Enzyme-assisted solid-phase microextraction fluorescence response sensor, method of making and use

Abstract

The application discloses an enzyme-assisted solid-phase microextraction fluorescence response sensor, a preparation method and application, and the sensor comprises a quartz rod with a surface sequentially subjected to hydroxylation and amination, and a 2G DNA sequence modified by a carboxyl group; wherein the 2G DNA sequence is coupled to the surface of the quartz rod, and the 2G DNA sequence is SEQ ID NO. 1. Advantages are as follows: (1) the application designs two modules of a reaction unit and a fluorescence detection unit on a solid-phase microextraction probe, which is beneficial to reduce experimental interference, remove matrix effects and improve a reaction rate; (2) the method requires a smaller amount of sample and does not need a complicated pretreatment extraction process; (3) the application converts the content of antibiotics in a sample into a fluorescence intensity value of a fluorescence instrument, the detection method is simple and convenient to operate, rapid in detection, applicable to high-sensitivity rapid detection and analysis, does not need single-fluorescent modification, does not need modification of a quenching agent, and has the advantage of low cost. The method uses a fluorescence spectrometer to detect a fluorescence signal, and the detection limit of streptomycin is 0.01084 nM.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food safety analysis, and relates to a nucleic acid aptamer affinity capture reaction and Nt.BstNBI enzyme cleavage and hybridization chain reaction initiated fluorescence signal detection method, in particular to an enzyme-assisted solid-phase microextraction fluorescence response sensor, a preparation method and application. BACKGROUND

[0002] Commonly used antibiotics in the beekeeping industry include chloramphenicol, tetracycline, streptomycin, etc. While antibiotics can treat bee diseases, incomplete metabolized antibiotics will remain in honey, affecting the quality of honey food.

[0003] The existing antibiotic analysis methods include fluorescence method, chromatography-mass spectrometry method, colorimetric method, chemiluminescence method, enzyme-linked immunosorbent method, immunochromatography method and electrochemical method. In recent years, biosensors combined with surface plasmon resonance (SPR), fluorescence, colorimetric, electrochemical sensor technology are considered as a powerful analysis testing tool, and many biosensor technologies have been developed to provide an economic, convenient and sensitive antibiotic detection platform. Although these analysis methods have achieved detection of antibiotics, these methods still have many limitations, such as complex operation process, low sensitivity, and insufficient specificity of the detection method. It is crucial to develop high selectivity and high sensitivity antibiotic detection methods for honey food safety. Nucleic acids have good functionalization and biocompatibility, and they can be used as substrates or biosensor signal mediators. Nucleic acid sensing usually utilizes strict base complementary pairing and specific affinity between oligonucleotides, for example, nucleic acid aptamers selected by SELEX technology, for specific binding of clinical monitoring, food contaminant detection, and environmental pollution analytes. Nucleic acids are often used in high-throughput, high-sensitivity and high-specificity analysis and detection, which can be divided into enzymatic signal conversion and non-enzymatic signal conversion. Enzymatic signal release methods have the characteristics of strong specificity and high stability, for example, Nt.BstNBI cleavage endonuclease specifically cleaves one single strand of double-stranded DNA substrate as a functional signal molecule; non-enzymatic signal release can realize signal release without the participation of biological enzyme molecules by using the entropy difference between nucleic acid analysis, for example, the binding competition between DNA.

[0004] Quartz glass material has stability and specific functionalization because it belongs to SiO2 material, and application in nucleic acid biosensor probe preparation can significantly improve the performance of nucleic acid biosensor probes, which can be used to prepare solid-phase microextraction probes. Solid-phase microextraction probes have good compatibility in the laboratory, have the advantages of low reagent and sample consumption, real-time detection, small analysis platform, convenient detection and low price, and have important significance in biosensor research. SUMMARY

[0005] Technical problems solved: In order to overcome the deficiencies of the prior art, the application utilizes the difference in the binding fluorescence signal of G-quadruplex dimer sequence and ThT, when STR exists, the STR is captured by the aptamer, and the remaining aptamer is complementarily combined with the nucleic acid sequence on the surface of the quartz rod sensor. The specific site of the complementary double-stranded enzyme Nt.BstNBI is cut and the G-quadruplex dimer sequence is released for high-sensitivity detection. In the detection strategy of the application, only the G-quadruplex dimer serves as a fluorescent marker, without other quenchers, greatly reducing the economic cost. In view of this, the application provides an enzyme-assisted solid-phase microextraction fluorescence response sensor, a preparation method and an application.

[0006] Technical scheme: The enzyme-assisted solid-phase microextraction fluorescence response sensor comprises a quartz rod with a surface sequentially subjected to hydroxylation and amination, and a carboxyl-modified 2G DNA sequence; wherein the 2G DNA sequence is coupled to the surface of the quartz rod, and the 2G DNA sequence is SEQ ID NO. 1: 5'-TTTTTGAGTCACGACCCGACAGAGGGCGGGATGGGGGGTTGGGCGGGATGGG-3'.

[0007] Preferably, the sensor is in an environment where streptomycin exists, the streptomycin is captured by the nucleic acid aptamer SEQ ID NO. 2 (sequence: 5'-GGGGTCTGGTGTTCTGCTTTGTTCTGTTTCTGTCGGGTCGTGACTC-3'), the excess aptamer is complementarily combined with the 2G DNA sequence on the surface of the sensor, the complementary double-stranded enzyme Nt.BstNBI is used to cut and release the G-quadruplex dimer sequence, the G-quadruplex dimer sequence is combined with ThT, and a fluorescence enhancement response is realized.

[0008] Among them, the 2G DNA and the nucleic acid aptamer are prepared in advance in a PBS buffer to a concentration of 1 μM. And heated at 95℃ for 5min, then cooled at room temperature for 1h to form a stable structure of DNA, ready for subsequent dilution.

[0009] The preparation method of the enzyme-assisted solid-phase microextraction fluorescence response sensor described above comprises the following steps:

[0010] S1, hydroxylated quartz rod

[0011] The quartz rod is ultrasonically cleaned in deionized water, wiped clean with dust-free paper, and then placed in an arowana solution, shaken at 400-600 rpm at room temperature for 12-16h, washed with deionized water and dried with nitrogen;

[0012] S2, aminated quartz rod

[0013] The quartz rod after hydroxylation of S1 is placed in the amination solution, 400-600 rpm oscillation on constant temperature shaker at 30℃ overnight, and then washed with ethanol and water, and dried with nitrogen;

[0014] S3, solid phase microextraction fluorescence response sensor

[0015] The 2G DNA with 5' end modified carboxyl is activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride for 30-45 min at room temperature, N-hydroxysuccinimide is added and oscillated for 5-10 min, and then incubated overnight at 4℃ on constant temperature shaker.

[0016] Preferably, the preparation of the Piranha solution in S1 is 30% hydrogen peroxide and 98% concentrated sulfuric acid, and the volume ratio of the preparation is 1:3-5, and the reaction temperature is room temperature for 12-16 h.

[0017] Preferably, the amination solution in S2 is composed of 3-aminopropyltriethoxysilane (APTES): water: ethanol = 1:0.8-1:30-48 by volume ratio, the treatment temperature is 30℃, and the reaction time is 12-16 h.

[0018] Preferably, the mass concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) in S3 is 10-20 mg / mL, the mass concentration of N-hydroxysuccinimide (NHS) is 10-20 mg / mL, and the volume ratio of the two is 1:0.8-1.

[0019] Preferably, the concentration of 2G DNA used in S3 is 750-1000 nM, and the volume ratio with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:1.

[0020] The above-mentioned enzyme-assisted solid phase microextraction fluorescence response sensor is used for detecting streptomycin in food.

[0021] Preferably, the detection method is: adding 50-100 μL of streptomycin aptamer solution into the sample to be detected, oscillating and incubating in the inner lining tube at room temperature for 90-120 min, inserting the solid phase microextraction fluorescence response sensor, oscillating and incubating at 40℃ for 120-150 min, then taking out and transferring to the inner lining tube containing Nt.BstNBI enzyme solution, oscillating and incubating at 55℃ for 60-90 min, then adding 10-20 μL of THT solution for continuous oscillation and incubation, measuring the fluorescence intensity of the reaction solution after incubation, and calculating the concentration of streptomycin in the sample to be detected according to the fluorescence intensity and the standard curve of the fluorescence intensity and the streptomycin concentration. The THT solution has a concentration of 6.5-8.5 μM and a volume of 10-15 μL, and the final determination solution has a fluorescence intensity at 485 nm. The Nt.BstNBI enzyme solution comprises: commercially available Nt.BstNBI enzyme diluted 100 times, reaction buffer and deionized water mixed in a volume ratio of 4:20:25.

[0022] Preferably, the streptomycin aptamer and the nucleic acid sequence on the surface of the sensor are complementary and combined to form specific cleavage sites SEQ ID NO. 3 (sequence: 5'…G AG T C N N N N▼N…3') and SEQ ID NO. 4 (sequence: 3'…CT C AGN N N N N…5'); after oscillation and incubation with the Nt.BstNBI enzyme solution, a G-quadruplex dimer with the sequence of SEQ ID NO. 5 (sequence: 5'-ACGACCCGACAGAGGGCGGGATGGGGGGTTGGGCGGGATGGG-3') is cleaved.

[0023] Preferably, the sensor has a detection limit of 0.01084 nM for streptomycin.

[0024] Advantages: (1) The traditional solid phase microextraction is usually a single analysis module, and the present application designs two modules of reaction unit and fluorescence detection unit on the solid phase microextraction probe, which is beneficial to reduce experimental interference, remove matrix effect and improve reaction rate; (2) Compared with the traditional method, the method requires less sample and does not need complicated pretreatment extraction process; (3) A novel method for detecting antibiotics in nucleic acid probes is designed, which converts the content of antibiotics in the sample into the fluorescence intensity value of the fluorescence instrument, and provides a more convenient and accurate biological detection platform compared with the traditional method; (4) The detection method is simple and convenient, rapid in detection, suitable for high-sensitivity rapid detection and analysis, does not need single-fluorescent modification and quenching agent modification, and has the advantage of low cost. The method uses a fluorescence spectrometer to detect the fluorescence signal, and the accurate quantitative streptomycin detection concentration range is 0.05 nM-500 nM, R 2 =0.9983, and the detection limit is 0.01084 nM. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure 1 is a schematic diagram of the solid phase microextraction nucleic acid sensor structure and detection process, wherein (A) is the process of amino-modification of the quartz rod, (B) is the solid phase microextraction nucleic acid sensor, and (C) is the process of STR detection.

[0026] Figure 2 Figure 2 is a schematic diagram of the process of modification of the solid phase microextraction probe, wherein a) is the XPS full spectrum of the process of modification of the quartz rod, b) is the XPS fine spectrum of the process of amino-modification of the quartz rod, and c) is the XPS fine spectrum of the process of nucleic acid modification of the quartz rod.

[0027] Figure 3 Figure 3 is a schematic diagram of the surface morphology and height of the process of modification of the solid phase microextraction probe characterized by atomic force microscopy (AFM), wherein a) is the AFM image of the untreated quartz rod, b) is the AFM image of the quartz rod after treatment with the Piranha solution, c) is the AFM image of the quartz rod after amino-modification, and d) is the AFM image of the quartz rod after nucleic acid modification.

[0028] Figure 4 Figure 4 is a CD spectrum of the G-quadruplex dimer in the process of detection of the solid phase microextraction nucleic acid sensor.

[0029] Figure 5 Figure 5 is an agarose gel electrophoresis image, wherein M is a DNA marker, 1 is an aptamer, 2 is 2G DNA, 3 is an aptamer + 2G DNA, 4 is an aptamer + 2G DNA + STR, and 5 is an aptamer + 2G DNA + STR + Nt. BstNBI.

[0030] Figure 6 Figure 6 is a result of fluorescence signal detection in the process of detection of the solid phase microextraction nucleic acid sensor, wherein a) is the result of optimization of the concentration of 2G DNA, b) is the result of optimization of the concentration of the aptamer, c) is the result of optimization of the extraction time, d) is the result of optimization of the concentration of the enzyme, e) is the result of optimization of the concentration of THT, f) is the result of optimization of the incubation temperature, and g) is the result of optimization of the incubation time.

[0031] Figure 7 Figure 7 is a result of the process of detection of the solid phase microextraction nucleic acid sensor, wherein a) is the fluorescence change value corresponding to different concentrations of STR, and b) is the calibration curve thereof.

[0032] Figure 8 Figure 8 is a mechanism diagram of a method for detecting the fluorescence response of a G-quadruplex dimer based on enzyme-assisted solid phase microextraction against streptomycin. DETAILED DESCRIPTION

[0033] The following examples further illustrate the present application, but should not be construed as limiting the present application. Modifications and substitutions to the methods, steps or conditions described herein can be made by one of skill in the art without departing from the spirit and scope of the present application. If not specifically mentioned, the technical means used in the examples are conventional means known to those skilled in the art.

[0034] Example 1

[0035] Based on enzyme-assisted solid-phase microextraction fluorescence response sensor, the following method is used for preparation:

[0036] (1) Preparation of hydroxylated quartz rod

[0037] After ultrasonic cleaning in deionized water, the quartz rod is wiped clean with a dust-free paper, and piranha solution is prepared. 1.5 mL is added to the liquid chromatography bottle, and the cleaned quartz rod is immersed in the chromatography bottle with piranha solution, treated with 400 rpm shaking at room temperature for 12 h. After treatment, the hydroxylated quartz rod is taken out, washed with a large amount of deionized water, and then dried with nitrogen for standby;

[0038] (2) Preparation of amino-terminated quartz rod

[0039] Amino-terminated solution is prepared, 1.5 mL of amino-terminated solution is added to the chromatography bottle, and the quartz rod is immersed in the solution. Shake at 400 rpm overnight at 30°C constant temperature shaker. The amino-terminated quartz rod is first washed with 10 mL of ethanol water, then washed with 30 mL of water three times, and then dried with nitrogen. The preparation of amino-terminated quartz rod is completed;

[0040] (3) Preparation of solid-phase microextraction nucleic acid quartz rod sensor

[0041] Add 50 μL of 1 μM 5'-modified carboxyl 2G DNA to 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (EDC) for activation of carboxyl on 2G DNA. After activation at room temperature 25°C for 30 min, add N-hydroxysuccinimide (NHS), shake well for 5 min, and then incubate overnight at 4°C constant temperature shaker to complete the preparation of solid-phase microextraction nucleic acid sensor.

[0042] The sensor prepared in this example is used for detection of streptomycin sulfate (STR) in food. The detection method is as follows: all DNA hairpins (2G DNA, aptamer) are prepared in advance with PBS buffer to a concentration of 1 μM, then heated at 95°C for 5 min, and then cooled at room temperature for 1 h to stabilize the structure of the DNA for subsequent dilution.

[0043] The solid phase microextraction nucleic acid sensor was inserted into a standard solution containing 50 μL of different concentrations of STR (0.05, 0.01, 0.5, 1, 5, 10, 50, 100, 500 nM) and 50 μL of 1 μM aptamer. After slow shaking at 40°C for 2 h and incubation at 40°C for 120 min, the quartz probe was removed and transferred to 100 μL of Nt.BstNBI enzyme solution, incubated at 55°C for 1 h to cleave the G4 quadruplex dimer and release it into the solution. Then, 10 μL of 7.5 μM THT solution was added to the bottle and incubated at 25°C for 50 min. Finally, the solution was added to a cuvette and the fluorescence signal value was detected by a fluorescence spectrophotometer with an emission wavelength of 485 nm. A standard curve of fluorescence intensity and STR was established.

[0044] The prepared solid phase microextraction nucleic acid sensor was used to detect actual honey samples containing streptomycin sulfate. Three different honeys were spiked with standard samples, and the final concentrations of the antibiotic streptomycin sulfate in the actual samples were 1, 10, and 100 nM, respectively. The detection process was as follows: the spiked samples were pretreated with PBS solution, and the extract was used for subsequent detection. The solid phase microextraction nucleic acid sensor was inserted into an actual sample extraction solution containing 50 μL of different concentrations of STR (1, 10, 100 nM) and 50 μL of 1 μM aptamer. After slow shaking at 40°C for 2 h and incubation at 40°C for 120 min, the quartz probe was removed and transferred to 100 μL of Nt.BstNBI enzyme solution, incubated at 55°C for 1 h to cleave the G4 quadruplex dimer and release it into the solution. Then, 10 μL of 7.5 μM THT solution was added to the bottle and incubated at 25°C for 50 min. The fluorescence signal value was detected by a fluorescence spectrophotometer, and the supernatant was measured for fluorescence signal. The content of STR in the actual sample was calculated by the standard curve.

[0045] To evaluate the detection effect of the established method on actual samples, three different honey samples (locust honey, milk vetch honey, and rape flower honey) were selected for detection of the spiked content of STR. The test results are shown in Table 1, and good relative recoveries were obtained.

[0046] Table 1: Spiked recovery rate of STR in actual samples

[0047]

[0048]

[0049] a Relative recovery rate = detected concentration / original concentration

[0050] Result analysis:

[0051] Figure 2 Figure 11. XPS spectra of the solid-phase microextraction probe modification process: a) Quartz rod modification process XPS full spectrum, b) XPS fine spectrum of the quartz rod before and after the amino modification and nucleic acid modification process. There is no N element after hydroxylation, and N element appears after amination, which proves that amination is complete; b) After nucleic acid modification, the relative intensity of N element increases, because there is N element in the nucleic acid backbone, which proves that the nucleic acid is successfully modified on the quartz rod; there is no P element after amination, and P element appears after nucleic acid modification, because there is P element in the nucleic acid backbone, which proves that the nucleic acid is successfully modified on the quartz rod.

[0052] Figure 3 Figure 12. AFM images of the surface morphology and height of the solid-phase microextraction probe modification process: a) Untreated, columnar graph height is -5 nm to 20 nm. b) After treatment with Piranha solution, the quartz rod surface is smoother, and the height does not change. c) After amination treatment, the quartz rod surface has some protrusions, and the height does not change. d) After nucleic acid modification, the quartz rod surface is rougher, and the height is -5 nm to 36 nm, the height increases, which proves that the nucleic acid chain 2G DNA is successfully modified on the quartz rod surface.

[0053] Figure 4 Figure 13. G-quadruplex dimer CD spectra during the detection process of the solid-phase microextraction nucleic acid sensor, verifying the successful formation of G4 dimer.

[0054] Figure 5 Figure 14. Agarose gel electrophoresis image M: DNA marker, 1 Aptamer, 2 2G DNA, 3 Aptamer + 2G DNA, 4 Aptamer + 2G DNA + STR, 5 Aptamer + 2G DNA + STR + Nt. BstNBI. The first column of bands is the DNA marker; the second column of bands is the aptamer Apt; the third column of bands is the partial base complementary pairing of Aptamer and 2G DNA; the fourth column of bands is the result of co-incubation after adding STR analyte to Aptamer and 2G DNA, which forms a displacement 2G DNA band, proving that the analyte responds to nucleic acid; the fifth column of bands is the result of co-incubation after adding STR analyte to Aptamer and 2G DNA, then adding Nt. BstNBI for shearing, a tailing band appears, proving that the double strand is sheared.

[0055] Figure 6For the optimization of the reaction conditions of the solid phase microextraction nucleic acid sensor detection process, (a) The fluorescence intensity diagram shows that 1 μM 2G DNA is the best modification concentration. (b) The fluorescence intensity diagram shows that 1.5 μM aptamer is the extraction concentration. (c) The fluorescence intensity diagram shows that 120 min is the best extraction time. (d) The fluorescence intensity diagram shows that 0.8 U / mL Nt.BstNBI is the best reaction concentration. (e) The fluorescence intensity diagram shows that 0.75 μM THT is the best reaction concentration. (f) The fluorescence intensity diagram shows that 25°C is the best incubation temperature of THT. (g) The fluorescence intensity diagram shows that 50 min THT is the best incubation time.

[0056] Figure 7 For the corresponding a) fluorescence change value and b) its calibration curve under different STR concentrations. The linear range is 0.05 nM-500 nM, the determination coefficient R2 of the curve is 0.9983, and the detection limit is 0.0108 nM.

Claims

1. An enzyme-assisted solid phase microextraction based fluorescent response sensing system for detection of streptomycin, characterized in that, The sensing system comprises a sensor, a nucleic acid aptamer with a sequence of SEQ ID NO. 2, a nicking endonuclease Nt.BstNBI and ThT, the sensor comprises a quartz rod with a surface sequentially hydroxylated and aminated, a 2G DNA sequence modified by a carboxyl group; wherein the 2G DNA sequence is coupled to the surface of the quartz rod, and the 2G DNA sequence is SEQ ID NO. 1; the sensor is in an environment with streptomycin, the streptomycin is captured by the nucleic acid aptamer, the excess aptamer is complementarily combined with the 2G DNA sequence on the surface of the sensor, the G-quadruplex dimer sequence is released by using the nicking endonuclease Nt.BstNBI to assist in cutting the complementary double-stranded sequence, the G-quadruplex dimer sequence is combined with ThT, and a fluorescence enhancement response is realized.

2. The method for preparing the enzyme-assisted solid phase microextraction fluorescence response sensing system according to claim 1, characterized in that, The method comprises the following steps: S1, preparing a hydroxylated quartz rod The quartz rod is ultrasonically cleaned in deionized water, wiped clean with dust-free paper, and then placed in an arowana solution, shaken at 400-600 rpm at room temperature for 12-16 h, washed with deionized water and dried with nitrogen; S2, preparing an aminated quartz rod The hydroxylated quartz rod in S1 is placed in an amination solution, shaken at 400-600 rpm overnight on a constant-temperature shaker at 30 DEG C, sequentially washed with ethanol and water, and dried with nitrogen; S3, preparing a solid-phase microextraction fluorescence response sensor including: activating the 2G DNA with a 5' end modified carboxyl group by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride for 30-45 min at room temperature.

3. The preparation method according to claim 2, characterized in that, The amination solution in S2 is composed of 3-aminopropyl triethoxysilane, water and ethanol, and the volume ratio is 3-aminopropyl triethoxysilane: water: ethanol = 1: 0.8-1: 30-48, the treatment temperature is 30 DEG C, and the reaction time is 12-16 h.

4. The production method according to claim 2, characterized by, The concentration of the 2G DNA used in S3 is 750-1000 nM, and the volume ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1:

1.

5. The application of the enzyme-assisted solid-phase microextraction fluorescence response sensing system according to claim 1 in the detection of streptomycin in food.

6. Use according to claim 5, characterized in that, The detection method is as follows: 50-100 muL of streptomycin nucleic acid aptamer solution is added to the sample to be detected, shaken and incubated in an inner liner tube at room temperature for 90-120 min, a solid-phase microextraction fluorescence response sensor is inserted, shaken and incubated at 40 DEG C for 120-150 min, then transferred to an inner liner tube containing Nt.BstNBI enzyme solution and shaken and incubated at 55 DEG C for 60-90 min, then 10-20 muL of ThT solution is added for continued shaking and incubation, the fluorescence intensity of the reaction solution after incubation is determined, and the concentration of streptomycin in the sample to be detected is calculated according to the fluorescence intensity and the standard curve of the fluorescence intensity and the concentration of streptomycin.

7. Use according to claim 6, characterized in that, The streptomycin nucleic acid aptamer is complementarily combined with the nucleic acid sequence on the surface of the sensor to form specific cleavage sites SEQ ID NO. 3 and SEQ ID NO. 4; after shaking and incubation with the Nt.BstNBI enzyme solution, the G-quadruplex dimer with a sequence of SEQ ID NO. 5 is cut.

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