CN110715967A - TiO2Photoelectric method for detecting adenosine triphosphate by-CdS-CdSe - Google Patents
TiO2Photoelectric method for detecting adenosine triphosphate by-CdS-CdSe Download PDFInfo
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- CN110715967A CN110715967A CN201911177441.8A CN201911177441A CN110715967A CN 110715967 A CN110715967 A CN 110715967A CN 201911177441 A CN201911177441 A CN 201911177441A CN 110715967 A CN110715967 A CN 110715967A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The invention discloses TiO2A method for detecting adenosine triphosphate by a photoelectric method of-CdS-CdSe. By TiO2The specific reaction of-CdS nano composite material modified ITO surface, adenosine triphosphate and aptamer-blocked probe combined release blocked probe, and the characteristic of utilizing the blocked probe to promote hybridization of h1 and h2 to form DNA nano-wire enable TiO to be prepared2The photoelectrochemical signal of the sensor is enhanced by the compounding of the-CdS-CdSe nanometer material, so that the ultrasensitive detection of adenosine triphosphate is realized. Compared with other traditional methods, the sensing system effectively reduces the detection limit of adenosine triphosphate, and has simple operation and high sensitivity.
Description
Technical Field
The invention relates to the technical field of adenosine triphosphate detection, in particular to TiO2The photoelectric method of-CdS-CdSe detects adenosine triphosphate.
Background
Adenosine triphosphate is a small molecule that is widely present in cells as a helper enzyme, and plays an important metabolic role in the metabolism of intracellular chemical energy. In addition, adenosine triphosphate is also considered as an indicator for monitoring cellular functions such as viability and damage. Thus, the detection of adenosine triphosphate has proven to be of significant biological significance.
Photoelectrochemical (PEC) analysis is an emerging and rapidly developing biometric detection technique that has attracted attention worldwide due to its advantages of simplicity of operation, inexpensive equipment, portability, and the like. Due to the different energy forms of the excitation source/light and the readout signal/electricity, the PEC method shows lower background signal and higher sensitivity compared to the traditional electrochemical method.
Disclosure of Invention
The invention aims to solve the technical problem of providing a portable high-sensitivity strong-specificity photoelectrochemical analysis detection method which can be easily realized in most laboratories without complex and fussy large-scale equipment support. The specific preparation scheme is as follows:
(1) preparation of TiO2CdS nanocomposite weighing 1 ~ 2 g TiO2 g separately2And 0.05 ~ 0.2.2 g CdCl2The powder is put in secondary water and stirred evenly, nitrogen is introduced into the solution for bubbling, 1M NaOH solution is slowly dripped to adjust the pH value of the solution to be alkaline, 20 ~ 60 ml of 20mM TAA is measured and added into the solution, the solution is refluxed for 2 ~ 5 hours at 80 ℃ under the protection of nitrogen, the solution is washed by absolute ethyl alcohol, centrifuged and washed by distilled water for a plurality of times, and finally TiO is obtained2the/CdS nano composite material is dissolved in secondary water and stored at 4 ℃ for later use;
(2) preparation of ITO-TiO2-a CdS electrode: cutting ITO glass into strips of 5 mm multiplied by 9 mm, then carrying out ultrasonic treatment in distilled water, acetone and ethanol in turn, and uniformly coating 2% of poly dimethyl diallyl ammonium chloride on the stripsOn the treated ITO glass, after 1 ~ 2 h, washing the ITO glass with distilled water for several times and drying in nitrogen atmosphere, and measuring 20 ~ 40 muL TiO2Dropwise adding the CdS nano composite material solution on the ITO surface, and standing for 1 ~ 2 h;
(3) synthesis of ITO-TiO2-CdS @ h1 complex: mixing ITO-TiO2-CdS electrode is immersed in 3 mM MPA solution at 4 ℃ for 5 ~ 8h, the electrode is rinsed with pH = 7.4 Tris-HCl buffer to remove unbound MPA, 10 ~ 25 μ L of 10 mM EDC and 20mM NHS solution is measured dropwise on the electrode surface to activate the modified carboxyl groups, 20 ~ 30 μ L of 1 μ M h1 probe solution is measured dropwise on the electrode surface and left at 4 ℃ for 10 ~ 15 h;
(4) preparation of CdSe nanomaterial by weighing 0.01 ~ 0.2.2 g of CdCl2Adding 0.05 ~ 0.1.1 mL of 3-mercaptopropionic acid into 30 mL of secondary water and stirring uniformly, adding 0.05 ~ mL of 3-mercaptopropionic acid into the solution and continuously stirring for 5 ~ 10 min to obtain a CdO precursor, slowly dropwise adding 1M NaOH solution to adjust the pH of the solution to be neutral, respectively weighing 0.05 ~ 0.2.2 g of Se powder and 0.3 ~ 0.8.8 g of sodium borohydride into the secondary water, stirring under a nitrogen atmosphere until transparent sol is obtained to obtain a NaHSe precursor, quickly injecting the NaHSe precursor into the CdO solution to obtain a CdSe nano material, dissolving the CdSe nano material into the secondary water and storing the CdSe nano material at 4 ℃ for later use;
(5) the CdSe @ h2 compound is synthesized by measuring 0.5 ~ 1 mL of 3 mM MPA solution in the CdSe solution and uniformly stirring at 4 ℃, measuring 10 ~ 25 μ L of 10 mM EDC and 20mM NHS solution and dropwise adding the solution on the surface of an electrode to activate modified carboxyl, measuring 20 ~ 30 μ L of 1 μ M h2 probe solution and dropwise adding the solution into the solution and keeping the solution at 4 ℃ for 12 hours;
(6) constructing an adenosine triphosphate ultrasensitive photoelectric detection sensor, measuring 2 ~ 5 microliter aptamer-enclosed probe solution, and dropwise adding the ITO-TiO prepared in the step (2)2Incubating the surface of the CdS electrode, dripping 8 ~ 10 muL adenosine triphosphate solution, 2 ~ 5 mu L h1 probe solution and 2 ~ 5 mu L h2 probe solution on the surface of the electrode for incubating when the solution is not completely dried, inserting the electrode into 20mM Tris-HCl buffer solution to detect a photoelectric signal, wherein the electrode treated in the step (6) is used as a working electrode, the counter electrode is a platinum wire electrode, the reference electrode is an Ag/AgCl electrode, the bias voltage value is 0V, and a xenon lamp is used as a xenon lampIs stimulated by light source.
The invention has the beneficial effects that:
(1) the method has the advantages of low cost, simple experimental operation and easily controlled reaction conditions;
(2) compared with the traditional large instrument, the sensor is portable, light in weight and low in cost;
(3) in TiO2Hybridization of h1 and h2 probes on the basis of-CdS nanocomposite to enable TiO2The recombination of CdS-CdSe nanomaterials further increases the photoelectrochemical signals of the sensor;
(4) compared with other traditional adenosine triphosphate detection methods, the detection limit of the sensor is lower, and the sensitivity is higher.
Detailed Description
The following is a description of detailed embodiments of the present invention: TiO22The photoelectric method of-CdS-CdSe detects adenosine triphosphate.
Example 1
(1) Preparation of TiO2-CdS nanocomposite: 2 g of TiO are weighed separately2And 0.15 g CdCl2The powder is put into 40 mL of secondary water and stirred uniformly, nitrogen is introduced into the dispersion liquid for bubbling for 15 min, 2 mL of 1M NaOH solution is added dropwise to adjust the pH value to 11, 40 mL of 20mM TAA is measured and added into the solution, the solution is refluxed at 80 ℃ for 2 h under the protection of nitrogen, the solution is treated by absolute ethyl alcohol, centrifuged at 8500 rpm for 10 min and washed by distilled water for 3 times, and finally TiO is obtained2the/CdS nano composite material is dissolved in secondary water and stored at 4 ℃ for later use.
(2) Preparation of ITO-TiO2-a CdS electrode: cutting ITO glass into small strips of 5 mm multiplied by 9 mm, then carrying out ultrasonic treatment in distilled water, acetone and ethanol in sequence, uniformly coating 30 mu L2% of poly dimethyl diallyl ammonium chloride 4 on the treated ITO glass, washing the ITO glass with distilled water for 3 times after 1 h and drying in a nitrogen atmosphere, and measuring 30 mu LTiO2Dropwise adding the-CdS nano composite material solution on the ITO surface, and standing for 1.5 h.
(3) Synthesis of ITO-TiO2-CdS @ h1 complex: mixing ITO-TiO2CdS electrode was immersed in 0.5 mL 4 ℃ 3 mM mcpa solution for 5h, the electrode was rinsed with 10 mM pH = 7.4 Tris-HCl buffer to remove unbound MPA, 25 μ L of 10 mM EDC and 20mM NHS solution was measured dropped on the electrode to activate the modified carboxyl group and held at room temperature for 50min, and 20 μ L of 1 μ M h1 probe solution was measured dropped on the electrode surface and held at 4 ℃ for 12 h.
(4) Preparing the CdSe nano material: 0.09 g of CdCl was weighed out2Uniformly stirring the solution in 30 mL of secondary water, measuring 0.056 mL of 3-mercaptopropionic acid, adding the solution into the solution, continuously stirring the solution for 5 min to obtain a CdO precursor, and slowly dropwise adding 2 mL of 1M NaOH solution to adjust the pH of the CdO precursor to 7; respectively weighing 0.06 g of Se powder and 0.38 g of sodium borohydride in 5 ml of secondary water, and stirring the solution under nitrogen atmosphere until transparent sol is obtained to obtain NaHSe precursor; and (3) quickly injecting the NaHSe precursor into the CdS solution to obtain the CdSe nano material, dissolving the CdSe nano material in secondary water, and storing the CdSe nano material at 4 ℃ for later use.
(5) Synthesis of CdSe @ h2 complex: 0.5 mL of 3 mM MPA solution was measured in CdSe solution and stirred at 4 ℃ for 5h, 25. mu.L of 10 mM EDC and 20mM NHS solution was measured and dropped to the electrode to activate the modified carboxyl group and kept at room temperature for 50min, and 20. mu.L of 1. mu. M h2 probe solution was measured and dropped to the above solution and kept at 4 ℃ for 12 h.
(6) Constructing an adenosine triphosphate ultrasensitive photoelectric detection sensor: measuring 5 mu L aptamer-enclosed probe solution, and dropwise adding the ITO-TiO prepared in the step (2)2Incubating the surface of the CdS electrode for 30min, dripping 10 mu L of adenosine triphosphate solutions with different concentrations, 5 mu L h1 probe solutions and 5 mu L h2 probe solutions on the surface of the electrode when the solutions are not completely dried, incubating for 30min, sequentially inserting the electrodes containing the adenosine triphosphate with different concentrations into 20mM Tris-HCl buffer solution to detect photoelectric signals, finding out a linear relation according to the change of the photoelectric signals, listing a linear equation and drawing a working curve; and (4) taking the electrode processed in the step (6) as a working electrode, taking the counter electrode as a platinum wire electrode, taking the reference electrode as an Ag/AgCl electrode, taking the bias voltage value as 0V, and taking a xenon lamp as a light source for stimulation.
Sequence listing
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Claims (1)
1.TiO2-photoelectric detection of adenosine triphosphate by CdS-CdSe, characterized in that it comprises the following steps:
(1) preparation of TiO2CdS nanocomposite weighing 1 ~ 2 g TiO2 g separately2And 0.05 ~ 0.2.2 g CdCl2The powder is put in secondary water andstirring uniformly, introducing nitrogen into the solution for bubbling, slowly dropwise adding 1M NaOH solution to adjust the pH value of the solution to be alkaline, measuring 20 ~ 60 ml of 20mM TAA, adding the 20mM TAA into the solution, refluxing for 2 ~ 5h at 80 ℃ under the protection of nitrogen, washing the solution with absolute ethyl alcohol, centrifuging, washing with distilled water for several times, and finally obtaining TiO2the/CdS nano composite material is dissolved in secondary water and stored at 4 ℃ for later use;
(2) preparation of ITO-TiO2Cutting ITO glass into strips of 5 mm multiplied by 9 mm, then carrying out ultrasonic treatment in distilled water, acetone and ethanol in sequence, uniformly coating 2% of poly dimethyl diallyl ammonium chloride on the treated ITO glass, washing the ITO glass with distilled water for a plurality of times after 1 ~ 2 h, drying in nitrogen atmosphere, measuring 20 ~ 40 mu L TiO22Dropwise adding the CdS nano composite material solution on the ITO surface, and standing for 1 ~ 2 h;
(3) synthesis of ITO-TiO2-CdS @ h1 complex: mixing ITO-TiO2-CdS electrode is immersed in 3 mM MPA solution at 4 ℃ for 5 ~ 8h, the electrode is rinsed with pH = 7.4 Tris-HCl buffer to remove unbound MPA, 10 ~ 25 μ L of 10 mM EDC and 20mM NHS solution is measured dropwise on the electrode surface to activate the modified carboxyl groups, 20 ~ 30 μ L of 1 μ M h1 probe solution is measured dropwise on the electrode surface and left at 4 ℃ for 10 ~ 15 h;
(4) preparation of CdSe nanomaterial by weighing 0.01 ~ 0.2.2 g of CdCl2Adding 0.05 ~ 0.1.1 mL of 3-mercaptopropionic acid into 30 mL of secondary water and stirring uniformly, adding 0.05 ~ mL of 3-mercaptopropionic acid into the solution and continuously stirring for 5 ~ 10 min to obtain a CdO precursor, slowly dropwise adding 1M NaOH solution to adjust the pH of the solution to be neutral, respectively weighing 0.05 ~ 0.2.2 g of Se powder and 0.3 ~ 0.8.8 g of sodium borohydride into the secondary water, stirring under a nitrogen atmosphere until transparent sol is obtained to obtain a NaHSe precursor, quickly injecting the NaHSe precursor into the CdO solution to obtain a CdSe nano material, dissolving the CdSe nano material into the secondary water and storing the CdSe nano material at 4 ℃ for later use;
(5) the CdSe @ h2 compound is synthesized by measuring 0.5 ~ 1 mL of 3 mM MPA solution in the CdSe solution and uniformly stirring at 4 ℃, measuring 10 ~ 25 μ L of 10 mM EDC and 20mM NHS solution and dropwise adding the solution on the surface of an electrode to activate modified carboxyl, measuring 20 ~ 30 μ L of 1 μ M h2 probe solution and dropwise adding the solution into the solution and keeping the solution at 4 ℃ for 12 hours;
(6) constructing an adenosine triphosphate ultrasensitive photoelectric detection sensor, measuring 2 ~ 5 microliter aptamer-enclosed probe solution, and dropwise adding the ITO-TiO prepared in the step (2)2And (4) incubating the surface of the CdS electrode, dripping 8 ~ 10 muL adenosine triphosphate solution, 2 ~ 5 mu L h1 probe solution and 2 ~ 5 mu L h2 probe solution on the surface of the electrode for incubating when the solution is not completely dried, inserting the electrode into 20mM Tris-HCl buffer solution to detect a photoelectric signal, wherein the electrode treated in the step (6) is used as a working electrode, the counter electrode is a platinum wire electrode, the reference electrode is an Ag/AgCl electrode, the bias voltage value is 0V, and a xenon lamp is used as a light source for stimulation.
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| CN114250224A (en) * | 2021-12-16 | 2022-03-29 | 福建和瑞基因科技有限公司 | Nucleic acid composition for extracting or detecting small-molecule RNA in sample, kit and method thereof |
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| CN114324264B (en) * | 2021-11-24 | 2023-07-21 | 中国科学院理化技术研究所 | Fluorescence ratio sensor for detecting ATP (adenosine triphosphate) and preparation method and application thereof |
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