A method for detecting mycotoxin A based on DNA motor

Abstract

The present invention provides a method for detecting ochratoxin A based on a DNA motor. The method is based on an autonomously operating DNA motor and provides a one-step amplified method for programmed automatic detection of ochratoxin A. Ochratoxin A can bind to an aptamer and induce the opening of a molecular hairpin structure. Then, a magnesium ion-specific DNA enzyme can cyclically shear a fluorophore-labeled DNA on a gold nanoparticle, thereby causing autonomous and processive movement of the DNA motor. Finally, the cut fluorophore-labeled DNA fragment can leave the surface of the gold nanoparticle, resulting in a significant recovery of the fluorescence signal. The good linear range is from 0.01 to 5 nM, and the detection limit is 2 pM. The method is automatic, and the entire process only requires a one-step operation, showing great potential in food safety supervision or ochratoxin A bioimaging.

Description

Technical Field

[0001] The present invention relates to the field of ochratoxin A detection, in particular to the field of ochratoxin A detection methods based on DNA motors and fluorescence. Background Art

[0002] Ochratoxin A is a toxic metabolite produced primarily by several species of Aspergillus and Penicillium. Ochratoxin A may contaminate agricultural products during improper storage or processing. Due to its chemical stability and long half-life, it has been widely found in food or feed, as well as in meat products and human blood serum. Ochratoxin A is also classified as a probable human carcinogen. Several international agencies are trying to establish safe legal limits for ochratoxin A concentrations in feed, raw food, and processed foods. To further ensure food safety, it is imperative to develop a simple and accurate method to detect its presence in food and feed.

[0003] Existing technologies Many standard methods for the detection of ochratoxin A have been established in well-equipped laboratories, including HPLC, GC-MS / MS, HPLC-MS / MS and ELISA. These methods are generally regarded as accurate and sensitive methods for the detection of ochratoxin A in various authentic foods. However, these techniques require expensive instruments, time-consuming pretreatment and experienced operators.

[0004] In recent years, aptamers have attracted widespread attention as an emerging molecular recognition element. Aptamers are functional single-stranded DNAs that can bind to various targets, including small ions, small molecules, nucleic acids, proteins and even cells, with high specificity and strong binding affinity. Aptamers show advantages over antibodies due to their unique advantages, such as easy chemical synthesis, easy modification and ideal storage conditions, and have been used for target detection by combining with colorimetry, fluorescence, electrochemistry, SERS, etc.

[0005] In order to further improve the sensitivity of aptamer-based detection methods, a variety of signal amplification methods have been developed, including nanomaterials, enzyme-based or enzyme-free amplification DNA technology, etc. The amplification strategy based on DNA motors is one of the most promising tools. Synthetic DNA motors are three-dimensional DNA nanomachines for DNA walking, which can mimic natural protein motors in cells and move continuously and autonomously on various tracks. In addition, DNA motors can release multiple substrates from DNA motors by cutting DNA substrates along the track through deoxyribozymes, resulting in a strong signal amplification effect. At present, it has been used to detect DNA, miRNA, proteins, cells, etc. However, it is still challenging to detect ochratoxin A in food samples with simple operation and high sensitivity. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a method for detecting mycotoxin A based on DNA motor.

[0007] The present invention comprises the following steps:

[0008] 1) Synthesis of gold nanoparticles;

[0009] 2) Preparation of DNA motors: Incubate the aptamer hairpin structure locked by fluorophore-labeled substrate strand DNA (S-DNA) and enzyme strand DNA (E-DNA) with gold nanoparticles overnight, and quantify the surface concentration of fluorophore-labeled S-DNA according to the prior art. Preparation of DNA motors: Incubate the aptamer hairpin structure locked by fluorophore-labeled substrate strand DNA (S-DNA) and enzyme strand DNA with gold nanoparticles overnight, and quantify the surface concentration of fluorophore-labeled S-DNA according to the prior art.

[0010] 5′HS-TTTTTTTTTGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACAACTATTTTTTTCTCTTCTCCGAGCCGGTCGAAAATAGTTGTCCG-3′, fluorophore-labeled substrate strand DNA sequence: 5′HS-TTTTTTTTTTTTTTTTACTATrAGGAAGAGAT-FAM 3′;

[0011] 3) Detecting ochratoxin A signals: incubating the sample containing ochratoxin A with the DNA motor in a PBS solution, and measuring the fluorescence signal;

[0012] 4) Quantitative calculation: Ochratoxin A was quantified using the fluorescence intensity at 517 nm using the standard curve method.

[0013] Preferably, the above step 2) is specifically as follows: incubate 1 μM fluorophore-labeled S-DNA and 60 nM E-DNA-locked aptamer hairpin with 10 nM gold nanoparticles overnight at room temperature. 0.05% Tween 20 is used to prevent nonspecific adsorption. After centrifugation at 12,000 rpm for 10 minutes, the precipitate is washed and redispersed in 10 mM PBS solution (pH 7.5). Finally, the surface concentration of fluorophore-labeled S-DNA is quantified according to the prior art. .

[0014] Preferably, the above step 3) is specifically as follows: 20 μL of ochratoxin A sample is incubated with 300 μL of DNA motor in 10 mM PBS (pH 7.5) containing 10 mM MgCl2 and 0.1 M NaCl for 80 minutes.

[0015] Preferably, the above step 4) is specifically as follows: the fluorescence spectrum is scanned from 500 nm to 600 nm. Then, ochratoxin A is quantified using the fluorescence intensity at 517 nm by a standard curve method.

[0016] The present invention provides a programmable, autonomous and automatically operating DNA motor for one-step detection of ochratoxin A. Ochratoxin A can bind to apatmer, which then causes the molecular hairpin to open. The opened E-DNA binds to the substrate chain on the gold nanoparticle to form a DNA enzyme structure. The DNA motor automatically catalyzes the cutting of the substrate chain by the DNA enzyme. It can cause the continuous release of fluorescently labeled DNA chains from the gold nanoparticles. Therefore, the fluorescence intensity is significantly restored. The fluorescent signal can be used for the quantification of ochratoxin A. This method is simple and sensitive, and only one-step operation is required for ochratoxin A detection. Importantly, this DNA motor model can accurately locate and quantify ochratoxin A in tissues or living cells. In addition, this method has been successfully applied to the detection of ochratoxin A in different food matrices with good specificity and selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the detection process of the present invention.

[0018] Figure 2 Fluorescence intensity of different samples: (1) DNA motor + magnesium ions (without ochratoxin A), (2) DNA motor + ochratoxin A (without magnesium ions), (3) DNA motor + ochratoxin A + magnesium ions (fluorophore-labeled S-DNA: E-DNA locking aptamer hairpin = 1:1), (4) DNA motor + ochratoxin A + magnesium ions (incubated for 40 minutes), (5) DNA motor with E'-DNA + ochratoxin A + magnesium ions, (6) DNA motor + ochratoxin A + magnesium ions.

[0019] Figure 3 (A) is the fluorescence emission spectra of different concentrations of ochratoxin A (0.01, 0.5, 1, 2, 5, 10, 15 nM) using this method under optimized experimental conditions. Figure 3 (B) Relationship between fluorescence intensity and ochratoxin A concentration. Inset: Calibration curve of fluorescence intensity versus ochratoxin A concentration. Figure 3 (C) Selectivity and anti-interference of this method for ochratoxin A (1 nM and 5 nM). Specific embodiments

[0020] The present invention will be further described in detail below in conjunction with the embodiments.

[0021] Prepare materials and reagents

[0022] Prepare mycotoxins (including ochratoxin A, ochratoxin B (OTB), zearalenone (ZEA), deoxynephellandrene (DON), aflatoxin B1 (AFB1), aflatoxin M1 (AFM1)), phosphate buffered saline (PBS), tetrachloroauric acid, sodium citrate and 6-mercaptohexanol (MCH), ultrapure water, DNA sequences (enzyme chain DNA (E-DNA) aptamer molecule hairpin sequence 5'HS-TTTTTTTTTGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACAACTATTTTTTTCTCTTCTCCGAGCCGGTCGAAAATAGTTGTCCG-3', fluorophore-labeled substrate chain DNA sequence: 5'HS-TTTTTTTTTTTTTTTTACTATrAGGAAGAGAT-FAM 3'.

[0023] Prepare the instrument: The fluorescence signal of FAM was excited at 492 nm and the signal intensity was recalculated at the emission wavelength of 517 nm using an LS 55 fluorometer.

[0024] The detection method and steps are as follows:

[0025] Synthesis of gold nanoparticles and preparation of DNA motors: According to the prior art, gold nanoparticles were synthesized by reducing tetrachloroauric acid with sodium citrate in aqueous solution. The concentration was determined according to the Lambert-Beer law, and the extinction coefficient was 2.7×108M-1·cm-1.

[0026] At room temperature, 1 μM fluorophore-labeled S-DNA and 60 nM E-DNA-locked aptamer hairpins were incubated with 10 nM gold nanoparticles overnight. 0.05% Tween 20 was used to prevent nonspecific adsorption. After centrifugation at 12,000 rpm for 10 minutes, the precipitate was washed and redispersed in 10 mM PBS solution (pH 7.5). Finally, the surface concentration of fluorophore-labeled S-DNA was quantified according to the prior art.

[0027] One-step detection of ochratoxin A: 20 μL of ochratoxin A sample was incubated with 300 μL of DNA motor in 10 mM PBS (pH 7.5) containing 10 mM MgCl2 and 0.1 M NaCl. After incubation for 80 minutes, the solution was transferred to a quartz cuvette for fluorescence signal measurement. The fluorescence spectrum was scanned from 500 nm to 600 nm. Then, ochratoxin A was quantified by the fluorescence intensity at 517 nm.

[0028] The above method is the best embodiment of the present invention.

[0029] Figure 1The detection principle of this method is shown in . The DNA motor system mainly consists of two parts: (1) the E-DNA-locked aptamer molecule hairpin sequence and (2) the fluorophore-labeled S-DNA sequence. They are both modified on gold nanoparticles through Au-S bonds. The E-DNA-locked aptamer sequence contains the ochratoxin A aptamer sequence and the E-DNA sequence (catalytic core and two binding arms). The E-DNA function is locked by the designed hairpin structure. In addition, the fluorescence signal of the fluorophore on S-DNA is severely quenched by gold nanoparticles, and the determined quenching efficiency is about 98%. In the presence of ochratoxin A, the aptamer sequence can fold and bind to it with strong affinity, resulting in the opening of the hairpin structure and the restoration of E-DNA function. Therefore, the two arm parts of E-DNA can hybridize with S-DNA to form a magnesium ion-specific DNA enzyme structure. The DNA enzyme catalyzes the hydrolysis of S-DNA at the ribonucleotide site. Therefore, the two arms of the DNA enzyme are dehybridized from E-DNA due to the lower melting temperature, resulting in the release of the fluorophore fragments from the surface of the gold nanoparticles. The released E-DNA can then hybridize with another nearby fluorophore-labeled S-DNA, resulting in autonomous and processive movement on the gold nanoparticles. This progress is powered by the cyclic cleavage reaction of DNA enzymes, the amount of fluorophore fragments produced, and the strong fluorescence signal.

[0030] Stability of DNA motors

[0031] The stability of this DNA motor is very important for the execution of this strategy. Stability can greatly affect the fluorescence intensity. Therefore, the fluorescence signal is used to evaluate the stability of this DNA motor. The fluorescence intensity of the DNA motor was monitored for 3 hours. The fluorescence signal of FAM was weak and stable over 3 hours, indicating that the hairpin structure was still intact and the DNA motor could not start itself. Therefore, the motor was stable enough throughout the analysis process and met the requirements of this experiment.

[0032] Feasibility study of DNA motors

[0033] Several different samples were used to study the feasibility of this DNA motor. Figure 2As shown. Sample 1 without ochratoxin A exhibited a background fluorescence signal, indicating that the molecular hairpin could not be opened without ochratoxin A, and the DNA enzyme structure could not be formed to start the DNA motor. Sample 2 without Mg 2+ also showed a fluorescence intensity similar to that of sample 1, indicating that there was no cyclic shearing reaction of DNA enzyme, thus causing a low fluorescence signal. When the molar ratio of fluorophore-labeled S-DNA to the aptamer molecular hairpin locked by E-DNA was changed to 1:1, the fluorescence intensity in sample 3 was relatively strong. The results show that the cleavage of DNA enzyme cannot be cycled at a molar ratio of 1:1, resulting in a decrease in the number of fluorophore-labeled S-DNA fragments. The incubation time of sample 4 was reduced to half, resulting in a decrease in the fluorescence signal. This may be caused by the incomplete cyclic cleavage reaction of DNA enzyme. When the E'-DNA (5'HS-TTT TTT TTT-GAT CGG GTG TGG GTG GCGTAA AGG GAG CAT CGG ACA ACTA TTT TTT TCT CTT CAT AAC TTC AAT CAT GGC TAGT TGTCCG-3') in sample 5 is different from the E-DNA sequence, the fluorescence signal becomes weak again because DNA enzyme is not formed. The DNA motor cannot be started to cause the departure of the fluorophore fragment. The fluorescence signal in sample 6 is the strongest, which is attributed to the cyclic shearing reaction of the DNA motor and causes the recovery of the fluorescence signal. All results are in accordance with the principle, which indirectly proves the feasibility of the strategy.

[0034] Analytical performance of DNA motors

[0035] To investigate the analytical performance of this single-step DNA motor, different concentrations of ochratoxin A were used. The fluorescence intensity increased significantly with increasing ochratoxin A concentration. Figure 3 As shown in A and B, there is a good linear trend between ochratoxin A concentration and fluorescence intensity in the range of 0.01 to 5 nM (R2 = 0.992). The calculated limit of detection (LOD) is 2 pM. Compared with the ochratoxin A detection methods in the prior art, our method shows comparable or even better performance. This can be attributed to the efficient amplification of the DNA motor. Importantly, our DNA motor method only requires a one-step operation and does not require complicated pretreatment or detection procedures.

[0036] The selectivity of the method was evaluated using several relevant mycotoxins including OTB, ZEA, DON, AFB1, and AFM1. Figure 3As shown in Figure C, a strong fluorescence signal was only shown on the sample with ochratoxin A. The other mycotoxin samples showed weak fluorescence signals. In addition, the fluorescence intensity of the mixed sample was similar to that of the ochratoxin A sample. These results demonstrate the high specificity of this DNA motor for ochratoxin A detection.

[0037] Figure 3 (A) is the fluorescence emission spectra of different concentrations of ochratoxin A (0.01, 0.5, 1, 2, 5, 10, 15 nM) using this method under optimized experimental conditions. Figure 3 (B) Relationship between fluorescence intensity and ochratoxin A concentration. Inset: Calibration curve of fluorescence intensity and ochratoxin A concentration. Figure 3 (C) shows the selectivity and anti-interference of this method for ochratoxin A (1 nM and 5 nM).

[0038] Detection of Ochratoxin A in Authentic Foods

[0039] Practical application of the method of the present invention in food: Some food samples including peanuts, grains, soybeans, coffee beans, wine and peanut oil were randomly collected from the local market. Before use, the solid samples were collected in sterilized polyethylene bags and the liquid samples were stored in glass bottles. First, the solid samples were ground with an electric grinder, and then the liquid samples were used directly. Then, these samples were mixed with methanol deionized water (70:30 v / v) and shaken for 30 minutes for ochratoxin A extraction. After centrifugation for 10 minutes, the supernatant was collected with a pipette and filtered with a 0.45 μm membrane, and then detected using the above-mentioned best embodiment of the present invention.

[0040] Some positive samples provided by Chongqing Institute of Metrology and Quality Inspection were also used to evaluate the practical application capability of the method. The results of the positive samples were also similar to those obtained by HPLC with post-column derivatization. In addition, some spiked foods were used to evaluate the recovery of the method. Samples with different concentrations were spiked into food matrices for ochratoxin A detection. The fluorescence signals of these spiked samples were recorded, and the concentrations were calculated by the linear equation obtained above. The recoveries of this method changed from 94.8% and 109.1% to RSD, and from 4.9% and 9.5% to RSD. All the results showed that it has great potential in food safety detection and control.

[0041] In summary, the present invention provides a designable, autonomous and self-operating DNA motor for one-step detection of ochratoxin A. The method provides high specificity through aptamer recognition and high sensitivity through cyclic cleavage reaction of DNA enzyme. In addition, it shows satisfactory performance in the detection of ochratoxin A in actual food. Importantly, this DNA motor is self-operating and can detect ochratoxin A in only one step without further purification or derivatization. It shows great promise in food safety regulation and precise localization of ochratoxin A in tissues or living cells.

Claims

1. A method for detecting ochratoxin A, comprising the following steps: 1) Synthesis of gold nanoparticles; 2) Preparation of DNA motor: The fluorophore-labeled substrate chain DNA and the aptamer hairpin locked by the enzyme chain DNA were incubated with gold nanoparticles overnight, and the surface concentration of the fluorophore-labeled substrate chain DNA was quantified. The sequence of the aptamer hairpin locked by the enzyme chain DNA was: 5'HS-TTTTTTTTTGATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACAACTATTTTTTTCTCTTCTCCGAGCCGGTCGAAAATAGTTGTCCG-3', and the sequence of the fluorophore-labeled substrate chain DNA was: 5'HS-TTTTTTTTTTTTTTACTATrAGGAAGAGAT-FAM 3'; 3) Detecting ochratoxin A signal: incubating the sample containing ochratoxin A and the DNA motor prepared in step 2) in a PBS solution, and measuring the fluorescence signal; 4) Quantitative calculation: Ochratoxin A was quantified using the fluorescence intensity at 517 nm using the standard curve method.

2. The method according to claim 1, characterized in that: Step 2) Specifically, 1 μM fluorophore-labeled substrate DNA and 60 nM enzyme DNA-locked aptamer hairpins were incubated with 10 nM gold nanoparticles overnight at room temperature, 0.05% Tween 20 was added to prevent nonspecific adsorption, and after centrifugation at 12,000 rpm for 10 minutes, the precipitate was washed and redispersed in 10 mM pH 7.5 PBS solution to quantify the surface concentration of fluorophore-labeled substrate DNA.

3. The method according to claim 1 or 2, characterized in that: Step 3) is as follows: 20 μL of ochratoxin A sample is incubated with 300 μL of the DNA motor prepared in step 2) in 10 mM PBS with pH 7.5 containing 10 mM MgCl2 and 0.1 M NaCl for 80 minutes, and the fluorescence signal is measured.

4. The method according to claim 1 or 2, characterized in that: Step 4) is specifically as follows: the fluorescence spectrum is scanned from 500 nm to 600 nm, and then ochratoxin A is quantified using the fluorescence intensity at 517 nm by a standard curve method.

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