CN113899724B - Method for high-sensitivity detection of ofloxacin based on nucleic acid aptamer sensor - Google Patents
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Abstract
The invention discloses a method for detecting ofloxacin with high sensitivity based on a nucleic acid aptamer sensor, and relates to the field of detection of fluoroquinolone antibiotics by a biosensor. The method comprises the following steps: 1) cy 3-aptamer binds to BHQ2-cDNA, BHQ2 quenches the fluorescence of Cy 3; 2) adding a sample to be detected, standing for reaction, and when a target object OFL exists, performing affinity competition to form an aptamer/OFL mixture, recovering fluorescence through the release of a Cy3 fluorescent group, and meanwhile, the OFL also enhances the fluorescence of Cy 3; 3) and determining the concentration of the OFL in the sample to be detected according to the recovery and enhancement degree of the fluorescence. The OFL has an enhancement effect on the fluorescence intensity of a fluorescent dye Cy3, the sensitivity of the method can be obviously enhanced, and the detection limit is as low as 0.2 nM; and the detection is quick and convenient, does not need to depend on large-scale equipment and instruments, and has no strict requirements on detection environment and time.
Description
Technical Field
The invention belongs to the technical field of biochemistry, molecular biology and analytical chemistry, relates to a biosensor for detecting fluoroquinolone antibiotics, and particularly relates to a method for detecting ofloxacin at high sensitivity based on a nucleic acid aptamer sensor.
Background
Quinolone drugs constitute a large class of synthetic antibiotic drugs with significant effects in the treatment of a variety of diseases, particularly their fluorinated derivatives, known as fluoroquinolones (fluoroquinolones, FQs). FQs is a potentially highly polluting substance which, at low concentrations, not only pollutes the aqueous environment but also has a toxic effect on the surface water. Ofloxacin (OFL), a typical, widely used fluoroquinolone antibiotic, has a wide range of pharmaceutical activities against most gram-negative bacteria and many gram-positive bacteria. The extensive use of ofloxacin in the body leads to incomplete metabolism in the body, has degradation resistance and high absorption properties, and accumulates in the environment, particularly in the soil environment. More seriously, the emergence of microbial resistance (AMR) is being accelerated by the abuse of antibiotics, which makes microbial-induced infections difficult to treat, and sometimes even incurable.
Currently, the detection methods of fluoroquinolone antibiotics mainly include high performance liquid chromatography, electrochemical methods, fluorescence detection, extraction spectrophotometry, liquid chromatography mass spectrometry, and the like. Despite their high sensitivity and good selectivity, expensive instrumentation, professional operators, complex pre-treatments and time-consuming procedures limit their application in the field of wastewater or detection of ofloxacin in food products. Standards for the concentration of ofloxacin in aquatic and animal products have also been defined in many countries, with the highest residual amount of ofloxacin (MRL) being determined in japan to be 10ppb (about 30nM), whereas european standards only specify a total limit of FQs between 300 and 800 ppb. Therefore, it is necessary and important to establish an effective and rapid method for detecting the OFL in water environment and food.
Although the inversion of a Truncated Aptamer for Ofloxacin Detection use a Rapid FRET-Based Apta-Assay [ J ]. Antibiotics, 2020, 9 (12): 860. discloses a method for detecting ofloxacin, but the 100 mu M OFL of the method can only recover the fluorescence of a modified fluorescent group FAM in a nucleic acid aptamer by about 40 percent, and the sensitivity is not high enough; furthermore, in the prior art, the fluorescence of FAM labeled on the aptamer cannot be enhanced by the OFL, and the detection of trace amounts of OFL in the sample is limited.
Disclosure of Invention
In view of the above problems in the prior art, the technical problem to be solved by the present invention is to provide a method for detecting ofloxacin with high sensitivity based on an aptamer sensor; detecting ofloxacin OFL based on the combination and separation of the fluorescent dye Cy3 and the fluorescence quencher BHQ2 resulting in Cy3 fluorescence quenching-recovery (turn-off/on) and an enhanced aptamer sensor; another technical problem to be solved by the present invention is to provide an aptamer sensor for detecting ofloxacin by the above method.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a nucleic acid aptamer sensor for detecting ofloxacin comprises a Cy3 fluorescence labeled OFL nucleic acid aptamer and BHQ2 modified complementary strand cDNA, wherein the Cy3 fluorescence labeled OFL nucleic acid aptamer is modified with a fluorescent group Cy3 at the 3' end of a sequence shown in SEQ ID NO. 1; the complementary strand cDNA modified by BHQ2 is modified with a quenching group BHQ2 at the 5' end of the sequence shown in SEQ ID NO. 2.
Cy3 fluorescently labeled OFL aptamers, herein designated Cy 3-aptamers;
BHQ 2-modified complementary strand cDNA, herein designated BHQ 2-cDNA;
the nucleotide sequence of the Cy 3-aptamer is:
5′-AAGTGAGGTTCGTCCCTTTAATAAACTCGATTAGGATCTCGTGAGGT GTGCTCTACAATCGTAATCAGTTAG-Cy3-3′；
the nucleotide sequence of the BHQ2-cDNA is: 5 '-BHQ 2-CTAACTGATTAC-3'.
The method for detecting ofloxacin at high sensitivity based on the aptamer sensor comprises the following steps:
1) cy 3-aptamer was bound to BHQ2-cDNA to form a mixture, BHQ2 quenched the fluorescence of Cy 3;
2) adding a sample to be detected into the mixture obtained in the step 1), standing for reaction, when a target object OFL exists in the sample to be detected, performing affinity competition, combining a Cy 3-aptamer and the OFL to form a Cy 3-aptamer/OFL mixture, unwinding the base pair of the aptamer/cDNA, recovering fluorescence through Cy3 fluorophore release, and enhancing the fluorescence of Cy3 by the OFL;
3) and establishing a linear regression equation between the OFL concentration and the fluorescence recovery and enhancement degree, and determining the concentration of the OFL in the sample to be detected according to the fluorescence recovery degree.
Further, in the step 1), the Cy 3-aptamer is a fluorescent group Cy3 modified at the 3' end of the sequence shown in SEQ ID NO. 1; the BHQ2-cDNA is a quenching group BHQ2 modified at the 5' end of the sequence shown in SEQ ID NO. 2.
Further, in step 1), Cy 3-aptamer and BHQ2-cDNA were combined in Tris-HCl buffer solution.
Further, the Tris-HCl buffer solution contains 1mM Mg2+And 2mMK+(ii) a The concentration of Tris-HCl buffer solution was 10mM, pH 7.4.
Further, in the step 1), the molar ratio of the Cy 3-aptamer to the BHQ2-cDNA is 1: 1-10; the concentration of Cy 3-aptamer was 50-600 nM.
Further, in the step 1), the molar ratio of the Cy 3-aptamer to the BHQ2-cDNA is 1: 4; the concentration of Cy 3-aptamer was 200 nM.
Further, in the step 2), the reaction temperature is 37 ℃ and the reaction time is 1 h.
Furthermore, the detection range of ofloxacin is 0-100 μ M, the linear detection range is 0-500nM, the linear regression equation is 0.0526x +5.8391, and the detection limit is 0.2 nM.
The aptamer sensor is used for detecting ofloxacin in milk and water.
Compared with the prior art, the invention has the beneficial effects that:
1) the fluorescent dye Cy 3-aptamer and quencher BHQ2-cDNA selected by the invention are combined, so that the target object OFL can be effectively detected;
2) the target OFL of the present invention has an enhancing effect on the fluorescence intensity of the fluorescent dye Cy 3: the combination of OFL and FAM-aptamer at the concentration of 100 mu M can recover the fluorescence of FAM in FAM-aptamer/TAMRA-cDNA by 40%, compared with OFL which can enhance the fluorescence recovery rate of Cy3 in Cy 3-aptamer/BHQ 2-cDNA to more than 70%; the sensitivity of the method can be obviously enhanced, and the detection limit is as low as 0.2 nM;
3) the method is simple to operate, rapid and convenient to detect, does not need to depend on large-scale equipment and instruments, and has no strict requirements on detection environment and time;
4) the method can be used for detecting trace OFL in milk, meat products and water samples with high sensitivity.
Drawings
FIG. 1 is a schematic diagram of the operation of an aptamer sensor for detecting OFL;
FIG. 2 is a graph of the effect of OFL binding to Cy 3-aptamer on Cy3 fluorescence intensity;
FIG. 3 is a graph showing the effect of molar concentration ratio of Cy 3-aptamer to BHQ2-cDNA on Cy3 fluorescence intensity;
FIG. 4 is a graph of the effect of Cy 3-aptamer concentration on the recovery of Cy3 fluorescence intensity by OFL;
FIG. 5 is a graph of the effect of OFL on Cy3 fluorescence intensity by specific binding to aptamers over a series of concentration gradients;
FIG. 6 is a standard graph showing the difference between the fluorescence intensity of Cy3 added to the OFL system solution and the fluorescence intensity of OFL in the blank control system solution as a function of the OFL concentration;
FIG. 7 is a specific detection chart of this method.
Detailed Description
The invention is further described with reference to specific examples.
The aptamers used in the following examples were Cy3 fluorescently labeled OFL nucleic acid aptamers, denoted Cy 3-aptamer;
the nucleotide sequence of Cy 3-aptamer (Cy3-Apt) is as follows:
5′-AAGTGAGGTTCGTCCCTTTAATAAACTCGATTAGGATCTCGTGAGGTGTGCTCTACAATCGTAATCAGTTAG-Cy3-3′；
the complementary cDNA is BHQ2 modified complementary strand cDNA, and is named as BHQ 2-cDNA;
the nucleotide sequence of the BHQ2-cDNA is: 5 '-BHQ 2-CTAACTGATTAC-3'.
Example 1
First, feasibility analysis
Cy 3-aptamer was taken at a molar concentration in Tris-HCl (10mM, pH 7.4, 2mM K+And 1mM Mg2+) In the buffer solution, OFL, BHQ2-cDNA and a mixture of BHQ2-cDNA and OFL with certain concentration are respectively added in sequence. Standing and reacting for a period of time under a certain temperature condition, and measuring fluorescence at the excitation wavelength of 535nm by using a Perkinelmer fluorescence spectrophotometer, wherein the width of a slit is 10nm x 10 nm. As can be seen from FIG. 2, the fluorescence intensity at 565nm was taken to plot a histogram, and when OFL was added to Cy 3-aptamer solution, OFL enhanced the fluorescence of Cy 3; BHQ2-cDNA efficiently quenched fluorescence of Cy3 in the absence of OFL and in the presence of BHQ 2-cDNA; when OFL exists, the OFL is combined with Cy 3-aptamer through competition reaction, so that BHQ2-cDNA is shed, and the fluorescence of Cy3 is recovered and enhanced.
Secondly, determination of the molar concentration ratio of aptamer to cDNA
Cy 3-aptamer and BHQ2-cDNA were added at different molar ratios to Tris-HCl (10mM, pH 7.4, 2mM K)+And 1mM Mg2+) Adding target object OFL with a certain concentration into the buffer solution, standing at a certain reaction temperature for reaction for a period of time, and measuring fluorescence at 535nm of excitation wavelength by using a PerkinElmer fluorescence spectrophotometer, wherein the width of a slit is 10nm x 10 nm. As can be seen in FIG. 3, with Captamer：CcDNAThe ratio decreased, the recovery degree of the fluorescence intensity of Cy3 increased first and then decreased, when C wasaptamer：CcDNAThe recovery of the fluorescence intensity of Cy3 was the greatest at a ratio of 1: 4. Thus, the optimal molar concentration ratio of aptamer to complementary DNA is 1: 4.
Determination of aptamer concentration
Varying concentrations of Cy 3-aptamer and BHQ2-cDNA (C)aptamer：CcDNAThe ratio is 1: 4) in Tris-HCl (10mM, pH 7.4, 2mM K)+And 1mM Mg2+) Adding target object OFL with a certain concentration into the buffer solution, standing at a certain reaction temperature for reaction for a period of time, and measuring fluorescence at 535nm of excitation wavelength by using a PerkinElmer fluorescence spectrophotometer, wherein the width of a slit is 10nm x 10 nm. As can be seen from FIG. 4, when the adapter is usedWhen OFL was added at a concentration of 200nM, the degree of recovery of the fluorescence intensity of Cy3 was good. Thus, 200nM and 800nM were chosen as the optimal Cy 3-aptamer and BHQ2-cDNA concentrations, respectively.
Fourth, establishment of standard curve
Ofloxacin solutions were prepared at concentrations of 0, 10, 50, 100, 200, 500, 1000, 5000, 10000, 20000, 50000 and 100000nM, respectively, and Tris-HCl (10mM, pH 7.4, 2mM K2-cDNA) containing 200nM Cy 3-aptamer and BHQ2-cDNA was added+And 1mM Mg2+) In a buffer solution, wherein the molar concentration ratio of Cy 3-aptamer to BHQ2-cDNA is 1: 4. standing at 37 ℃ for 1h, placing the solution in a fluorescence spectrophotometer to obtain fluorescence spectrograms under different concentrations, designing three groups of parallel tests for each concentration, and setting the width of a slit to be 10nm x 10 nm. And finally, selecting a fluorescence spectrogram with the excitation wavelength of 535nm, drawing a standard curve by taking the fluorescence intensity at 565nm, taking the OFL concentration as a horizontal coordinate and the relative fluorescence value as a vertical coordinate as the standard curve, establishing a linear regression equation of the ofloxacin, and calculating the regression equation and the correlation coefficient. As shown in FIGS. 5 and 6, in the OFL concentration range of 0-100000 nM, the linear relationship between the relative fluorescence intensity and the ofloxacin concentration is 0.0526x +5.8391, R2The linear range is 0-500nM and the detection limit is 0.2nM, which indicates the high sensitivity of the method to OFL.
Fifth, specific detection
Cy 3-aptamer (200nM) and BHQ2-cDNA at a molar ratio of 1:4 were added to Tris-HCl (10mM, pH 7.4, 2mM K)+And 1mM Mg2+) In the buffer solution, fluoroquinolone antibiotics (ofloxacin (OFL), Ciprofloxacin (CIP), Pefloxacin (PEF)), tetracycline antibiotics (tetracycline (TC), aureomycin (CTC), Oxytetracycline (OTC), Minocycline (MIN), Metacycline (METC)) and glycine (Gly) were sequentially added, respectively, so that the final concentration of each antibiotic in the reaction system was 1. mu.M. Three replicates of each antibiotic were run at 37 ℃ for 1 h. Fluorescence was measured at an excitation wavelength of 535am using a PerkinElmer fluorescence spectrophotometer with a slit width of 10nm x 10 nm. As can be seen from FIG. 7, the fluorescence intensity at 565nm was plotted to obtain a histogram, and other antibiotics were used in the present inventionThe interference is not significant, and the detection method has good specificity on the detection of OFL.
Sixth, recovery rate in actual water sample
The ofloxacin solution with the concentration of 500nM is prepared in five water qualities of ultrapure water, drinking water, tap water, lake water and river water, and three groups of experiments are repeated. Cy 3-aptamer (200nM) and BHQ2-cDNA at a molar ratio of 1:4 were added to Tris-HCl (10mM, pH 7.4, 2mM K)+And 1mM Mg2+) To the buffer solutions, different water qualities containing 500nM OFL were added, respectively, so that the OFL concentration in each reaction system was 50nM, and the reaction was allowed to stand at 37 ℃ for 1 hour, and fluorescence was measured at an excitation wavelength of 535nM with a PerkinElmer fluorescence spectrophotometer.
As shown in table 1, in the experiment of labeling the OFLs of the actual four water samples, such as drinking water, tap water, lake water, and river water, the recovery rates of the OFLs were all about 90 to 110% compared to ultrapure water. The specific recovery rates of the OFL in different water samples were respectively: baisuishan natural mineral water 95.63%, laboratory tap water 110.75%, basalt lake 105.67%, purple lake stream 91.39%. The result shows that the detection method has accuracy and reliability in detecting the OFL in the actual water sample.
TABLE 1 recovery of OFL in different water samples
| Type of water sample | Drinking water | Tap water | Lake water | River water |
| Source | Baisuishan natural mineral water | Laboratory | Basalt lake | Zi Huxi |
| Recovery rate | 95.63％ | 110.75％ | 105.67％ | 91.39％ |
It is to be noted that the above-mentioned list is only a few specific embodiments of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Sequence listing
<110> Nanjing university of forestry
<120> method for detecting ofloxacin with high sensitivity based on aptamer sensor
<130> 1
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 72
<212> DNA
<213> OFL nucleic acid aptamer (artificial)
<400> 1
aagtgaggtt cgtcccttta ataaactcga ttaggatctc gtgaggtgtg ctctacaatc 60
gtaatcagtt ag 72
<210> 2
<211> 12
<212> DNA
<213> complementary strand cDNA (artificial)
<400> 2
ctaactgatt ac 12
Claims (7)
1. The method for detecting ofloxacin at high sensitivity based on the aptamer sensor is characterized in that the aptamer sensor comprises Cy 3-aptamer and BHQ 2-cDNA; the Cy 3-aptamer is an OFL aptamer fluorescently labeled with Cy3, and a fluorophore Cy3 is modified at the 3' end of the sequence shown in SEQ ID NO. 1; the BHQ2-cDNA is a complementary strand cDNA modified by BHQ2, and a quenching group BHQ2 is modified at the 5' end of the sequence shown in SEQ ID NO. 2; the method specifically comprises the following steps:
1) cy 3-aptamer was bound to BHQ2-cDNA to form a mixture, BHQ2 quenched the fluorescence of Cy 3;
2) adding a sample to be detected into the mixture obtained in the step 1), standing for reaction, when a target object OFL exists in the sample to be detected, performing affinity competition, combining a Cy 3-aptamer and the OFL to form a Cy 3-aptamer/OFL mixture, unwinding the base pair of the aptamer/cDNA, recovering fluorescence through the release of a Cy3 fluorophore, and enhancing the fluorescence of Cy3 by the adjacent OFL;
3) and establishing a linear regression equation between the OFL concentration and the fluorescence recovery and enhancement degree, and determining the concentration of the OFL in the sample to be detected according to the fluorescence recovery and enhancement degree.
2. The method for detecting ofloxacin with high sensitivity based on aptamer sensor of claim 1, wherein in step 1), Cy 3-aptamer and BHQ2-cDNA are combined in Tris-HCl buffer solution.
3. The method for detecting ofloxacin with high sensitivity based on aptamer sensor of claim 1, wherein in the step 1), the molar ratio of Cy 3-aptamer to BHQ2-cDNA is 1: 1-10; the concentration of Cy 3-aptamer was 50-600 nM.
4. The method for detecting ofloxacin with high sensitivity based on aptamer sensor of claim 3, wherein in the step 1), the molar ratio of Cy 3-aptamer to BHQ2-cDNA is 1: 4; the concentration of Cy 3-aptamer was 200 nM.
5. The method for detecting ofloxacin with high sensitivity based on aptamer sensor of claim 2, wherein Tris-HCl buffer solution contains 1mM Mg2+And 2mM K+(ii) a The concentration of Tris-HCl buffer solution was 10mM, pH = 7.4.
6. The method for detecting ofloxacin with high sensitivity based on aptamer sensor according to claim 1, wherein in the step 2), the reaction temperature is 37 ℃ and the reaction time is 1 h.
7. The method for detecting ofloxacin with high sensitivity based on aptamer sensor of claim 1, wherein the linear detection range of OFL is 0-500nM, the linear regression equation is y =0.0526x +5.8391, and the detection limit is 0.2 nM.
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