CN108333239A - A kind of biosensor detecting kanamycins based on aptamer - Google Patents

Abstract

The present invention provides a kind of biosensor detecting kanamycins based on aptamer, including gold electrode, primer layers of HAP2 layers and HAP1 aptamer on gold electrode are modified successively from inside to outside；HAP2 layers of thickness is 15 ± 2 nm, and the thickness that primer layers of HAP1 aptamer is 10 ± 2 nm.HAP1 in primer layers of the HAP1 aptamer（Mole）、aptamer（Mole）、primer（Mole）、ExoIII（Activity）Mole with it is active than be 1:1:1:250.The electrode of the biosensor of the present invention is easy, minimizes, is portable, being used multiple times；Reaction condition is mild, and speed is fast, easy to operate；Preparation method is simple, and process costs are low, performance stablize, electrode it is reproducible.

Description

A biosensor for detection of kanamycin based on nucleic acid aptamer

technical field

The invention belongs to the technical field of biosensors, in particular to a biosensor for detecting kanamycin based on nucleic acid aptamers, and also relates to a preparation method thereof.

Background technique

Antibiotics have the function of killing and destroying microbial cell structures, and are widely used in the preparation of various antibacterial drugs worldwide. At the same time, the abuse of antibiotics has become a problem that needs to be solved urgently worldwide. Antibiotic overuse can spur infection-causing bacteria to become more resistant. Antibiotics are also used in the food industry, such as milk, honey, meat products and other foods. Antibiotic residues in food will pose a serious threat to human health. Therefore, it is urgent and important to develop a method that can detect trace antibiotics in food and the environment to protect our health.

Kanamycin is an aminoglycoside antibiotic. If it is not used in an appropriate amount, it may cause allergic reactions and damage the nervous system, urinary system, and digestive system. At present, commonly used kanamycin detection methods include fluorescence detection, high performance liquid chromatography (HPLC), capillary electrophoresis (CE), enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), etc., these methods There are often problems such as expensive instruments, long analysis period, complicated sample pretreatment, and expensive detection costs, which have made it difficult to meet the requirements of convenience, speed, and sensitivity of antibiotic detection.

Therefore, there is an urgent need to establish a rapid, accurate, sensitive and highly specific detection method to detect kanamycin residues in food.

Contents of the invention

In order to solve the problems of relatively low specificity and sensitivity and high cost of the method for detecting kanamycin in the prior art, the present invention provides a nucleic acid-based adaptation method with high specificity and sensitivity, low cost and fast detection speed. A biosensor for the detection of kanamycin by conformational changes and ExoIII-assisted cycle amplification.

The present invention also provides a method for detecting kanamycin by the biosensor.

In order to achieve the above object, the present invention adopts the following technical solutions.

A biosensor for detecting kanamycin based on a nucleic acid aptamer, including a gold electrode, sequentially modifying the HAP2 layer and the HAP1-aptamer-primer layer on the gold electrode from the inside to the outside;

The sequence of the HAP2 is shown in SEQ No.1, and its 5' end is modified with a sulfhydryl group (-SH);

The sequence of the HAP1 is shown in SEQ No.2, the sequence of the aptamer is shown in SEQ No.3; the sequence of the primer is shown in SEQ No.4;

The HAP1-aptamer-primer layer contains HAP1, aptamer, primer and ExoIII.

In the biosensor, the thickness of the HAP2 layer is 15 ± 2 nm, and the thickness of the HAP1-aptamer-primer layer is 10 ± 2 nm.

The mole-to-activity ratio of HAP1 (mole), aptamer (mole), primer (mole), and ExoIII (activity) in the HAP1-aptamer-primer layer is 1:1:1:250.

A method for detecting kanamycin of the above-mentioned biosensor, comprising the following steps:

(1) Polish the electrode;

(2) Add the HAP2 solution dropwise to the electrode surface and incubate at a constant temperature of 37 °C;

(3) Incubate the mixture of HAP1, aptamer, primer and ExoIII with the solution to be tested or a series of concentrations of kanamycin solution at 37 °C, and then add the above mixture dropwise to the solution obtained in step (2). Electrode surface, incubate at 37 ℃, and then wash the electrode;

(4) Add heme dropwise to the electrode obtained in step (3), incubate at a constant temperature of 37 ℃, and then wash the electrode;

(5) Using Ag/AgCl as a reference electrode, using a Pt electrode as a counter electrode, and using the electrode obtained in step (4) as a working electrode, the signal is detected by differential pulse voltammetry in a solution containing H ₂ O ₂ ; Calculate the regression equation according to the electrical signal, and calculate the content of kanamycin in the liquid to be tested.

In the above method, the final concentration of heme is 1-30mM, preferably 1-20mM. The final concentration of H ₂ O ₂ is 1-3.5 mM, preferably 1-2.5 mM. The reaction time is 0.5-3h, preferably 0.5-2h.

The polishing treatment method in the step (1) is as follows: the electrode is polished in 0.3 µm and 0.05 µm alumina slurry until it becomes a mirror surface, and rinsed with phosphate buffered saline (PBS) and secondary water for 3-5 days. Second-rate.

In the step (5), the potential is set to -0.1~-1 V, the scan rate is 0.06 s, and the pulse width is 0.05 V.

The principle of this biosensor is as follows:

The aptamer and primer of kanamycin can form an arched probe. In the presence of kanamycin, the aptamer in the arched probe combines with the antibiotic to release the primer.

HAP1 contains a sequence that can form a hairpin structure. In the presence of primer, it can open the hairpin to form a double-stranded structure. Under the action of ExoIII, HAP1 is cut to release the trigger chain (5'-CCC AGT TGG CCC TAT T-3').

HAP2 contains a sequence capable of forming a hairpin structure, and its 5' end is modified with a sulfhydryl group (-SH), which can form a stable Au-S bond with the gold electrode, thereby fixing HAP2 on the electrode surface; the trigger chain opens the HAP2 hairpin to form a double Chain structure, under the action of ExoIII, HAP2 is cut, and the released part of the HAP2 sequence contains a large amount of guanine G. In the presence of potassium ions, adding heme can form a G-quadruplex/heme complex. It can act as horseradish peroxidase to oxidize and reduce hydrogen peroxide to obtain obvious electrical signals, and achieve the purpose of quantitative detection of target substances by detecting the redox electrical signals.

The present invention has the following advantages:

The electrode of the biosensor of the present invention is simple, miniaturized, easy to carry, and can be used multiple times; the main process of detection is realized on the electrode, which improves the reaction speed, reduces the complexity of operation, and has mild reaction conditions. Fast, simple and sensitive detection of target objects; the biosensor of the present invention has simple preparation method, low process cost, stable performance, good electrode repeatability, and is suitable for the detection of kanamycin in food safety and the industrialization of biosensors practical application.

Description of drawings

Fig. 1 is the working principle of this biosensor;

Figure 2 is the impact of different heme concentrations on the detection of kanamycin;

Fig. 3 is the influence of different H ₂ O ₂ concentrations on the detection of kanamycin;

Figure 4 is the impact of different reaction times on the detection of kanamycin;

Fig. 5 is the current scanning figure of different kanamycin concentrations;

Figure 6 is a standard curve for the detection of kanamycin.

Detailed ways

The present invention will be further described below in conjunction with the embodiments and accompanying drawings, but the present invention is not limited by the following embodiments.

In the example, the PBS buffer contains Na ₂ HPO ₄ (10 mM), NaH ₂ PO ₄ (10 mM), NaCl (140 mM), KCl (1 mM), MgCl ₂ (1 mM), CaCl ₂ (1 mM ), the pH value is 7.4.

The prepared PBS buffer and ultrapure water were sterilized at 120 °C for 20 min.

Each DNA sequence is as follows:

HAP2: 5'-SH-GGG TTG GGC GGG ATG GGG GGC CAA CTG GG-3';

HAP1: 5'-CCC AGT TGG CCC TAT TCC CGC AAC TAG CCG-3';

aptamer: 5'-GCG ACC CGT GGG GGT TGA GGC TAA GCC G-3';

primer: 5'-CGG CTA GTT GCG GGT CGC-3'.

Example 1 Effect of different hemoglobin concentrations on the detection of kanamycin.

(1) Polish the electrode: the electrode is polished in 0.3 µm and 0.05 µm alumina slurry until it becomes a mirror surface, and then rinsed three times with phosphate-buffered saline (PBS) and ultrapure water;

(2) Add 10 μL of 1.0 μM HAP2 dropwise to the electrode surface and incubate at 37 °C for 2 h;

(3) Mix sterile water, 2 μL 1× ExoIII buffer, 2 μL HAP1 (10 μM), 2 μL aptamer (10 μM), 2 μL primer (10 μM), 2 μL ExoIII (100 U/μL) , 8 μL of sterilized water and 2 μL of 10 nM kanamycin solution were added to a pre-prepared sterilized centrifuge tube; shaken for 30 s to mix well, and then put the mixture into a 37 ℃ incubator and incubated for 2 h ;Then the above mixture was added dropwise to the surface of the electrode obtained in step (2), incubated at 37°C for 2 h, and then rinsed with PBS for 3 times;

(4) Add 10 μL of heme (final concentration of 1.0 mM, 5.0 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM) onto the electrode obtained in step (3), and then place the electrode on Incubate in an incubator at 37 °C for 20 min, then rinse the electrode 3 times with PBS;

(5) With Ag/AgCl as the reference electrode, the Pt electrode as the counter electrode, and the electrode obtained in step (4) as the working electrode, differential pulse voltammetry was used in 15 mL of PBS containing 2.5 mM H ₂ O ₂ The signal was detected by the method; the potential was set to -0.1~-1 V, the scan rate was 0.06 s, and the pulse width was 0.05 V.

Draw a curve with the hemoglobin concentration as the abscissa and the current signal as the ordinate, as shown in Figure 2. According to Figure 2, the detected current signal increases as the concentration of hemoglobin increases in the range of 0-20 mM, and when the concentration exceeds 20 mM, the current tends to be stable.

Example 2 Effects of different H ₂ O ₂ concentrations on the detection of kanamycin.

(1) Polish the electrode: the electrode is polished in 0.3 µm and 0.05 µm alumina slurry until it becomes a mirror surface, and then rinsed three times with phosphate-buffered saline (PBS) and ultrapure water;

(2) Add 10 μL of 1.0 μM HAP2 dropwise to the electrode surface and incubate at 37 °C for 2 h;

(3) Mix sterile water, 2 μL 1× ExoIII buffer, 2 μL HAP1 (10 μM), 2 μL aptamer (10 μM), 2 μL primer (10 μM), 2 μL ExoIII (100 U/μL) , 8 μL of sterilized water and 2 μL of 10 nM kanamycin solution were added to a pre-prepared sterilized centrifuge tube; shaken for 30 s to mix well, and then put the mixture into a 37 ℃ incubator and incubated for 2 h ;Then the above mixture was added dropwise to the surface of the electrode obtained in step (2), incubated at 37°C for 2 h, and then rinsed with PBS for 3 times;

(4) Add 10 μL of heme (final concentration 20 mM) dropwise onto the electrode obtained in step (3), then place the electrode in a 37°C incubator and incubate for 20 min, then rinse the electrode with PBS for 3 times;

(5) With Ag/AgCl as the reference electrode, the Pt electrode as the counter electrode, and the electrode obtained in step (4) as the working electrode, 15 mL contains 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM , 3.5 mM H ₂ O ₂ in PBS using differential pulse voltammetry for signal detection; the potential was set at -0.1 to -1 V, the scan rate was 0.06 s, and the pulse width was 0.05 V.

Take the H ₂ O ₂ concentration as the abscissa and the current signal as the ordinate to draw a curve, as shown in Fig. 3 . It can be seen from Figure 3 that the detected current signal increases as the concentration of H ₂ O ₂ increases in the range of 0-2.5 mM, and when the concentration exceeds 2.5 mM, the current tends to be stable.

Example 3 Effects of different reaction times on the detection of kanamycin.

(1) Polish the electrode: the electrode is polished in 0.3 µm and 0.05 µm alumina slurry until it becomes a mirror surface, and then rinsed three times with phosphate-buffered saline (PBS) and ultrapure water;

(2) Add 10 μL of 1.0 μM HAP2 dropwise to the electrode surface and incubate at 37 °C for 2 h;

(3) Mix sterile water, 2 μL 1× ExoIII buffer, 2 μL HAP1 (10 μM), 2 μL aptamer (10 μM), 2 μL primer (10 μM), 2 μL ExoIII (100 U/μL) , 8 μL of sterilized water and 2 μL of 10 nM kanamycin solution were added to a pre-prepared sterilized centrifuge tube; shaken for 30 s to mix well, and then placed the mixture in a 37 ℃ incubator and incubated for 0.5 h , 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.0 h; then drop the above mixture onto the surface of the electrode obtained in step (2), incubate at 37 °C for 2 h, and then rinse the electrode with PBS for 3 times;

(4) Add 10 μL of heme (final concentration 20 mM) dropwise onto the electrode obtained in step (3), then place the electrode in a 37°C incubator and incubate for 20 min, then rinse the electrode with PBS for 3 times;

(5) With Ag/AgCl as the reference electrode, the Pt electrode as the counter electrode, and the electrode obtained in step (4) as the working electrode, differential pulse voltammetry was used in 15 mL of PBS containing 2.5 mM H ₂ O ₂ The signal was detected by the method; the potential was set to -0.1~-1 V, the scan rate was 0.06 s, and the pulse width was 0.05 V.

Take the response time as the abscissa and the current signal as the ordinate to draw a curve, as shown in Figure 4. It can be seen from Figure 4 that the detected current signal increases as the concentration of H ₂ O ₂ increases in the range of 0-2.5 mM, and when the concentration exceeds 2.5 mM, the current tends to be stable.

Example 4 Detection of Kanamycin.

(1) Polish the electrode: the electrode is polished in 0.3 µm and 0.05 µm alumina slurry until it becomes a mirror surface, and then rinsed three times with phosphate-buffered saline (PBS) and ultrapure water;

(2) Add 10 μL of 1.0 μM HAP2 dropwise to the electrode surface and incubate at 37 °C for 2 h;

(3) Mix sterile water, 2 μL 1× ExoIII buffer, 2 μL HAP1 (10 μM), 2 μL aptamer (10 μM), 2 μL primer (10 μM), 2 μL ExoIII (100 U/μL) , 8 μL sterile water and 2 μL kanamycin solution (concentrations are 0.5 pM, 1 pM, 5 pM, 10 pM, 50 pM, 100 pM, 500 pM, 1 nM, 5 nM, 10 nM, 50 nM ) into a pre-prepared sterilized centrifuge tube; shake for 30 s to mix well, then put the mixture in a 37 ℃ incubator and incubate for 2 h; then add the above mixture dropwise to step (2) to obtain The surface of the electrode was incubated at 37 °C for 2 h, and then the electrode was rinsed 3 times with PBS;

(4) Add 10 μL of heme (final concentration 20 mM) dropwise onto the electrode obtained in step (3), then place the electrode in a 37°C incubator and incubate for 20 min, then rinse the electrode with PBS for 3 times;

(5) With Ag/AgCl as the reference electrode, the Pt electrode as the counter electrode, and the electrode obtained in step (4) as the working electrode, differential pulse voltammetry was used in 15 mL of PBS containing 2.5 mM H ₂ O ₂ The signal was detected by the method; the potential was set to -0.1~-1 V, the scan rate was 0.06 s, and the pulse width was 0.05 V.

The results are shown in Figure 5 and Figure 6. It can be seen from Figure 5 that the detected current signal increases with the increase of the target concentration in the range of 1 pM -10 nM, and Figure 6 shows the effect of the kanamycin concentration on The number is proportional to the magnitude of the current peak, and the fitting curve is I=0.73101+ 0.47343 lg(C/pM), where C is the concentration of kanamycin (pM), and the correlation coefficient is 0.97959.

At the same time, it can be seen from Figure 5 and Figure 6 that on the basis of the concentration of 1 pM and 10 nM, the detection continues to lower and higher concentrations respectively. When the concentration is lower than 1 pM and higher than 10 nM, The logarithm of the kanamycin concentration and the magnitude of the current peak just no longer conform to the law of the fitting curve, that is, the lowest and highest values of the current peak in the figure. Therefore, the lower and upper limit of detection of the method can be obtained as 1 pM and 10 nM, respectively.

<110> Jinan University

<120> A biosensor for detection of kanamycin based on nucleic acid aptamer

<130> 20180117

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 30

<212>DNA

<213> Artificial Sequence

<220>

<223> HAP1

<400> 1

cccagttggc cctattcccg caactagccg 30

<210> 2

<211> 29

<212>DNA

<213> Artificial Sequence

<220>

<223> HAP2

<400> 2

gggttgggcg ggatgggggg ccaactggg 29

<210> 3

<211> 28

<212>DNA

<213> Artificial Sequence

<220>

<223> aptamer

<400> 3

gcgacccgtgggggttgagg ctaagccg 28

<210> 4

<211> 18

<212>DNA

<213> Artificial Sequence

<220>

<223> primer

<400> 4

cggctagttg cgggtcgc 18

Claims (7)

1. a kind of biosensor detecting kanamycins based on aptamer, which is characterized in that including gold electrode, by it is interior to Primer layers of the outer HAP2 layers modified successively on gold electrode and HAP1- aptamer-；

The sequence of the HAP2 is as shown in SEQ No.1,5 ' terminal modified sulfydryls（-SH）；

The sequence of the HAP1 as shown in SEQ No.2, the sequence of the aptamer is as shown in SEQ No.3；The primer's Sequence is as shown in SEQ No.4；

Contain HAP1, aptamer, primer, ExoIII in HAP1-aptamer-primer layers described.

2. biosensor according to claim 1, which is characterized in that HAP2 layers of thickness is 15 ± 2 nm, HAP1- Aptamer-primer layers of thickness is 10 ± 2 nm.

3. biosensor according to claim 1, which is characterized in that in HAP1-aptamer-primer layers described HAP1（Mole）、aptamer（Mole）、primer（Mole）、ExoIII（Activity）Mole with it is active than be 1:1:1: 250。

4. a kind of method of biosensor detection kanamycins as described in claim 1, which is characterized in that including following step Suddenly：

（1）Electrode is processed by shot blasting；

（2）HAP2 solution is added drop-wise to electrode surface, 37 DEG C of constant-temperature incubations；

（3）By the mixed liquor of HAP1, aptamer, primer and ExoIII kanamycins with prepare liquid or series concentration respectively Solution temperature at 37 DEG C is incubated, and above-mentioned mixed liquor is then added drop-wise to step respectively（2）The electrode surface of acquisition, temperature at 37 DEG C It is incubated, then cleaning electrode；

（4）Ferroheme is added drop-wise to step（3）On the electrode of acquisition, 37 DEG C of constant-temperature incubations, then cleaning electrode；

（5）It is to electrode, with step with Pt electrodes using Ag/AgCl as reference electrode（4）The electrode of acquisition is working electrode, Containing H₂O₂Solution in using differential pulse voltammetry carry out signal detection；Regression equation is calculated according to electric signal, is calculated to be measured Kanamycins content in liquid.

5. according to the method described in claim 4, it is characterized in that, the final concentration of 1-30mM of the ferroheme；The H₂O₂'s Final concentration of 1-3.5mM；The reaction time is 0.5-3h.

6. according to the method described in claim 4, it is characterized in that, the step（1）Middle polishing treatment method is：Electrode point It is not processed by shot blasting in 0.3 μm and 0.05 μm of oxidation aluminium paste, until being in minute surface, uses phosphate buffered saline （PBS）It is rinsed 3-5 times with secondary water.

7. according to the method described in claim 4, it is characterized in that, the step（5）Middle current potential is set as -0.1 ~ -1 V, Sweep speed is 0.06 s, and pulse width is 0.05 V.