WO2019010930A1 - Up-conversion material-based photoelectrochemical dna sensor and detection method therefor - Google Patents

Up-conversion material-based photoelectrochemical dna sensor and detection method therefor Download PDF

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WO2019010930A1
WO2019010930A1 PCT/CN2018/070010 CN2018070010W WO2019010930A1 WO 2019010930 A1 WO2019010930 A1 WO 2019010930A1 CN 2018070010 W CN2018070010 W CN 2018070010W WO 2019010930 A1 WO2019010930 A1 WO 2019010930A1
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conversion material
photoelectrochemical
ssdna
dna sensor
electrode
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PCT/CN2018/070010
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French (fr)
Chinese (zh)
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林鹏
刘红
陶文艳
宋家俊
胡进
柯善明
曾燮榕
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深圳大学
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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
    • G01N27/416—Systems
    • 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
    • G01N27/3276—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a hybridisation with immobilised receptors

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  • the invention relates to the field of biological material detection, in particular to a photoelectrochemical DNA sensor based on an up-conversion material and a detection method thereof.
  • Photoelectrochemical is a commonly used detection method in the field of biosensing. It has good effects in detecting ions, glucose, enzymes, DNA and antigen antibodies.
  • the commonly used PEC detection methods have certain limitations.
  • the light source commonly used in the process of detecting DNA by the PEC method is ultraviolet light, and the ultraviolet light excitation causes a certain damage to the DNA structure, thereby causing the DNA concentration detection result not to be accurate;
  • the up-conversion material is a luminescent material that emits visible light under infrared light excitation, that is, a material that converts infrared light into visible light, which is characterized in that the absorbed photon energy is lower than the emitted photon energy, which is contrary to Stokes' law, and thus It is called anti-Stokes law; the up-conversion material also has many applications in bio-fluorescence detection.
  • the up-conversion material can be immobilized on the DNA analyte to detect the DNA substance by detecting the fluorescence intensity.
  • this method of determining the concentration of DNA to be tested by fluorescence intensity has many problems such as many interference factors, long experimental period, and inaccurate results.
  • the object of the present invention is to provide a photoelectrochemical DNA sensor based on an up-conversion material and a detection method thereof, aiming at solving the inaccuracy of the prior art in detecting DNA concentration, the experimental period is long, and the interference factor More questions.
  • a photoelectrochemical DNA sensor based on an up-conversion material for detecting a concentration of a target ssDNA wherein the photoelectrochemical DNA sensor comprises an electrolytic cell containing an electrolyte, a reference electrode disposed inside the electrolytic cell, a counter electrode, and a working electrode, on which the photoelectrically active material layer is assembled, on which the probe ssDNA is fixedly disposed, the up-converting material is connected to the target ssDNA, and the target ssDNA is mixed with the probe ssDNA After the change, the photocurrent is changed, and the detection of ssDNA of different concentration targets is realized.
  • the photoelectrochemical DNA sensor based on an up-conversion material wherein the photoelectric active material layer is composed of one or more of a quantum dot material, TiO 2 , graphene or phthalocyanine.
  • the photoelectrochemical DNA sensor based on the up-conversion material wherein the reference electrode is one of an Ag/AgCl reference electrode, an Hg/HgCl reference electrode, or an Hg/HgO reference electrode.
  • the photoelectrochemical DNA sensor based on an up-conversion material, wherein the counter electrode is a platinum electrode.
  • the photoelectrochemical DNA sensor based on the up-conversion material wherein the substrate of the working electrode is one of ITO, FTO or AZO.
  • the photoelectrochemical DNA sensor based on the up-conversion material wherein the up-conversion material is one of NaYF4: Yb, Er, NaYF4: Yb, Tm or NaYF4: Yb, Ho.
  • a method for detecting a photoelectrochemical DNA sensor based on an up-conversion material comprising the steps of:
  • the target ssDNA to be tested is marked with an up-conversion material in advance;
  • the target ssDNA is dropped on the working electrode fixed with the probe ssDNA for hybridization reaction
  • the working electrode after the reaction is placed in an electrolytic cell, and the current-time curve is measured under the irradiation of infrared light, and the concentration of the target ssDNA is obtained according to the change of the current.
  • the method for detecting a photoelectrochemical DNA sensor based on an up-conversion material wherein the conditions of the hybridization reaction in the step B are: infiltrating the working electrode in a 20 mM MgCl 2 solution, and then dropping the target ssDNA to be tested, The reaction was carried out at 30-40 ° C for 40-100 min.
  • step B further comprises: rinsing the reacted working electrode with a PBS solution having a concentration of 10 mM.
  • the method for detecting a photoelectrochemical DNA sensor based on an up-conversion material wherein the infrared light has a wavelength ranging from 950 to 1000 nm.
  • the present invention comprises: assembling a photoactive material layer on a working electrode, and fixing a probe ssDNA on the photoelectric active material layer, and additionally modifying the up-conversion material to the target ssDNA; when the target ssDNA and the probe ssDNA occur After the hybridization reaction, a fluorescence resonance energy transfer effect (FRET) occurs between the up-conversion material and the photo-active material layer under the illumination of the infrared light wavelength, so the detected photocurrent changes; different concentrations of the up-conversion material The intensity of the generated fluorescence is different, and the photocurrent generated by the excitation is also different, thereby detecting different concentrations of DNA.
  • the photoelectrochemical DNA sensor based on the up-conversion material provided by the invention has high sensitivity, simple operation, low equipment requirement, short detection time, wide application range and good prospect.
  • FIG. 1 is a schematic structural view of a preferred embodiment of a photoelectrochemical DNA sensor based on an up-conversion material according to the present invention
  • FIG. 2 is a schematic diagram showing the principle of a photoelectrochemical DNA sensor detection method based on an up-conversion material according to the present invention
  • FIG. 3 is a flow chart of a preferred embodiment of a photoelectrochemical DNA sensor detection method based on an up-conversion material according to the present invention
  • Figure 4 is a schematic diagram showing the detection results of different concentrations of target ssDNA of the present invention.
  • FIG. 5 is an I-T graph of a device in which a working electrode is modified with CdS QDs under illumination in Embodiment 1 of the present invention
  • Figure 6 is a graph showing the I-T curve before and after DNA hybridization in the present invention.
  • the present invention provides a photoelectrochemical DNA sensor based on an up-conversion material and a method for detecting the same, and the present invention will be further described in detail below in order to make the objects, technical solutions and effects of the present invention more clear and clear. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
  • the present invention provides a photoelectrochemical DNA sensor based on an up-conversion material for detecting the concentration of a target ssDNA, as shown in the figure, wherein the photoelectrochemical DNA sensor comprises an electrolyte 10 An electrolytic cell 20, a reference electrode 30 disposed inside the electrolytic cell 20, a counter electrode 40, and a working electrode 50, on which the photovoltaic active material layer 60 is assembled, and the photoelectric active material layer 60 is fixedly disposed
  • the probe ssDNA70, the up-conversion material 80 is linked to the target ssDNA90, and the target ssDNA90 is hybridized with the probe ssDNA70 to cause a change in photocurrent, and detection of different concentrations of the target ssDNA90 is achieved.
  • the conventional PEC method generally uses ultraviolet light in the process of detecting living substances (DNA, protein, etc.), and ultraviolet light easily damages the structure of the living substance during the process of exciting the irradiation, destroying the chemical structure thereof. , resulting in inaccurate detection results.
  • the present invention fixes a known concentration of target ssDNA on the photoactive layer by assembling a photoactive material layer on the working electrode, and additionally modifies the target ssDNA to be measured by the up-conversion material, and the modified The target ssDNA is added to the target ssDNA for hybridization reaction. After the reaction is completed, the fluorescence resonance energy transfer effect (FRET) is generated between the up-converting nanoparticles and the photoelectric active material under the irradiation of infrared light, so the detected The photocurrent will change.
  • FRET fluorescence resonance energy transfer effect
  • the present invention establishes a three-electrode test system consisting of working electrodes, reference electrodes and counter electrodes to detect the magnitude of photocurrent; up-conversion nanometers with different concentrations
  • the fluorescence intensity produced by the particles is different, and the photocurrent generated by the excitation is also different, thereby detecting different concentrations of DNA.
  • This new detection method has high sensitivity, simple operation, low equipment requirements, miniaturization and batch detection, wide application range and good prospects.
  • the reference electrode is one of an Ag/AgCl reference electrode, an Hg/HgCl reference electrode or a Hg/HgO reference electrode;
  • the counter electrode is a platinum electrode;
  • the substrate of the electrode is one of ITO, FTO or AZO, and the photoelectrically active material layer on the substrate is composed of one or more of a quantum dot material, TiO 2 , graphene or phthalocyanine; preferably, preferably CdS quantum dots are used as photoelectrically active materials.
  • the photoelectrochemical DNA sensor based on the up-conversion material provided by the invention is a novel biosensing technology for detecting DNA, and after fixing the probe ssDNA on the photoelectric active material layer, the hybridization is performed by different concentrations and modified with the up-conversion material.
  • the target ssDNA is used to cause different increases in photocurrent, and the detection of different concentrations of target ssDNA is achieved by measuring changes in photocurrent.
  • the present invention also provides a detection method of a photoelectrochemical DNA sensor based on an up-conversion material, as shown in FIG. 3, which includes the steps of:
  • the up-conversion material is one of NaYF4:Yb, Er, NaYF4:Yb, Tm or NaYF4:Yb, Ho
  • the up-conversion material provided by the invention is composed of an inorganic matrix and rare earth doped therein.
  • Hetero-ion composition taking NaYF4:Yb,Er as an example, when bismuth double doping, Er as activator, Yb as sensitizer, NaYF4 is the most efficient matrix material for up-conversion luminescence; the up-conversion material can exist
  • the present invention is preferably in the form of spherical particles, the particle size of which can be changed, and the fluorescence wavelength can also be changed.
  • the target ssDNA is dropped on the working electrode fixedly provided with the probe ssDNA to perform a hybrid reaction
  • a target ssDNA with different concentrations of the up-conversion material is added to the working electrode, and the target ssDNA with the up-converting material is immobilized on the working electrode by hybridization between the DNA; further, the hybridization reaction
  • the process is as follows: the working electrode is infiltrated in a 20 mM MgCl 2 solution, and the target ssDNA is added dropwise, and the reaction is carried out at 30-40 ° C for 40-100 min; after the reaction is completed, the reaction is carried out by using a PBS solution having a concentration of 10 mM. The working electrode is rinsed to remove unhybridized target ssDNA.
  • the working electrode after the reaction is placed in an electrolytic cell, and the current-time curve is measured under irradiation of infrared light, and the concentration of the target ssDNA is obtained according to the change of the current.
  • the working electrode after the hybridization reaction is placed in an electrolytic cell, and the photocurrent is measured under infrared light having a wavelength range of 950-1000 nm, due to the relationship between the up-conversion material and the photoelectric active material (CdS QDs).
  • the fluorescence resonance energy transfer effect increases the photocurrent.
  • the present invention detects different concentrations of target ssDNA by detecting changes in photocurrent with different concentrations of target ssDNA. The detection results of different concentrations are shown in FIG. 4 .
  • the electrode was blocked with 1 mM MEA at 4 ° C for 2 hours, and then carefully rinsed with 10 mM PBS, and the IT curve was measured under infrared light of a wavelength of 980 nm, as shown by I 2 in FIG. Due to steric effects, the photocurrent decreases with respect to I 1, when the photocurrent I 1 is modified CdS QDs on ITO, the working electrode is produced under irradiation of infrared light wavelength of 980nm.
  • UC NPs up-converting nanoparticles
  • 50 uL of 25 uM UC NPs and 125 uL of 10 mM EDC were added in 1 mL of 10 mM PBS, and reacted at room temperature for 30 min; then 50 uL of 10 uM target SS DNA and 65 uL of 10 mM NHS were added. Reactive at room temperature for 2-4 h. After centrifugation, it was stored at 4 ° C until use.
  • Hybridization between target ssDNA and probe ssDNA 25 ⁇ L of different concentrations of UC NPs labeled target ssDNA were dropped on the surface of the probe ssDNA-modified gate electrode, and incubated at 37 ° C for 1 h at a concentration of 20 mM MgCl 2 . after rinsing with 10mM PBS to remove non-hybridized the DNA, and then, in the infrared light wavelength of 980nm, IT measurement curve, as shown in FIG I 3. Due to the fluorescence resonance energy transfer effect (FRET), the photocurrent will increase, offsetting the effects of steric hindrance.
  • FRET fluorescence resonance energy transfer effect
  • the present invention integrates a photo-active material layer on a working electrode, and fixes a probe ssDNA on the photo-active material layer, and additionally modifies the up-converting material onto the target ssDNA; when the target ssDNA and the probe After the hybridization reaction of ssDNA, under the illumination of infrared light, a fluorescence resonance energy transfer effect (FRET) occurs between the up-conversion material and the photoelectric active material layer, so the detected photocurrent changes; different concentrations The intensity of the fluorescence produced by the conversion material is different, and the photocurrent generated by the excitation is also different, thereby detecting different concentrations of DNA.
  • the photoelectrochemical DNA sensor based on the up-conversion material provided by the invention has high sensitivity, simple operation, low equipment requirement, short detection time, wide application range and good prospect.

Abstract

An up-conversion material-based photoelectrochemical DNA sensor and a detection method therefor, wherein the photoelectrochemical DNA sensor comprises an electrolytic cell (20) filled with an electrolytic solution (10), and a reference electrode (30), a counter electrode (40) and a working electrode (50) that are disposed in the electrolytic cell (20); a photoelectric active material layer (60) is assembled on the working electrode (50); a probe ssDNA (70) is fixedly arranged on the photoelectric active material layer (60); an up-conversion material (80) is connected to a target ssDNA (90); after hybridization of the target ssDNA (90) and the probe ssDNA (70), the photocurrent changes, which achieves the detection of the target ssDNA (90) having different concentrations.

Description

基于上转换材料的光电化学DNA传感器及其检测方法Photoelectrochemical DNA sensor based on up-conversion material and detection method thereof 技术领域Technical field
本发明涉及生物材料检测领域，尤其涉及基于上转换材料的光电化学DNA传感器及其检测方法。The invention relates to the field of biological material detection, in particular to a photoelectrochemical DNA sensor based on an up-conversion material and a detection method thereof.
背景技术Background technique
光电化学(Photoelectrochemical，PEC)是生物传感领域常用的检测方法，其在检测离子、葡萄糖、酶、DNA以及抗原抗体等方面均具有较好的效果。然而，常用的PEC检测方法都具有一定的局限，比如采用PEC方法检测DNA的过程中常用的光源为紫外光，而紫外光激发时容易对DNA结构造成一定的损伤，从而导致DNA浓度检测结果不精确；Photoelectrochemical (PEC) is a commonly used detection method in the field of biosensing. It has good effects in detecting ions, glucose, enzymes, DNA and antigen antibodies. However, the commonly used PEC detection methods have certain limitations. For example, the light source commonly used in the process of detecting DNA by the PEC method is ultraviolet light, and the ultraviolet light excitation causes a certain damage to the DNA structure, thereby causing the DNA concentration detection result not to be accurate;
上转换材料是一种红外光激发下能发出可见光的发光材料，即将红外光转换成可见光的材料，其特点是所吸收的光子能量低于发射的光子能量，这种现象违背Stokes定律，因此又被称为反Stokes定律；上转换材料在生物荧光检测中也有很多应用，例如，可将上转换材料固定在DNA待检测物上，通过检测荧光强度来检测DNA物质。然而这种通过荧光强度来判断检测待测DNA浓度的方法存在干扰因素多、实验周期长、结果不够精确等问题。The up-conversion material is a luminescent material that emits visible light under infrared light excitation, that is, a material that converts infrared light into visible light, which is characterized in that the absorbed photon energy is lower than the emitted photon energy, which is contrary to Stokes' law, and thus It is called anti-Stokes law; the up-conversion material also has many applications in bio-fluorescence detection. For example, the up-conversion material can be immobilized on the DNA analyte to detect the DNA substance by detecting the fluorescence intensity. However, this method of determining the concentration of DNA to be tested by fluorescence intensity has many problems such as many interference factors, long experimental period, and inaccurate results.
因此，现有技术还有待于改进和发展。Therefore, the prior art has yet to be improved and developed.
发明内容Summary of the invention
鉴于上述现有技术的不足，本发明的目的在于提供基于上转换材料的光电化学DNA传感器及其检测方法，旨在解决现有技术在检测DNA浓度时存在结果不精确、实验周期长、干扰因素多的问题。In view of the above deficiencies of the prior art, the object of the present invention is to provide a photoelectrochemical DNA sensor based on an up-conversion material and a detection method thereof, aiming at solving the inaccuracy of the prior art in detecting DNA concentration, the experimental period is long, and the interference factor More questions.
本发明的技术方案如下：The technical solution of the present invention is as follows:
一种基于上转换材料的光电化学DNA传感器，用于检测目标ssDNA的浓度，其中，所述光电化学DNA传感器包括装有电解液的电解池、设置在电解池内部的参比电极、对电极以及工作电极，所述工作电极上组装有光电活性材料层，所述光电活性材料层上固定设置有探针ssDNA，所述上转换材料与目标ssDNA连接，所述目标ssDNA与所述探针ssDNA杂化后引起光电流的变化，实现对不 同浓度目标ssDNA的检测。A photoelectrochemical DNA sensor based on an up-conversion material for detecting a concentration of a target ssDNA, wherein the photoelectrochemical DNA sensor comprises an electrolytic cell containing an electrolyte, a reference electrode disposed inside the electrolytic cell, a counter electrode, and a working electrode, on which the photoelectrically active material layer is assembled, on which the probe ssDNA is fixedly disposed, the up-converting material is connected to the target ssDNA, and the target ssDNA is mixed with the probe ssDNA After the change, the photocurrent is changed, and the detection of ssDNA of different concentration targets is realized.
所述的基于上转换材料的光电化学DNA传感器，其中，所述光电活性材料层由量子点材料、TiO 2、石墨烯或酞菁中的一种或多种组成。 The photoelectrochemical DNA sensor based on an up-conversion material, wherein the photoelectric active material layer is composed of one or more of a quantum dot material, TiO 2 , graphene or phthalocyanine.
所述的基于上转换材料的光电化学DNA传感器，其中，所述参比电极为Ag/AgCl参比电极、Hg/HgCl参比电极或Hg/HgO参比电极中的一种。The photoelectrochemical DNA sensor based on the up-conversion material, wherein the reference electrode is one of an Ag/AgCl reference electrode, an Hg/HgCl reference electrode, or an Hg/HgO reference electrode.
所述的基于上转换材料的光电化学DNA传感器，其中，所述对电极为铂电极。The photoelectrochemical DNA sensor based on an up-conversion material, wherein the counter electrode is a platinum electrode.
所述的基于上转换材料的光电化学DNA传感器，其中，所述工作电极的基底为ITO、FTO或AZO中的一种。The photoelectrochemical DNA sensor based on the up-conversion material, wherein the substrate of the working electrode is one of ITO, FTO or AZO.
所述的基于上转换材料的光电化学DNA传感器，其中，所述上转换材料为NaYF4:Yb，Er、NaYF4:Yb，Tm或者NaYF4:Yb，Ho中的一种。The photoelectrochemical DNA sensor based on the up-conversion material, wherein the up-conversion material is one of NaYF4: Yb, Er, NaYF4: Yb, Tm or NaYF4: Yb, Ho.
一种如上任一所述的基于上转换材料的光电化学DNA传感器的检测方法，其中，包括步骤：A method for detecting a photoelectrochemical DNA sensor based on an up-conversion material according to any of the above, comprising the steps of:
A、预先采用上转换材料对待测目标ssDNA进行标记；A. The target ssDNA to be tested is marked with an up-conversion material in advance;
B、将待测目标ssDNA滴在固定设置有探针ssDNA的工作电极上进行杂化反应；B, the target ssDNA is dropped on the working electrode fixed with the probe ssDNA for hybridization reaction;
C、将反应后的工作电极放入电解池中，在红外光的照射下，测量电流-时间曲线，根据电流的变化获得待测目标ssDNA的浓度。C. The working electrode after the reaction is placed in an electrolytic cell, and the current-time curve is measured under the irradiation of infrared light, and the concentration of the target ssDNA is obtained according to the change of the current.
所述的基于上转换材料的光电化学DNA传感器的检测方法，其中，所述步骤B中杂化反应的条件为：将工作电极在20mM的MgCl 2溶液中浸润后滴加待测目标ssDNA，在30-40℃的条件下反应40-100min。 The method for detecting a photoelectrochemical DNA sensor based on an up-conversion material, wherein the conditions of the hybridization reaction in the step B are: infiltrating the working electrode in a 20 mM MgCl 2 solution, and then dropping the target ssDNA to be tested, The reaction was carried out at 30-40 ° C for 40-100 min.
所述的基于上转换材料的光电化学DNA传感器的检测方法，其中，所述步骤B还包括：采用浓度为10mM的PBS溶液对反应后的工作电极进行冲洗。The method for detecting a photoelectrochemical DNA sensor based on an up-conversion material, wherein the step B further comprises: rinsing the reacted working electrode with a PBS solution having a concentration of 10 mM.
所述的基于上转换材料的光电化学DNA传感器的检测方法，其中，所述红外光的波长范围为950-1000nm。The method for detecting a photoelectrochemical DNA sensor based on an up-conversion material, wherein the infrared light has a wavelength ranging from 950 to 1000 nm.
有益效果：本发明通过在工作电极上组装光电活性材料层，并在所述光电活性材料层上固定设置探针ssDNA，另外将上转换材料修饰到目标ssDNA上；当 目标ssDNA与探针ssDNA发生杂化反应后，在红外光波长的光照下，上转换材料与光电活性材料层之间会产生荧光共振能量转移效应(FRET)，因此检测到的光电流会发生变化；不同浓度的上转换材料产生的荧光强度不同，激发产生的光电流也会不同，从而检测出不同浓度的DNA。本发明提供的基于上转换材料的光电化学DNA传感器具有高的灵敏度，操作简单，设备要求低，检测时间短，应用范围广，具有良好的前景。[Advantageous Effects] The present invention comprises: assembling a photoactive material layer on a working electrode, and fixing a probe ssDNA on the photoelectric active material layer, and additionally modifying the up-conversion material to the target ssDNA; when the target ssDNA and the probe ssDNA occur After the hybridization reaction, a fluorescence resonance energy transfer effect (FRET) occurs between the up-conversion material and the photo-active material layer under the illumination of the infrared light wavelength, so the detected photocurrent changes; different concentrations of the up-conversion material The intensity of the generated fluorescence is different, and the photocurrent generated by the excitation is also different, thereby detecting different concentrations of DNA. The photoelectrochemical DNA sensor based on the up-conversion material provided by the invention has high sensitivity, simple operation, low equipment requirement, short detection time, wide application range and good prospect.
附图说明DRAWINGS
图1为本发明一种基于上转换材料的光电化学DNA传感器较佳实施例的结构示意图；1 is a schematic structural view of a preferred embodiment of a photoelectrochemical DNA sensor based on an up-conversion material according to the present invention;
图2为本发明一种基于上转换材料的光电化学DNA传感器检测方法的原理示意图；2 is a schematic diagram showing the principle of a photoelectrochemical DNA sensor detection method based on an up-conversion material according to the present invention;
图3为本发明一种基于上转换材料的光电化学DNA传感器检测方法较佳实施例的流程图；3 is a flow chart of a preferred embodiment of a photoelectrochemical DNA sensor detection method based on an up-conversion material according to the present invention;
图4为本发明不同浓度的目标ssDNA的检测结果示意图；Figure 4 is a schematic diagram showing the detection results of different concentrations of target ssDNA of the present invention;
图5为本发明实施例1中光照下工作电极修饰有CdS QDs的器件的I-T曲线图；5 is an I-T graph of a device in which a working electrode is modified with CdS QDs under illumination in Embodiment 1 of the present invention;
图6为本发明中DNA杂化前后的I-T曲线图。Figure 6 is a graph showing the I-T curve before and after DNA hybridization in the present invention.
具体实施方式Detailed ways
本发明提供一种基于上转换材料的光电化学DNA传感器及其检测方法，为使本发明的目的、技术方案及效果更加清楚、明确，以下对本发明进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。The present invention provides a photoelectrochemical DNA sensor based on an up-conversion material and a method for detecting the same, and the present invention will be further described in detail below in order to make the objects, technical solutions and effects of the present invention more clear and clear. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
请参阅图1和图2，本发明提供一种基于上转换材料的光电化学DNA传感器，用于检测目标ssDNA的浓度，如图所示，其中，所述光电化学DNA传感器包括装有电解液10的电解池20、设置在电解池20内部的参比电极30、对电极40以及工作电极50，所述工作电极50上组装有光电活性材料层60，所述光电活性材料层60上固定设置有探针ssDNA70，所述上转换材料80与目标 ssDNA90连接，所述目标ssDNA90与所述探针ssDNA70杂化后引起光电流的变化，实现对不同浓度目标ssDNA90的检测。1 and 2, the present invention provides a photoelectrochemical DNA sensor based on an up-conversion material for detecting the concentration of a target ssDNA, as shown in the figure, wherein the photoelectrochemical DNA sensor comprises an electrolyte 10 An electrolytic cell 20, a reference electrode 30 disposed inside the electrolytic cell 20, a counter electrode 40, and a working electrode 50, on which the photovoltaic active material layer 60 is assembled, and the photoelectric active material layer 60 is fixedly disposed The probe ssDNA70, the up-conversion material 80 is linked to the target ssDNA90, and the target ssDNA90 is hybridized with the probe ssDNA70 to cause a change in photocurrent, and detection of different concentrations of the target ssDNA90 is achieved.
具体来说，常规PEC方法在检测生命物质(DNA、蛋白质等)的过程中通常采用的光源为紫外光，而紫外光在激发照射的过程中容易对生命物质的结构造成损伤，破坏其化学结构，从而导致检测结果不精确。Specifically, the conventional PEC method generally uses ultraviolet light in the process of detecting living substances (DNA, protein, etc.), and ultraviolet light easily damages the structure of the living substance during the process of exciting the irradiation, destroying the chemical structure thereof. , resulting in inaccurate detection results.
基于此，本发明通过在工作电极上组装光电活性材料层，并将已知浓度的目标ssDNA固定在所述光电活性层上；另外采用上转换材料对待测目标ssDNA进行修饰，并将修饰后的待测目标ssDNA滴加到目标ssDNA上进行杂化反应，反应完成后在红外光的照射下，上转换纳米颗粒与光电活性物质之间会产生荧光共振能量转移效应(FRET)，因此检测到的光电流会发生变化。Based on this, the present invention fixes a known concentration of target ssDNA on the photoactive layer by assembling a photoactive material layer on the working electrode, and additionally modifies the target ssDNA to be measured by the up-conversion material, and the modified The target ssDNA is added to the target ssDNA for hybridization reaction. After the reaction is completed, the fluorescence resonance energy transfer effect (FRET) is generated between the up-converting nanoparticles and the photoelectric active material under the irradiation of infrared light, so the detected The photocurrent will change.
对于PEC生物传感器中光电流信号的检测主要通过电化学工作站，本发明建立了一个由工作电极、参比电极、对电极组成的三电极测试系统来检测光电流的大小；不同浓度的上转换纳米粒子产生的荧光强度不同，激发产生的光电流也会不同，从而检测出不同浓度的DNA。这种新的检测方法具有高的灵敏度，操作简单，设备要求低，能实现微型化和批量检测，应用范围广，具有良好的前景。For the detection of photocurrent signals in PEC biosensors mainly through electrochemical workstations, the present invention establishes a three-electrode test system consisting of working electrodes, reference electrodes and counter electrodes to detect the magnitude of photocurrent; up-conversion nanometers with different concentrations The fluorescence intensity produced by the particles is different, and the photocurrent generated by the excitation is also different, thereby detecting different concentrations of DNA. This new detection method has high sensitivity, simple operation, low equipment requirements, miniaturization and batch detection, wide application range and good prospects.
进一步地，在本发明中，所述参比电极为Ag/AgCl参比电极、Hg/HgCl参比电极或Hg/HgO参比电极中的一种；所述对电极为铂电极；所述工作电极的基底为ITO、FTO或AZO中的一种，所述基底上的光电活性材料层由量子点材料、TiO 2、石墨烯或酞菁中的一种或多种组成；较佳地，优选CdS量子点作为光电活性材料。 Further, in the present invention, the reference electrode is one of an Ag/AgCl reference electrode, an Hg/HgCl reference electrode or a Hg/HgO reference electrode; the counter electrode is a platinum electrode; The substrate of the electrode is one of ITO, FTO or AZO, and the photoelectrically active material layer on the substrate is composed of one or more of a quantum dot material, TiO 2 , graphene or phthalocyanine; preferably, preferably CdS quantum dots are used as photoelectrically active materials.
本发明所提供的基于上转换材料的光电化学DNA传感器是一种新型检测DNA的生物传感技术，在光电活性材料层上固定探针ssDNA后，通过杂化不同浓度且修饰有上转换材料的目标ssDNA来引起光电流不同程度的增加，通过测量光电流的变化来实现对不同浓度目标ssDNA的检测。The photoelectrochemical DNA sensor based on the up-conversion material provided by the invention is a novel biosensing technology for detecting DNA, and after fixing the probe ssDNA on the photoelectric active material layer, the hybridization is performed by different concentrations and modified with the up-conversion material. The target ssDNA is used to cause different increases in photocurrent, and the detection of different concentrations of target ssDNA is achieved by measuring changes in photocurrent.
进一步，本发明还提供一种基于上转换材料的光电化学DNA传感器的检测方法，如图3所示，其中包括步骤：Further, the present invention also provides a detection method of a photoelectrochemical DNA sensor based on an up-conversion material, as shown in FIG. 3, which includes the steps of:
S10、预先采用上转换材料对待测目标ssDNA进行标记；S10, pre-using the up-conversion material to mark the target ssDNA;
具体来说，所述上转换材料为NaYF4:Yb，Er、NaYF4:Yb，Tm或者NaYF4:Yb，Ho中的一种，本发明提供的上转换材料是由无机基质以及镶嵌在其中的稀土掺杂离子组成；以NaYF4:Yb，Er为例，即镱铒双掺时，Er作为激活剂，Yb作为敏化剂，NaYF4是目前上转换发光效率最高的基质材料；所述上转换材料可以存在多种形态，本发明优选球形颗粒状，其粒径大小可变换，荧光波长也可改变。Specifically, the up-conversion material is one of NaYF4:Yb, Er, NaYF4:Yb, Tm or NaYF4:Yb, Ho, and the up-conversion material provided by the invention is composed of an inorganic matrix and rare earth doped therein. Hetero-ion composition; taking NaYF4:Yb,Er as an example, when bismuth double doping, Er as activator, Yb as sensitizer, NaYF4 is the most efficient matrix material for up-conversion luminescence; the up-conversion material can exist In various forms, the present invention is preferably in the form of spherical particles, the particle size of which can be changed, and the fluorescence wavelength can also be changed.
S20、将待测目标ssDNA滴在固定设置有探针ssDNA的工作电极上进行杂化反应；S20, the target ssDNA is dropped on the working electrode fixedly provided with the probe ssDNA to perform a hybrid reaction;
具体来说，在工作电极上加入接有上转换材料的不同浓度的目标ssDNA，通过DNA间的杂化，接有上转换材料的目标ssDNA会固定在工作电极上；进一步，所述杂化反应的过程为：将工作电极在20mM的MgCl 2溶液中浸润后滴加待测目标ssDNA，在30-40℃的条件下反应40-100min；反应完成后，采用浓度为10mM的PBS溶液对反应后的工作电极进行冲洗，以除去未杂化的目标ssDNA。 Specifically, a target ssDNA with different concentrations of the up-conversion material is added to the working electrode, and the target ssDNA with the up-converting material is immobilized on the working electrode by hybridization between the DNA; further, the hybridization reaction The process is as follows: the working electrode is infiltrated in a 20 mM MgCl 2 solution, and the target ssDNA is added dropwise, and the reaction is carried out at 30-40 ° C for 40-100 min; after the reaction is completed, the reaction is carried out by using a PBS solution having a concentration of 10 mM. The working electrode is rinsed to remove unhybridized target ssDNA.
S30、将反应后的工作电极放入电解池中，在红外光的照射下，测量电流-时间曲线，根据电流的变化获得待测目标ssDNA的浓度。S30. The working electrode after the reaction is placed in an electrolytic cell, and the current-time curve is measured under irradiation of infrared light, and the concentration of the target ssDNA is obtained according to the change of the current.
具体来说，将发生杂化反应后的工作电极放入电解池中，在波长范围为950-1000nm的红外光照射下，测量光电流，由于上转换材料与光电活性材料(CdS QDs)之间的荧光共振能量转移效应，使光电流增大，本发明通过检测光电流随不同浓度的目标ssDNA而引起的变化来检测不同浓度的目标ssDNA，不同浓度检测结果如图4所示。Specifically, the working electrode after the hybridization reaction is placed in an electrolytic cell, and the photocurrent is measured under infrared light having a wavelength range of 950-1000 nm, due to the relationship between the up-conversion material and the photoelectric active material (CdS QDs). The fluorescence resonance energy transfer effect increases the photocurrent. The present invention detects different concentrations of target ssDNA by detecting changes in photocurrent with different concentrations of target ssDNA. The detection results of different concentrations are shown in FIG. 4 .
下面通过具体实施例对本发明一种基于上转换材料的光电化学DNA传感器的检测方法做进一步的解释说明：The following further explains the detection method of the photoelectrochemical DNA sensor based on the up-conversion material of the present invention through a specific embodiment:
实施例1Example 1
(1)TGA修饰的CdS QDs的合成：在三口烧瓶中加入50mL 0.01M CdCl 2溶液，搅拌，通入氮气，升温至40℃后加入50μL TGA，反应30min。在此期间，使用1M的NaOH溶液调节混合液的pH到11。然后，加入5.0mL 0.1M Na 2S溶液，氮氛下110℃加热，回流4h，用水(体积比1﹕1)稀释后，保存于4℃冰 箱待用。 (1) Synthesis of TGA-modified CdS QDs: 50 mL of 0.01 M CdCl 2 solution was added to a three-necked flask, stirred, and nitrogen gas was introduced thereto. After heating to 40 ° C, 50 μL of TGA was added and reacted for 30 minutes. During this time, the pH of the mixture was adjusted to 11 using a 1 M NaOH solution. Then, 5.0 mL of a 0.1 M Na 2 S solution was added, heated at 110 ° C under a nitrogen atmosphere, refluxed for 4 hours, diluted with water (volume ratio 1:1), and stored in a refrigerator at 4 ° C until use.
(2)CdS QDs在ITO电极上的固定：将洗净干燥后的ITO电极依次浸入2％PDDA(0.5M NaCl溶液配制)和CdS QDs溶液中各10min，每次浸泡完用水清洗，该过程重复3次，得到所需的多层膜修饰工作电极。固定工作面积后，在980nm波长的红外光光照下，测量I-T曲线，如图5所示。(2) Fixation of CdS QDs on ITO electrode: The washed and dried ITO electrode was immersed in 2% PDDA (0.5 M NaCl solution) and CdS QDs solution for 10 min each time, and each time the water was washed, the process was repeated. Three times, the desired multilayer film modified working electrode was obtained. After fixing the working area, the I-T curve was measured under infrared light of 980 nm wavelength, as shown in FIG.
(3)探针ssDNA在CdS QDs修饰的栅电极表面的固定：通过探针ssDNA上的NH 2基团和CdS QDs上的COOH基团之间的偶联反应进行。将CdS QDs修饰的电极浸入20mg/mL EDC和10mg/mL NHS的溶液中1小时，随后用水小心冲洗，将25μL探针ssDNA(1μM)滴在电极表面并4℃孵化过夜后，使用10mM PBS小心冲洗，以便去除未固定的探针ssDNA。然后，使用1mM MEA于4℃封闭电极2小时，再用10mM PBS小心冲洗后，在980nm波长的红外光光照下，测量I-T曲线，如图6中I 2所示。由于位阻效应的影响，光电流相对于I 1会减小，所述I 1为在ITO上修饰CdS QDs时，工作电极在980nm波长红外光的照射下产生的光电流。 (3) Immobilization of the probe ssDNA on the surface of the CdS QDs-modified gate electrode: by a coupling reaction between the NH 2 group on the probe ssDNA and the COOH group on the CdS QDs. The CdS QDs-modified electrode was immersed in a solution of 20 mg/mL EDC and 10 mg/mL NHS for 1 hour, followed by careful washing with water, and 25 μL of probe ssDNA (1 μM) was dropped on the electrode surface and incubated at 4 ° C overnight, using 10 mM PBS. Rinse to remove unfixed probe ssDNA. Then, the electrode was blocked with 1 mM MEA at 4 ° C for 2 hours, and then carefully rinsed with 10 mM PBS, and the IT curve was measured under infrared light of a wavelength of 980 nm, as shown by I 2 in FIG. Due to steric effects, the photocurrent decreases with respect to I 1, when the photocurrent I 1 is modified CdS QDs on ITO, the working electrode is produced under irradiation of infrared light wavelength of 980nm.
(4)上转换纳米粒子(UC NPs)对目标ssDNA的标记：在1mL 10mM PBS中加入50uL 25uM的UC NPs和125uL 10mM的EDC，室温反应30min；然后再加入50uL 10uM目标SS DNA和65uL 10mM NHS，室温反应2-4h。离心收集后在4℃保存备用。(4) Labeling of target ssDNA by up-converting nanoparticles (UC NPs): 50 uL of 25 uM UC NPs and 125 uL of 10 mM EDC were added in 1 mL of 10 mM PBS, and reacted at room temperature for 30 min; then 50 uL of 10 uM target SS DNA and 65 uL of 10 mM NHS were added. Reactive at room temperature for 2-4 h. After centrifugation, it was stored at 4 ° C until use.
(5)目标ssDNA和探针ssDNA之间的杂化：25μL的不同浓度UC NPs标记的目标ssDNA滴在探针ssDNA修饰的栅电极表面，在浓度为20mM的MgCl 2条件下37℃孵化1h，之后用10mM PBS冲洗，去除未杂化DNA，然后，在980nm波长的红外光光照下，测量I-T曲线，如图6中I 3所示。由于荧光共振能量转移效应(FRET)，光电流会增大，抵消掉位阻效应的影响。 (5) Hybridization between target ssDNA and probe ssDNA: 25 μL of different concentrations of UC NPs labeled target ssDNA were dropped on the surface of the probe ssDNA-modified gate electrode, and incubated at 37 ° C for 1 h at a concentration of 20 mM MgCl 2 . after rinsing with 10mM PBS to remove non-hybridized the DNA, and then, in the infrared light wavelength of 980nm, IT measurement curve, as shown in FIG I 3. Due to the fluorescence resonance energy transfer effect (FRET), the photocurrent will increase, offsetting the effects of steric hindrance.
综上所述，本发明通过在工作电极上组装光电活性材料层，并在所述光电活性材料层上固定设置探针ssDNA，另外将上转换材料修饰到目标ssDNA上；当目标ssDNA与探针ssDNA发生杂化反应后，在红外光波长的光照下，上转换材料与光电活性材料层之间会产生荧光共振能量转移效应(FRET)，因此检测到的 光电流会发生变化；不同浓度的上转换材料产生的荧光强度不同，激发产生的光电流也会不同，从而检测出不同浓度的DNA。本发明提供的基于上转换材料的光电化学DNA传感器具有高的灵敏度，操作简单，设备要求低，检测时间短，应用范围广，具有良好的前景。In summary, the present invention integrates a photo-active material layer on a working electrode, and fixes a probe ssDNA on the photo-active material layer, and additionally modifies the up-converting material onto the target ssDNA; when the target ssDNA and the probe After the hybridization reaction of ssDNA, under the illumination of infrared light, a fluorescence resonance energy transfer effect (FRET) occurs between the up-conversion material and the photoelectric active material layer, so the detected photocurrent changes; different concentrations The intensity of the fluorescence produced by the conversion material is different, and the photocurrent generated by the excitation is also different, thereby detecting different concentrations of DNA. The photoelectrochemical DNA sensor based on the up-conversion material provided by the invention has high sensitivity, simple operation, low equipment requirement, short detection time, wide application range and good prospect.
应当理解的是，本发明的应用不限于上述的举例，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，所有这些改进和变换都应属于本发明所附权利要求的保护范围。It is to be understood that the application of the present invention is not limited to the above-described examples, and those skilled in the art can make modifications and changes in accordance with the above description, all of which are within the scope of the appended claims.

Claims (10)

  1. 一种基于上转换材料的光电化学DNA传感器，用于检测目标ssDNA的浓度，其特征在于，所述光电化学DNA传感器包括装有电解液的电解池、设置在电解池内部的参比电极、对电极以及工作电极，所述工作电极上组装有光电活性材料层，所述光电活性材料层上固定设置有探针ssDNA，所述上转换材料与目标ssDNA连接，所述目标ssDNA与所述探针ssDNA杂化后引起光电流的变化，实现对不同浓度目标ssDNA的检测。A photoelectrochemical DNA sensor based on an up-conversion material for detecting a concentration of a target ssDNA, characterized in that the photoelectrochemical DNA sensor comprises an electrolytic cell containing an electrolyte, a reference electrode disposed inside the electrolytic cell, and a pair An electrode and a working electrode, on which the photoelectrically active material layer is assembled, on which the probe ssDNA is fixedly disposed, the up-converting material is connected to the target ssDNA, the target ssDNA and the probe The hybridization of ssDNA causes photocurrent changes to achieve detection of ssDNA at different concentrations.
  2. 根据权利要求1所述的基于上转换材料的光电化学DNA传感器，其特征在于，所述光电活性材料层由量子点材料、TiO 2、石墨烯或酞菁中的一种或多种组成。 The up-conversion material-based photoelectrochemical DNA sensor according to claim 1, wherein the photoelectric active material layer is composed of one or more of a quantum dot material, TiO 2 , graphene or phthalocyanine.
  3. 根据权利要求1所述的基于上转换材料的光电化学DNA传感器，其特征在于，所述参比电极为Ag/AgCl参比电极、Hg/HgCl参比电极或Hg/HgO参比电极中的一种。The photoelectrochemical DNA sensor based on up-conversion material according to claim 1, wherein the reference electrode is one of an Ag/AgCl reference electrode, an Hg/HgCl reference electrode or a Hg/HgO reference electrode. Kind.
  4. 根据权利要求1所述的基于上转换材料的光电化学DNA传感器，其特征在于，所述对电极为铂电极。The up-conversion material-based photoelectrochemical DNA sensor according to claim 1, wherein the counter electrode is a platinum electrode.
  5. 根据权利要求1所述的基于上转换材料的光电化学DNA传感器，其特征在于，所述工作电极的基底为ITO、FTO或AZO中的一种。The up-conversion material-based photoelectrochemical DNA sensor according to claim 1, wherein the substrate of the working electrode is one of ITO, FTO or AZO.
  6. 根据权利要求1所述的基于上转换材料的光电化学DNA传感器，其特征在于，所述上转换材料为NaYF4:Yb，Er、NaYF4:Yb，Tm或者NaYF4:Yb，Ho中的一种。The up-conversion material-based photoelectrochemical DNA sensor according to claim 1, wherein the up-conversion material is one of NaYF4: Yb, Er, NaYF4: Yb, Tm or NaYF4: Yb, Ho.
  7. 一种如权利要求1-6任一所述的基于上转换材料的光电化学DNA传感器的检测方法，其特征在于，包括步骤：A method for detecting a photoelectrochemical DNA sensor based on an up-conversion material according to any one of claims 1 to 6, comprising the steps of:
    A、预先采用上转换材料对待测目标ssDNA进行标记；A. The target ssDNA to be tested is marked with an up-conversion material in advance;
    B、将待测目标ssDNA滴在固定设置有探针ssDNA的工作电极上进行杂化反应；B, the target ssDNA is dropped on the working electrode fixed with the probe ssDNA for hybridization reaction;
    C、将反应后的工作电极放入电解池中，在红外光的照射下，测量电流-时间曲线，根据电流的变化获得待测目标ssDNA的浓度。C. The working electrode after the reaction is placed in an electrolytic cell, and the current-time curve is measured under the irradiation of infrared light, and the concentration of the target ssDNA is obtained according to the change of the current.
  8. 根据权利要求7所述的基于上转换材料的光电化学DNA传感器的检测方法，其特征在于，所述步骤B中杂化反应的条件为：将工作电极在20mM的MgCl 2溶液中浸润后滴加待测目标ssDNA，在30-40℃的条件下反应40-100min。 The method for detecting a photoelectrochemical DNA sensor based on an up-conversion material according to claim 7, wherein the condition of the hybridization reaction in the step B is: instilling the working electrode in a 20 mM MgCl 2 solution and then adding the solution The target ssDNA to be tested is reacted at 30-40 ° C for 40-100 min.
  9. 根据权利要求7所述的基于上转换材料的光电化学DNA传感器的检测方法，其特征在于，所述步骤B还包括：采用浓度为10mM的PBS溶液对反应后的工作电极进行冲洗。The method for detecting a photoelectrochemical DNA sensor based on an up-conversion material according to claim 7, wherein the step B further comprises: rinsing the reacted working electrode with a PBS solution having a concentration of 10 mM.
  10. 根据权利要求7所述的基于上转换材料的光电化学DNA传感器的检测方法，其特征在于，所述红外光的波长范围为900-1000nm。The method for detecting a photoelectrochemical DNA sensor based on an up-conversion material according to claim 7, wherein the infrared light has a wavelength in the range of 900 to 1000 nm.
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