Preparation method of hydrogel-based fluorescent biosensor for rapidly and quantitatively detecting Hg < 2 + >

Abstract

The invention designs a preparation method of a hydrogel-based fluorescent biosensor for rapidly detecting the specific content of Hg < 2 + >. An RCA / G quadruplex product is synthesized in the rolling circle amplification process, the RCA / G quadruplex product is combined with ThT fluorescent dye, an RCA-ThT fluorescent probe material with excellent performance is obtained, and Hg < 2 + > can be efficiently and sensitively recognized. In addition, in order to realize on-site rapid detection, a designed RCA / G quadruplex product is introduced into the hydrogel for physical combination, and the product is packaged on a centrifugal tube cover to serve as a real-time detection sample. The hydrogel fluorescent biosensor packaged by the centrifugal tube can be used at any time, and is convenient to carry and low in cost. Meanwhile, a smart phone WeChat applet is developed, and the quantitative result of Hg < 2 + > can be obtained in real time through mobile phone photographing. The detection limit of the hydrogel-based fluorescent biosensor on Hg < 2 + > is 0.044 [mu] M, and the detection range is 0-100 [mu] M. Experimental results show that the sensor has good stability and instant detection capability, and has application prospects in food safety monitoring.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and in particular relates to a hydrogel-based fluorescent biosensor for rapid quantitative detection of Hg 2+ Preparation method of specific content. Background Art

[0002] Hg 2+ It is widely present in various environmental media and is currently considered to be one of the most toxic substances. 2+ It can participate in the complete cycle in the ecosystem, is difficult to be biodegraded, and will gradually accumulate in the ecosystem. 2+ Once it enters the human body through the food chain, it can induce a variety of diseases, such as cardiovascular disease, movement disorders, etc. In severe cases, it can lead to organ poisoning and failure, posing a serious threat to human health. 2+ The method of Hg 2+ The detection methods include atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, high performance liquid chromatography and electrochemical methods. Among them, fluorescent probes have attracted widespread attention due to their high sensitivity and strong anti-interference ability. The RCA / G quadruplex fluorescent probe is composed of G-quadruplex intercalated with ThT fluorescent dye and has high sensitivity. G-quadruplex is an atypical nucleic acid sequence rich in guanine with a unique secondary structure. It forms a quadruplex structure through base stacking and is currently widely used in the construction of label-free fluorescence detection platforms. A large number of studies have shown that ThT can induce G-rich DNA to fold into G-quadruplexes, undergo affinity interaction, and form fluorescent probes. Compared with the other two commonly used G-quadruplex selective dyes N-methylporphyrin dipropionic acid 1X (NMM) and PPIX, ThT can more effectively identify G-quadruplex TBA29, significantly enhance the fluorescence intensity, and bring great convenience to naked eye observation. Therefore, ThT is combined with G-quadruplex to build RCA / G quadruplex fluorescent probe. The constructed RCA-ThT quadruplex fluorescent aptamer sensor was introduced into the hydrogel, and then 2+ The solution was contacted with the hydrogel and it was found that it could significantly quench the fluorescence intensity of ThT. The color change of DNA hydrogel could be observed by the naked eye, and the overall fluorescence weakened, which could be used to reflect the Hg 2+ content.

[0003] The implementation of point-of-care testing is a trend in screening harmful substances in products. At present, the use of smartphones is increasing day by day. The combination of highly sensitive sensors and convenient and fast smartphones will develop into a promising tool. By utilizing the powerful photography and excellent computing capabilities of smartphones, the obtained fluorescent images can be immediately converted into digital color values ​​of red (R), green (G), and blue (B) (RGB) using color recognition software for on-site quantitative analysis, that is, the mercury content can be quantitatively calculated based on the RGB values. This method greatly shortens the detection time and avoids the cumbersome labeling process. It meets the "ASSURED" standard of point-of-care testing, namely affordable, sensitive, specific, user-friendly, rapid and repeatable, equipment-free, and delivered (to the end user). In summary, hydrogel-based fluorescent biosensors have high sensitivity and accuracy and have good application prospects. Summary of the invention

[0004] A hydrogel-based fluorescent biosensor for rapid quantitative detection of Hg 2+ The preparation method of the specific content comprises the following steps: (1) Construction of RCA-ThT fluorescent probe: Padlock and Primer were introduced into T4 Buffer, and long DNA chains were amplified by RCA reaction. After annealing, T4 DNA ligase was added, and the mixture was incubated overnight in a PCR instrument. Then, phi29 polymerase and dNTP were added to obtain RCA / G-quadruplex products. The RCA / G-quadruplex products were fully dispersed and dissolved in a neutral solution, and the mixture was incubated in a water bath thermostat oscillator. ThT fluorescent dye was added to obtain RCA-ThT fluorescent probe. (2) Preparation of a hydrogel-based fluorescent biosensor: agarose powder was dissolved in a buffer solution and heated to boiling, and then cooled to room temperature to obtain a preliminary hydrogel; then the incubated RCA-ThT fluorescent probe was added and poured into a culture dish to obtain a DNA nanoflower hydrogel. The centrifuge tube cap was embedded in the DNA nanoflower hydrogel for sampling, and different concentrations of Hg 2+ The solution is added and the centrifuge tube is placed upside down to obtain a hydrogel-based fluorescent biosensor that is ready for use; (3) Rapid quantitative detection of Hg using smartphones 2+ Content: After incubating the centrifuge tube cap with hydrogel, put it into the detection instrument and irradiate it with ultraviolet light. At this time, the hydrogel will show different degrees of fluorescence. Use a smartphone to take a photo and upload it to the mini program for recognition. Hg can be obtained in a short time.2+ The specific content.

[0005] It is further defined that in step (1), the neutral solution is one or more of ultrapure water, Tris-HCl buffer solution, and ethanol; the incubation time of the RCA / G-quadruplex product in a water bath constant temperature oscillator is 6 to 36 h; the ThT concentration is 0.1 to 10 mM; and the volume is 10 μL.

[0006] It is further defined that in step (2), the mass of the weighed agarose powder is between 0.18 and 0.24 g; the buffer solution is one or more of Tris-HCl buffer solution, PBS buffer solution, acetate buffer solution, HEPES solution, and Tris-EDTA; the volume of the buffer solution used is between 20 and 25 mL; and the cooling time is 2 to 3 min.

[0007] It is further defined that in step (3), the shaking incubation time is 45 min to 90 min; and the wavelength of the ultraviolet flashlight excitation light used is 365 nm. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 A hydrogel-based fluorescent biosensor for rapid quantitative detection of Hg 2+ Schematic diagram of the preparation method of specific content.

[0009] Figure 2 The fluorescent biosensor constructed in Example 1 of the present invention was subjected to the addition of 0 Mmol (line a), 10 Mmol (line b), and 100 Mmol Hg 2+ Fluorescence spectrum detection results of the solution (line c) and with only ThT added (line d).

[0010] Figure 3 The fluorescent biosensor constructed in Example 1 of the present invention detects Hg 2+ The standard curve of

[0011] Figure 4 Comparison of ΔGreen value (G value) of the fluorescent biosensor constructed in Example 1 of the present invention in the presence of other heavy metal ions Implementation

[0012] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and specific examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Example 1

[0013] A hydrogel-based fluorescent biosensor for rapid quantitative detection of Hg 2+ The preparation method of the specific content, the realization method is as follows Figure 1 shown.

[0014] A hydrogel-based fluorescent biosensor for rapid quantitative detection of Hg 2+ The preparation method of the specific content comprises the following steps: (1) Construction of RCA-ThT fluorescent probe: Padlock (100 μM, 1 μL) and primer (100 μM, 1 μL) were introduced into T4 DNA ligase buffer with a final volume of 200 μL, and then divided into two parts and placed in a PCR instrument for annealing. After completion, 2.5 μL of T4 DNA ligase was added to each centrifuge tube, and the tube was placed in a PCR instrument and incubated overnight at 16°C, and inactivated at 65°C for 10 min. Then, the solutions in the two centrifuge tubes were mixed, and 5 μL of phi29 polymerase (10 U / μL) and 50 μL of dNTP (10 mM) were added. Finally, a total volume of 400 μL of 1×phi29 polymerase buffer system was synthesized, and the tube was incubated at 30°C for 24 h for inactivation to obtain RCA / G-quadruplex products. The RCA / G-quadruplex product was fully dispersed at 50 °C and 800 rpm. 10 μL was dissolved in 180 μL ultrapure water, and then 10 μL 1 mM ThT was added. The product was placed in a water bath thermostat and incubated for 60 min to obtain the RCA-ThT fluorescent probe.

[0015] (2) Preparation of hydrogel-based fluorescent biosensor: Weigh 0.24 g agarose powder and dissolve it in 25 mL prepared Tris solution. Heat the solution to boiling, cool it slightly and place it in a 50 °C water bath and heat it at a constant temperature for 10 minutes to obtain a preliminary hydrogel. Immediately add the incubated RCA-ThT fluorescent probe, shake the conical flask to allow the fluorescent probe to fully contact the hydrogel, and pour it into a clean culture dish before the hydrogel solidifies to obtain a DNA nanoflower hydrogel with a uniform mixture of fluorescent probes. Insert the centrifuge tube cap into the DNA nanoflower hydrogel to take a sample, and add different concentrations of Hg 2+ The solution is added and the centrifuge tube is placed upside down to obtain a hydrogel-based fluorescent biosensor that is ready for use; (3) Rapid quantitative detection of Hg using smartphones 2+ Content: Place the centrifuge tube in a shaker and incubate for 1 hour. Place the centrifuge tube cap with hydrogel into the detection instrument and irradiate with ultraviolet light at a wavelength of 365 nm. The hydrogel will show different degrees of fluorescence. Take a photo with a smartphone and upload it to the mini program to identify and obtain the RGB value. Based on the RGB value, Hg can be quickly obtained in a short time. 2+ The specific content.

[0016] (4) Establishment of standard curve: Different concentrations of Hg 2+ The standard solution is added to the centrifuge tube and Hg is obtained through step (3). 2+ Different G / B values ​​at different concentrations, with Hg 2+ The logarithm of the concentration was used as the horizontal axis, and the G / B value was used as the vertical axis for linear fitting to establish a standard curve of the fluorescence signal.

[0017] like Figure 2 As shown, the fluorescent biosensor constructed in Example 1 of the present invention is subjected to the addition of 0 mM (line a), 10 mM (line b) and 100 mM Hg 2+ Fluorescence spectrum detection results of the solution (line c).

[0018] like Figure 3 As shown, it is a comparison of the Green value (G value) of the fluorescent biosensor constructed in Example 1 of the present invention in the presence of other heavy metal ions. Example 2

[0019] A hydrogel-based fluorescent biosensor for rapid quantitative detection of Hg 2+ The preparation method and its practical application comprise the following steps: (1) To verify the prepared hydrogel-based fluorescent biosensor for Hg 2+ We have conducted tests and analyses on several other common heavy metal ions. The heavy metal ions selected for analysis and comparison include Pb 2+ 、Cd 2+ , Cu 2+ , K + 、Zn 2+ and Sr 2+ The selected ion concentration is 10 mM. The above-mentioned several different interfering toxin standards were tested according to the detection system constructed in Example 1. The test results are as follows: Figure 4 As shown, it is shown that the method of the present invention has a great influence on Hg 2+ Has good selectivity. Example 3

[0020] A hydrogel-based fluorescent biosensor for rapid quantitative detection of Hg 2+ The preparation method and its practical application comprise the following steps: (1) Food sample processing: Take completely dried tea leaves and put them into a container to grind them into powder. Accurately weigh 5 g of tea powder and dissolve it in 100 mL of ultrapure water. Then, place it in a 90 °C water bath at a constant speed of 600 rpm / min for 30 min. After the solution cools to room temperature, place it in a centrifuge at 9000 rpm / min for 15 min to separate precipitated impurities. Then, filter it with a 0.45 μm syringe filter to obtain the required tea filtrate. The obtained tea filtrate is mixed with Hg 2+ Standard solution mixture, Hg 2+ The final concentrations were 10 mmol / mL, 30 mmol / mL, and 50 mmol / mL, respectively.

[0021] (2) Sample detection: Measure the RGB value according to the steps in Example 1 and substitute it into the standard curve to obtain the Hg 2+ concentration.

[0022] (3) When water is used as the actual sample for measurement, water from the laboratory tap is directly taken and the tap water sample is mixed with Hg 2+ Standard solution mixture, Hg 2+ The final concentrations were 10 mmol / mL, 30 mmol / mL and 50 mmol / mL, respectively. The Green value (G value) was determined according to steps (1) to (3) of Example 1, and the standard curve detected in Example 1 was used to obtain the Hg 2+ The concentration of each sample was measured three times and the average value was taken. The average recovery rate was calculated to be 96.37%~117.5%.

[0023] The prepared fluorescent biosensor was verified to be effective for Hg 2+ The detection showed high sensitivity, good selectivity, good reproducibility and stability, and real-time detection capability. The detection of actual samples showed that the prepared sensor has very good practical application value.

[0024] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

[0025] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A hydrogel-based fluorescent biosensor for rapid quantitative detection of Hg 2+ The preparation method is characterized in that The following steps are involved: (1) Construction of RCA-ThT fluorescent probe: Introduce template chain and primer into T4 buffer solution, amplify long DNA chain by RCA reaction, anneal, then add T4 DNA ligase, incubate overnight in PCR instrument, add phi29 polymerase and dNTP to obtain RCA / G-quadruplex product, fully disperse and dissolve the RCA / G-quadruplex product in neutral solution, incubate in water bath constant temperature oscillator, add ThT fluorescent dye, and obtain RCA-ThT fluorescent probe; (2) Preparation of hydrogel-based fluorescent biosensor: agarose powder was dissolved in a buffer solution and heated to boiling, and then cooled to room temperature to obtain a preliminary hydrogel; RCA-ThT fluorescent probe was added and poured into a culture dish to obtain DNA nanoflower hydrogel, the centrifuge tube cap was embedded in the DNA nanoflower hydrogel, and different concentrations of Hg 2+ Solution, the centrifuge tube is placed upside down to obtain the hydrogel-based fluorescent biosensor, which is ready for use; (3) Rapid quantitative detection of Hg using smartphones 2+ : After incubation, place the centrifuge tube cap with hydrogel in the detection instrument and irradiate it with ultraviolet light. At this time, the hydrogel will show different degrees of fluorescence. Use a smartphone to take a photo and upload it to the mini program to identify and obtain the RGB value. Based on the RGB value, Hg can be quickly obtained in a short time. 2+ The specific content.

2. A hydrogel-based fluorescent biosensor according to claim 1 for rapid quantitative detection of Hg 2+ The preparation method of the specific content is characterized in that In step (1), the neutral solution is one or more of ultrapure water, Tris-HCl buffer solution, and ethanol; the incubation time of the RCA / G-quadruplex product in a water bath constant temperature oscillator is 6 to 36 h; the ThT concentration is 0.1 to 10 mM; and the volume is 10 μL.

3. A hydrogel-based fluorescent biosensor according to claim 1 for rapid quantitative detection of Hg 2+ The preparation method of the specific content is characterized in that In step (2), the mass of agarose powder weighed is between 0.18 and 0.24 g; the buffer solution is one or more of Tris-HCl buffer solution, PBS buffer solution, acetate buffer solution, HEPES solution, and Tris-EDTA; the volume of the buffer solution used is between 20 and 25 mL; and the cooling time is 2 to 3 min.

4. A hydrogel-based fluorescent biosensor according to claim 1 for rapid quantitative detection of Hg 2+ The preparation method of the specific content is characterized in that In step (3), the shaking incubation time is 45 min to 90 min; the wavelength of the ultraviolet flashlight excitation light used is 365 nm.

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