A photoelectrochemical aptamer sensor for detecting potassium ions and its preparation method

Abstract

The present invention discloses a photoelectrochemical aptamer sensor for detecting potassium ions and a preparation method thereof. The sensor is based on Bi2S3 and CeO2. After the two are combined to form a composite material, due to the band gap matching between the materials, the visible light absorption is enhanced, the charge transfer rate is accelerated, and the photoelectrochemical response is significantly improved; the selected G-rich aptamer chain is sensitive to K + Specific recognition, K + When there is a G-rich aptamer chain and K + The formation of a quadruplex conformation forms a weak conductive layer on the electrode. The steric effect and the interface properties of the weak conductive layer have a significant impact on electron transfer, resulting in a decrease in the photocurrent response. + The method is simple, stable and easy to operate.

Description

Technical Field

[0001] The present invention relates to a method for detecting K + The present invention relates to a photoelectrochemical aptamer sensor and a preparation method thereof, and more specifically to a photoelectrochemical aptamer sensor based on Bi2S3 and CeO2 stepwise modified electrodes. Background Art

[0002] Guanine-rich DNA sequences can serve as aptamers. These sequences contain two or more G-tracts and loop regions, potentially forming G-rich quadruplexes. These structures are stabilized by Hoogsteenh bonds and central cations. Using the SELEX (systematic evolution of ligands by exponential enrichment) method, several guanine-rich DNA sequences, designated aptamers, were screened in vitro and found to exhibit specificity and high binding affinity for a variety of targets, including metal cations, small organic molecules, and proteins. Recently, photoelectrochemical biosensors based on G-rich aptamers have been extensively developed, with functional G-rich aptamers such as Hum, PW17, AGRO100, T30695, and PS2 being particularly widely used.

[0003] Photoelectrochemical (PEC) analysis, as an emerging detection technology, has developed rapidly. In the PEC detection process, light is used as the excitation signal and electricity as the response signal. The combination of light and electricity, two different substances, enables PEC technology to have the advantages of both electrochemical and optical methods, including fast response, simple operation, low instrument cost and high detection sensitivity. Therefore, PEC technology is widely used in biological analysis. However, the low light energy utilization rate and poor biocompatibility of traditional photoelectrochemical materials have limited their application in life analytical chemistry. Therefore, finding new photoelectrochemical electrode materials that are efficient, low-cost and biocompatible remains an important research goal for the construction of photoelectrochemical immunosensors.

[0004] As a metal sulfide, Bi2S3 has attracted widespread attention due to its wide visible light response range, simple preparation, high biocompatibility, and low toxicity. It can absorb light in the entire visible spectrum, accounting for about 45% of the solar spectrum. In addition, Bi2S3 has a wavelength of 104-105 cm in the visible light range. -1absorption coefficient and photoelectric conversion efficiency of about 5%. More importantly, the band gap of Bi2S3 is relatively narrow, varying between 1.3 and 1.7 eV, and the morphology is adjustable. However, the narrow band gap causes the electron holes in Bi2S3 to recombine rapidly, reducing the PEC response and resulting in low detection sensitivity. In order to overcome this challenge, different methods have been adopted, such as element doping, conductive material modification, and heterojunction construction. CeO2, as a typical metal oxide, is a common n-type semiconductor. Due to its good biocompatibility, stability, strong light collection ability and unique electronic structure, it has been widely used in biomedicine, photocatalysis and photovoltaic cells. In addition, because it has a wide band gap (2.5 eV), the recombination of electron holes is effectively improved after CeO2 is compounded with Bi2S3, thereby improving the PEC response effect.

[0005] Potassium is one of the essential elements for the human body. It plays an important role in maintaining extracellular permeability and regulating the concentration of other ions in the body. In particular, as a blood electrolyte, it has a direct impact on the cardiovascular system. Changes in the extracellular potassium level in the blood may lead to hyperkalemia or hypokalemia, causing arrhythmia and ultimately threatening heart function and blood pressure regulation. Therefore, it is necessary to develop a detection system that can reliably detect this ion. To date, there have been a lot of studies on K + Although studies on photoelectrochemical biosensing have been reported, photoelectrochemical biosensing systems are limited to a few examples. Summary of the Invention

[0006] In order to solve the above-mentioned shortcomings of the prior art, the present invention provides a detection method based on Bi2S3 and CeO2 step-by-step modification. + The photoelectrochemical aptamer sensor and its preparation method are used to realize the photoelectrochemical biosensor with high photocurrent response and high stability to detect K + Rapid and sensitive detection.

[0007] The present invention solves the technical problem by adopting the following technical solution:

[0008] The present invention first discloses a method for detecting K + The photoelectrochemical aptamer sensor is characterized in that: the photoelectrochemical aptamer sensor is to gradually add a dispersion of Bi2S3 material and CeO2 material on the surface of an ITO glass electrode, then add a chitosan solution, and finally incubate a G-rich aptamer on the electrode by electrostatic adsorption. The G-rich aptamer can bind to the target K +A specific recognition reaction occurs. The photoelectrochemical aptamer sensor prepared by the present invention uses Bi2S3 and CeO2 as photoelectroactive materials, which are typical metal sulfides and metal oxides respectively. After the two are combined to form a composite material, due to the band gap matching between the materials, the visible light absorption is enhanced, the charge transfer rate is accelerated, and the photoelectrochemical response is significantly improved. The selected G-rich aptamer chain (name: Hum; base sequence: 5'-GGGTTAGGGTTAGGGTTAGGG-3') is sensitive to K + Specific recognition, in K + When there is a G-rich aptamer chain and K + The formation of a quadruplex conformation forms a weakly conductive layer on the electrode. The steric effect and the interfacial properties of the weakly conductive layer have a significant impact on electron transfer, resulting in a decrease in the photocurrent response.

[0009] Furthermore, the preparation method of the Bi2S3 material is as follows: 0.08 mmol of Bi(NO)3·5H2O and 0.14 mmol of Na2S·9H2O are respectively dissolved in 20 mL of ethanol; then the dissolved Bi(NO)3 solution is poured into the Na2S solution, and the mixed solution is continuously stirred for 2 hours. The product is washed with deionized water and ethanol in sequence, and then placed in a 60°C oven for drying to obtain the Bi2S3 material.

[0010] Furthermore, the preparation method of the CeO2 material is as follows: weigh 1 mmol of Ce(NO)3·6H2O and 1 mmol of 1,2,4,5-benzenetetracarboxylic acid in 20 mL of ethanol, stir until dissolved, and then put into a reactor to react at 80°C for 1 hour; after the reaction is completed, the product is alternately centrifuged and washed three times with deionized water and ethanol, and finally dried at 60°C; the dried product is fully ground and calcined at 600°C for 120 minutes to obtain the CeO2 material.

[0011] The detection K of the present invention + The preparation method of the photoelectrochemical aptamer sensor is carried out according to the following steps:

[0012] Step 1: ultrasonically clean the ITO glass electrode with water and ethanol in sequence, and then dry it at 60°C for later use;

[0013] Step 2: Add 3 mg of Bi2S3 to 3 mL of deionized water and disperse them ultrasonically for 1.5 h to obtain a Bi2S3 dispersion; add 5 mL of deionized water to 5 mg of CeO2 and disperse them ultrasonically for 1 h to obtain a CeO2 dispersion;

[0014] Step 3, evenly add 25 μL to 30 μL of Bi2S3 dispersion onto the surface of the ITO glass electrode cleaned and dried in step 1, and dry it at room temperature; then add 30 μL to 35 μL of CeO2 dispersion to obtain a Bi2S3 / CeO2 stepwise modified electrode;

[0015] Step 4: dissolving chitosan in glacial acetic acid to obtain a chitosan solution with a mass concentration of 0.2%; diluting the G-rich aptamer to 1 μM to 1.2 μM with TE buffer to obtain a G-rich aptamer solution;

[0016] 8 μL of chitosan solution was added to the surface of the Bi2S3 / CeO2 stepwise modified electrode prepared in step 3. Chitosan was used to immobilize the aptamer. Then 30 μL of G-rich aptamer solution was added to the surface and incubated at 4°C for 12 h. After the incubation, the electrode was removed and rinsed with TE buffer with a pH of 7.2-7.4 to obtain the detection K + Photoelectrochemical aptasensors.

[0017] The above aptamer sensor was used to detect K + The method is:

[0018] Step A: add 30 μL of K to be tested to the surface of the photoelectrochemical aptamer sensor. + Solution, incubate at 37℃ for 40-50min; rinse slowly with TE buffer to obtain the electrode to be tested, and let it stand for use;

[0019] Step B: The electrode to be tested obtained in step 1 is subjected to photoelectrochemical testing in a PBS buffer solution containing 0.1 mol / L AA and a pH of 7.4 to obtain the K + The photocurrent response value of the solution is calculated by comparing the photocurrent response value with K + The standard relationship curve of concentration is used to determine the K + K in solution + concentration.

[0020] Furthermore, the standard relationship curve is obtained by combining a series of standard K + The solutions (0 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL and 10 ng / mL) were prepared into electrodes according to step A, and then the photoelectrochemical test was performed according to step B to obtain the K + The photocurrent response value corresponding to the sample is then expressed in K + The logarithm of the sample concentration is the horizontal axis, and the photocurrent response value is the vertical axis. Figure 1As shown, the standard relationship curve is I (μA) = -5.56lgC + 57.87. The test shows that when K + The photocurrent response value increases with K in the sample concentration range of 10fg / mL to 10ng / mL. + The detection limit was 0.4 pg / mL.

[0021] The photoelectrochemical test is a three-electrode system with the photoelectrochemical aptamer sensor as the working electrode, the Pt electrode as the counter electrode, and the saturated silver chloride electrode as the reference electrode. A 250W xenon lamp is used as the light source, the wavelength range is 280-1000nm, the external voltage is 0V, and the current changes are recorded using a CHI660D electrochemical workstation.

[0022] Compared with the prior art, the beneficial effects of the present invention are embodied in:

[0023] 1. The present invention realizes K + The detection method is simple, with a wide detection range, high sensitivity and easy operation;

[0024] 2. The present invention is effective for K + The detection method requires a small amount of sample and has low detection cost;

[0025] 3. The present invention prepares a photoelectrochemical aptamer sensor by stepwise modification of Bi2S3 and CeO2, which has a high photocurrent response value, good biocompatibility, and excellent stability.

[0026] 4. The present invention combines Bi2S3 and CeO2 to prepare Bi2S3 and CeO2 step-by-step modified electrodes, which expands the spectral absorption range and improves the photoelectric conversion efficiency. It is a new strategy for preparing photoelectrochemical biosensors. + The specific binding of the two molecules improves the sensitivity and specificity, providing a new idea for clinical diagnosis and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The present invention is to analyze the concentrations of K of 10fg / mL, 100fg / mL, 1pg / mL, 10pg / mL, 100pg / mL, 1ng / mL and 10ng / mL respectively. + The standard relationship curve obtained by photoelectrochemical testing of standard samples.

[0028] Figure 2 These are the transmission electron microscopy (TEM) characterization results of Bi2S3 in the present invention.

[0029] Figure 3This is the scanning electron microscope (SEM) characterization result of CeO2 in the present invention.

[0030] Figure 4 These are the X-ray diffraction (XRD) characterization results of Bi2S3 and CeO2 in the present invention. DETAILED DESCRIPTION

[0031] To make the above-mentioned objects, features, and advantages of the present invention more clearly understood, the following detailed description of specific embodiments of the present invention is provided in conjunction with the accompanying drawings. The following is merely an example and illustration of the concept of the present invention. Any modification, supplement, or substitution of the described specific embodiments by a person skilled in the art without departing from the concept of the invention or exceeding the scope defined by the claims shall fall within the scope of protection of the present invention.

[0032] The Bi2S3 material used in the following examples was prepared by dissolving 0.08 mmol of Bi(NO)3·5H2O and 0.14 mmol of Na2S·9H2O in 20 mL of ethanol. The dissolved Bi(NO)3 solution was then poured into the Na2S solution, and the mixture was stirred for 2 hours. The product was washed three times with deionized water and then ethanol, followed by drying in a 60°C oven to obtain the Bi2S3 material for future use.

[0033] The CeO2 material used in the following examples was prepared as follows: 1 mmol of Ce(NO)3·6H2O and 1 mmol of 1,2,4,5-benzenetetracarboxylic acid were weighed and dissolved in 20 mL of ethanol. The mixture was stirred for 10 minutes to dissolve the raw materials. The stirred solution was placed in a reactor and reacted at 80°C for 1 hour. After the reaction, the product was washed three times by alternating centrifugation with deionized water and ethanol, and finally dried at 60°C. The dried product was thoroughly ground and calcined at 600°C for 120 minutes. The resulting product was the CeO2 material required for this experiment.

[0034] The TE buffer solution with a pH of 7.2 to 7.4 and the G-rich aptamers used in the following examples were purchased from Shanghai Sangon Biotechnology Service Co., Ltd.; the PBS buffer solution with a pH of 7.4 and 0.1 mol / L ascorbic acid (LAA) used in the following examples was prepared as follows: 1.1496 g of Na2HPO4, 0.2964 g of NaH2PO4·2H2O, 0.8 g of NaCl, and 1.7613 g of AA were weighed and prepared into 100 mL of aqueous solution.

[0035] Example 1

[0036] In this embodiment, the detection K is first prepared according to the following steps + Photoelectrochemical aptasensors:

[0037] Step 1: ultrasonically clean the ITO glass electrode with water and ethanol four times, and then dry it at 60°C for later use;

[0038] Step 2: Add 3 mg of Bi2S3 to 3 mL of deionized water and ultrasonically disperse for 1.5 h to obtain a Bi2S3 dispersion; add 5 mL of deionized water to 5 mg of CeO2 and ultrasonically disperse for 1 h to obtain a CeO2 dispersion.

[0039] Step 3: Evenly add 30 μL of Bi2S3 dispersion onto the surface of the ITO glass electrode cleaned and dried in step 1 and let it dry at room temperature. Then, add 30 μL of CeO2 dispersion onto the surface of the electrode to obtain a Bi2S3 / CeO2 graded modified electrode.

[0040] Step 4: dissolving chitosan in glacial acetic acid to obtain a chitosan solution with a mass concentration of 0.2%; diluting the G-rich aptamer to 1.2 μM with TE buffer at a pH of 7.2 to 7.4 to obtain a G-rich aptamer solution.

[0041] 8 μL chitosan solution was added to the surface of the modified electrode prepared in step 3. Then 30 μL 1.2 μM G-rich aptamer solution was added to the surface and incubated at 4°C for 12 h. After the incubation, the electrode was removed and rinsed with TE buffer solution with a pH of 7.2-7.4 to obtain the detection K + The photoelectrochemical aptamer sensor was kept aside for future use.

[0042] The above aptamer sensor was used to detect K + The method is:

[0043] Step A: Add 30 μL of K to be tested to the surface of the photoelectrochemical aptamer sensor. + The solution was incubated at 37°C for 40 minutes. After slowly rinsing with TE buffer solution, the electrode to be tested was obtained and allowed to stand for use.

[0044] Step B: The electrode to be tested obtained in step 1 is subjected to photoelectrochemical testing in a PBS buffer solution containing 0.1 mol / L AA and a pH of 7.4 to obtain the K + The photocurrent response value of the solution is calculated by comparing the photocurrent response value with K + The standard relationship curve of concentration is used to determine the K + K in solution + concentration.

[0045] To verify the feasibility of the method of this embodiment, the K +The concentrations of the samples were detected and calculated using the aptamer sensor of this embodiment according to the above method, which were 1.18 ng / mL, 11.09 ng / mL and 99.59 ng / mL, respectively. It can be seen that the prepared aptamer sensor has a good sensitivity to the target K + There are fast, sensitive, accurate and efficient tests.

[0046] Example 2

[0047] In this example, the steps of "adding 30 μL Bi2S3 dispersion" and "adding 30 μL CeO2 dispersion" in step 3 of the preparation method of the photoelectrochemical aptamer sensor in Example 1 were changed to "adding 25 μL Bi2S3 dispersion" and "adding 35 μL CeO2 dispersion". The remaining conditions and steps were the same as those in Example 1. The morphology and properties of the obtained aptamer sensor were similar to those of the aptamer sensor obtained in Example 1. + The samples were tested and similar test results were obtained.

[0048] Example 3

[0049] In this example, the "1.2 μM G-rich aptamer" in step 4 of the photoelectrochemical aptamer sensor preparation method of Example 1 was changed to "1 μM G-rich aptamer". The remaining conditions and steps were the same as those of Example 1. The morphology and properties of the obtained aptamer sensor were similar to those of the aptamer sensor obtained in Example 1. + The samples were tested and similar test results were obtained.

[0050] Example 4

[0051] In this example, the aptamer sensor of Example 1 detects K + The "incubation for 40 min" in step A of the method was changed to "incubation for 50 min", and the other conditions and steps were the same as those in Example 1. The morphology and properties of the obtained aptamer sensor were similar to those of the aptamer sensor obtained in Example 1. + The samples were tested and similar test results were obtained.

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A photoelectrochemical aptamer sensor for detecting potassium ions, characterized in that: The photoelectrochemical aptamer sensor is to gradually add a dispersion of Bi2S3 material and CeO2 material on the surface of an ITO glass electrode, then add a chitosan solution, and finally incubate the G-rich aptamer on the electrode by electrostatic adsorption. The G-rich aptamer can bind to the target K + A specific recognition reaction occurs.

2. The photoelectrochemical aptasensor according to claim 1, wherein: The preparation method of the Bi2S3 material is as follows: 0.08 mmol of Bi(NO)3·5H2O and 0.14 mmol of Na2S·9H2O are respectively dissolved in 20 mL of ethanol; then the dissolved Bi(NO)3 solution is poured into the Na2S solution, and the mixed solution is continuously stirred for 2 hours. The product is washed with deionized water and ethanol in sequence, and then placed in a 60°C oven for drying to obtain the Bi2S3 material.

3. The photoelectrochemical aptasensor according to claim 1, wherein: The preparation method of the CeO2 material is as follows: weighing 1 mmol of Ce(NO)3·6H2O and 1 mmol of 1,2,4,5-benzenetetracarboxylic acid in 20 mL of ethanol, stirring until dissolved, and then placing the mixture in a reactor at 80°C for reaction for 1 hour; after the reaction is completed, the product is alternately centrifuged and washed three times with deionized water and ethanol, and finally dried at 60°C; the dried product is fully ground and calcined at 600°C for 120 minutes to obtain the CeO2 material.

4. A method for preparing the photoelectrochemical aptasensor according to any one of claims 1 to 3, characterized in that: The steps include: Step 1: ultrasonically clean the ITO glass electrode with water and ethanol in sequence, and then dry it at 60°C for later use; Step 2: Add 3 mg of Bi2S3 to 3 mL of deionized water and disperse them ultrasonically for 1.5 h to obtain a Bi2S3 dispersion; add 5 mL of deionized water to 5 mg of CeO2 and disperse them ultrasonically for 1 h to obtain a CeO2 dispersion; Step 3, evenly add 25 μL to 30 μL of Bi2S3 dispersion onto the surface of the ITO glass electrode cleaned and dried in step 1, and dry it at room temperature; then add 30 μL to 35 μL of CeO2 dispersion to obtain a Bi2S3 / CeO2 stepwise modified electrode; Step 4: dissolving chitosan in glacial acetic acid to obtain a chitosan solution with a mass concentration of 0.2%; diluting the G-rich aptamer to 1 μM to 1.2 μM with TE buffer to obtain a G-rich aptamer solution; 8 μL chitosan solution was added to the surface of the Bi2S3 / CeO2 stepwise modified electrode prepared in step 3, and then 30 μL G-rich aptamer solution was added to the surface and incubated at 4°C for 12 h. After the incubation, the electrode was taken out and rinsed with TE buffer with a pH of 7.2-7.4 to obtain the detection K + Photoelectrochemical aptasensors.

5. A method for detecting K using the photoelectrochemical aptamer sensor according to any one of claims 1 to 3 + The method is characterized in that The steps include: Step A: add 30 μL of K to be tested to the surface of the photoelectrochemical aptamer sensor. + Solution, incubate at 37℃ for 40-50min; rinse slowly with TE buffer to obtain the electrode to be tested, and set aside for use; Step B: The electrode to be tested obtained in step 1 is subjected to photoelectrochemical testing in a PBS buffer solution containing 0.1 mol / L AA and a pH of 7.4 to obtain the K + The photocurrent response value of the solution is calculated by comparing the photocurrent response value with K + The standard relationship curve of concentration is used to determine the K + K in solution + concentration.

6. The detection method according to claim 5 + The method is characterized in that: The standard relationship curve is obtained by adding a series of standard K + After the solution is prepared into the electrode to be tested according to step A, the photoelectrochemical test is performed according to step B to obtain the K + The photocurrent response value corresponding to the sample is then expressed in K + The logarithm of the sample concentration is the horizontal axis, and the photocurrent response value is the vertical axis for fitting.

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