of AuNPs (15nM,2.5nM) was added 10. mu. LBSPP (20mg/mL) (final concentration 0.2 mg/mL). The mixture was incubated overnight at room temperature in the dark or at 50 ℃ for 1 h. Centrifugation (12000rpm, 25 ℃, 12-15min) removes supernatant and removes excess BSPP and sodium citrate. 1mL of water (deionized water can be added according to the initial volume of the nano gold) is added to re-dissolve the AuNPs of the lower layer material.
HS-DNA and AuNPs are introduced according to the ratio of 1000:1 (the amount ratio of substances, according to the situation), and incubated for more than 6h at room temperature. 5M NaCl was added stepwise to the mixture to give a final solution concentration of 50 mM. Aging the mixture by standing the mixture at room temperature for more than 1 h. The above step 5 was repeated, and the mixture was continuously treated with 5M NaCl, and gradually added dropwise to increase the salt concentration of the reaction solution to 100mM, 150mM, 200mM, 250mM, 300mM in this order at intervals of 1 hour or more per step, and finally to 300 mM. (Note: high concentration of NaCl minimizes the repulsive force of the high density immobilized DNA). The 300mM mixture was incubated overnight. The mixture was then centrifuged 2 times to remove unligated DNA and NaCl from the solution and redispersed in water. The method comprises the following specific steps: centrifuging for the first time (12000rpm, 25 ℃, 30min), discarding the supernatant, and adding 1mL of water for redissolving; centrifuging for the second time (12000rpm, 25 deg.C, 30min), discarding supernatant, and adding water for redissolving (the amount of water can be determined according to the required concentration).
Fifth, the OTA detection system of the invention is as follows
Adding 2.5 muL PB solution, 2.5 muL magnesium chloride solution and 1.75 muL apt solution into a 200 muL EP tube in sequence, adding water to supplement to a final volume of 31.175 muL, uniformly mixing by vortex, adding 2.5 muL OTA solutions with different concentrations after reacting for 5min, uniformly mixing by vortex, adding 13.3 muL HS-DNA-AuNPs solution and 6.5 muL sodium chloride solution after reacting for 5min, uniformly mixing by vortex, immediately adding a liquid transfer gun into a cuvette, measuring spectral data by an ultraviolet spectrophotometer after reacting for 5min, and processing the spectral data by origin. The final concentration of HS-AuNPs in the reaction system is 5nM, the final concentration of PB is 10mM, the final concentration of magnesium chloride is 1mM, the final concentration of apt is 0.35. mu.M, the final concentration of sodium chloride is 650mM, and the final concentration of OTA is to be detected.
The OTA detection system of the invention combines the aptamer with the OTA or the complementary sequence thereof, modifies the HS-DNA with a specific sequence and short enough length on the AuNPs surface, optimizes the reaction condition, ensures that the combination acting force of the aptamer and the OTA is larger than the hydrogen bond acting force of the aptamer and the complementary chain, ensures that the distance between the agglomerated AuNPs is close enough, generates corresponding color change through surface plasma resonance, and realizes the purpose of detecting the OTA.
As shown in FIG. 5, FIG. 5a shows the non-crosslinked aggregates detected by the HS-DNA-AuNPs colorimetric sensor. Firstly, components except HS-DNA-AuNPs and sodium chloride are added into a detection system, then apt to be combined with OTA first without external interference is obtained, and then HS-DNA-AuNPs and sodium chloride are added, and apt not to be combined with OTA is combined with HS-DNA in a complementary pairing mode. The scheme is verified, as shown in figure 5b, because OTA does not exist, apt and HS-DNA are in a large number of complementary pairing, the non-crosslinking agglomeration degree of the HS-DNA-AuNPs is obvious, the absorption peak of the spectrum is red-shifted to 550nm, the absorption value is reduced to 0.6, and the color of the colloidal solution is changed into purple within 5min, as shown in the color of the tube 1 at the left side of the upper inset of figure 5 b. When detecting OTA with the concentration of 1.2 mu M, 12.4 mu M and 49.6 mu M, the experimental result of the scheme shows that when the concentration of the OTA is 1.2 mu M, the absorption peak of the corresponding ultraviolet spectrum only red shifts to about 530nm, the absorption value is far higher than that of a blank group, and the color of the solution is red close to that of nanospheres. At 12.4 μ M, 49.6 μ M OTA concentration, the corresponding absorption peak in the UV spectrum is also at 530nm, and the absorption value shows regular gradual increase relative to the blank group, and the solution color is red, as shown in the 2 nd, 3 rd and 4 th tubes on the left side of the upper inset of FIG. 5 b. This shows that as the concentration of OTA increases, more apt is combined in the solution, and the apt which can be freely and complementarily paired with HS-DNA-AuNPs in the solution decreases with the increase of the concentration of OTA, which means that the quantity of double-stranded DNA complementary to the surface of HS-DNA-AuNPs decreases and the hydrophobic force of terminal base pairs decreases, and finally the degree of non-crosslinking aggregation of HS-DNA-AuNPs is reduced.
Sixthly, the colorimetric sensor of the invention detects the sensitivity of OTA
As shown in FIG. 7, FIG. 7a shows that at an OTA concentration of 12.4nM, the solution color is pale red, visually distinguishable from the purple of the blank, and as the concentration of OTA increases, the solution color gradually shifts from the purple at an OTA concentration of 0nM to red, thus allowing semi-quantitative detection of OTA without the need for equipment. Meanwhile, as the concentration of OTA increases, the ultraviolet absorption peak gradually blueshifts from 550nM to 525nM (FIG. 7b), and the OTA spectrum change of 6.2nM can be detected at the lowest. And the maximum absorption value is gradually increased overall, a fitted curve between the maximum absorption value and the OTA concentration is shown in FIG. 7c, and in the OTA concentration range of 6.2-620nM, the fitted linear equation is that y is 6.735 multiplied by 10 < -4 > x +0.765, R2 is 0.9914, x is the OTA concentration, and y is the maximum absorption value, and the two show good correlation. Although the colorimetric detection capability of the sensor for OTA is equivalent to that of other colorimetric methods, the HS-DNA-AuNPs colorimetric sensor has the greatest advantage of fast reaction time of 5min, and the sensor provides a feasible method for fast detection of OTA on site.
Seventhly, the colorimetric sensor of the invention detects the specificity of OTA
The selected toxins are AFB1, AFB2, OTB, DON, and the results are shown in FIG. 8. Different toxins were added to the assay system instead of OTA and the final concentration was determined to be 620 nM. From FIG. 8a, it can be seen that the extinction spectrum of HS-DNA-AuNPs is 525nm only when OTA is added, and the extinction spectrum is blue-shifted to 550nm and the maximum absorption value is reduced to 0.8 when other toxins are added and the HS-DNA-AuNPs are not cross-linked and aggregated. FIG. 8b is a histogram showing the difference between the maximum absorbance of the HS-DNA-AuNPs UV spectrum and the maximum absorbance of the blank control in the presence of different toxins, and it can be seen that the OTA value is significantly different from other toxins. The color change picture can also see that only the solution containing OTA is red, other solutions are purple, and the color difference is obvious. The reason is that the apt has high specificity to OTA, and the addition of other toxins does not influence the non-crosslinking aggregation of the apt and HS-DNA complementary pairing HS-DNA-AuNPs. Therefore, the HS-DNA-AuNPs colorimetric sensor has good detection specificity for OTA.
The HS-DNA sequence design and condition optimization method is also suitable for detecting other toxins in corresponding food, and the aim of detecting corresponding harmful substances can be fulfilled by the non-crosslinking agglomeration reaction tool of the HS-DNA-AuNPs and the aptamer.
The above-described embodiments should not be construed as limiting the scope of the invention, and any alternative modifications or alterations to the embodiments of the present invention will be apparent to those skilled in the art.
The present invention is not described in detail, but is known to those skilled in the art.
Sequence listing
<110> China oceanic university
<120> colorimetric sensor for mycotoxin detection in food, detection system and application
<141> 2021-04-30
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 1
tgtccgatgc tctttccaca cccgatc 27
Claims (9)
1. A colorimetric sensor for mycotoxin detection in food comprises an aptamer sequence and is characterized by also comprising a nano material for modifying HS-DNA; the HS-DNA sequence is a sequence which is complementary with the aptamer sequence and has mismatched bases in the middle;
the mycotoxin is ochratoxin A; the HS-DNA sequence is as follows: HS- (CH)2)6-TGT CCG ATG CTC TTT CCA CAC CCG ATC；
The aptamer sequence is as follows; GAT CGG GTG TGG GTG GCG TAA AGG GAG CATC GGA CA are provided.
2. The colorimetric sensor for mycotoxin detection in food according to claim 1, wherein the nano-material is a nano-gold particle, a nano-gold rod, or a nano-gold sheet.
3. The colorimetric sensor for mycotoxin detection in food according to claim 2, wherein the nano-material is gold nanoparticles, and the gold nanoparticles have a diameter of 15-50 nm.
4. The colorimetric sensor for mycotoxin detection in food products according to claim 3, wherein the gold nanoparticles are 20nm in diameter.
5. The colorimetric sensor for mycotoxin detection in food products according to claim 1, wherein the HS-DNA sequence Tm value is less than 35 ℃.
6. The colorimetric sensor for mycotoxin detection in food products according to any one of claims 1 to 5, comprising a nano-material solution for modifying HS-DNA, a nucleic acid aptamer solution, a PB solution, a magnesium chloride solution, and a sodium chloride solution; the nano material solution for modifying the HS-DNA is an HS-DNA-AuNPs solution.
7. The colorimetric sensor for mycotoxin detection in food products according to claim 6, wherein the HS-DNA-AuNPs solution is prepared by the following process:
(1) preparing AuNPs with the diameter of 15-50nm into a solution for later use;
(2) preparing a reduced HS-DNA solution for later use;
(3) mixing the reduced HS-DNA solution and the AuNPs solution according to a certain mass ratio, and incubating for more than 6 hours at room temperature; gradually adding sodium chloride solution to make the final concentration of the solution be 50 mM; standing at room temperature for aging; repeatedly treating the mixture with a sodium chloride solution, gradually dropwise adding, sequentially increasing the salt concentration of the reaction solution to 300mM finally, and incubating overnight; centrifuging, removing the unconnected DNA and NaCl in the solution, and re-dispersing in water to obtain the final product.
8. The colorimetric sensor for mycotoxin detection in food products according to claim 7, wherein the reduced HS-DNA solution prepared in step (2) is prepared as follows:
a, mixing TCEP and HS-DNA according to a certain molar ratio, and incubating at room temperature to obtain a mixed solution;
b, performing the following operations on the ice box: will CH3Adding COONa into the mixed solution of the DNA containing TCEP, adding glacial ethanol, incubating for a certain time, centrifuging at 10 deg.C, removing supernatant, adding glacial ethanol, vortexing, continuing centrifuging, removing most of supernatant, and standing the residual liquid at room temperature to volatilize ethanol; adding water, re-dissolving, and mixing.
9. The detection system using the colorimetric sensor according to any one of claims 6 to 8, wherein the modified HS-DNA nanomaterial solution, the aptamer solution, the PB solution, the magnesium chloride solution, and the sodium chloride solution are prepared to have final concentrations of 5nM, 0.35. mu.M, 10mM, 1mM, and 650mM, respectively.
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