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CN109444117A - A kind of electrochemical luminescence biosensor and its application based on silver nanoclusters quenching effect and multiple amplifying technique - Google Patents

A kind of electrochemical luminescence biosensor and its application based on silver nanoclusters quenching effect and multiple amplifying technique Download PDF

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CN109444117A
CN109444117A CN201811306301.1A CN201811306301A CN109444117A CN 109444117 A CN109444117 A CN 109444117A CN 201811306301 A CN201811306301 A CN 201811306301A CN 109444117 A CN109444117 A CN 109444117A
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dna
atp
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接贵芬
葛君君
王卫
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Qingdao University of Science and Technology
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Abstract

本发明公开了一种基于银纳米簇双重猝灭效应和多重循环放大技术的电化学发光生物传感器及其制法和应用。本发明的技术方案是通过适体的特异性识别作用，目标ATP与适体结合后打开颈环，暴露出可以与模板DNA结合的序列，在聚合酶和剪切酶的作用下实现多重循环放大反应，生成大量DNA产物链。同时，在DNA模板原位合成的Ag NCs通过循环产物DNA链接到电极上，由于电极上CdS量子点与Ag NCs与之间的ECL共振能量转移以及Ag NCs对电极表面附近的共反应剂的消耗，实现了对电极上ECL信号的双重猝灭，根据ECL信号的变化对ATP进行超灵敏检测。

The invention discloses an electrochemiluminescence biosensor based on silver nano-cluster double quenching effect and multiple cycle amplification technology, and a preparation method and application thereof. The technical scheme of the present invention is that through the specific recognition of the aptamer, the target ATP is combined with the aptamer to open the neck loop, exposing the sequence that can be combined with the template DNA, and realize multiple cyclic amplification under the action of polymerase and cleavage enzyme. The reaction produces a large number of DNA product chains. Meanwhile, the Ag NCs synthesized in situ on the DNA template were linked to the electrode through the cycle product DNA, due to the ECL resonance energy transfer between the CdS quantum dots on the electrode and the Ag NCs and the consumption of the co-reactants near the electrode surface by the Ag NCs. , the double quenching of the ECL signal on the electrode is realized, and the ultrasensitive detection of ATP is carried out according to the change of the ECL signal.

Description

It is a kind of raw based on the electrochemical luminescence of silver nanoclusters quenching effect and multiple amplifying technique Object sensor and its application
Technical field:
The present invention relates to a kind of electrochemical luminescences based on silver nanoclusters dual quenching effect and Multiple Cycle amplifying technique Biosensor；The invention further relates to the analysis applications of the preparation method of the biosensor and its detection ATP.
Background technique:
Atriphos (ATP) is a kind of multi-functional biomolecule, is continuously every vital movement of cell Energy [Knowles J R.Annual Review of Biochemistry, 2003,49 (1): 877- required for providing 919.]、[Chen J,Liu Y,Ji X,et al.Biosensors&Bioelectronics,2016,83:221-228.]、 [Fu P,Sun M,Xu L,et al.Nanoscale,2016,8(32):15008-15015.].Research has shown that ATP can make For a biochemical indicator, the ATP and cardiovascular disease of various concentration, cell viability and cellular damage, Parkinson's disease and A Erci The silent disease in sea have close connection [Li X, Peng Y, Chai Y, et al.Chemical Communications, 2016,52 (18):3673-3676.]、[Lu L,Jing C S,Zhong F G,et al.Biosensors&Bioelectronics, 2015,63:14-20.], sensitive ATP recognition detection method is developed to have great importance.
Electrogenerated chemiluminescence (ECL) is as a kind of powerful analytical technology, since it has high sensitivity, low background, behaviour Make simply to receive more and more attention [Liu Y, Lei J, Huang Y, et with the advantages that excellent controllability al.Analytical Chemistry,2014,86(17):8735-8741.]、[Ma W,Xu L,de Moura A F,et Al.Chemical Reviews, 2017,117 (12): 8041-8093.], and it is applied to a variety of biological marker analyte detections In.At the same time, many has and is also constantly opened simultaneously compared with the ECL luminescent material of high luminous performance and good electrochemical stability It is applied among bio-sensing, such as luminol [Xu L, Sun M, Ma W, et al.Materials Today, 2016, 19:595-606.], quantum dot [Rohrbach F, F,Fichte M A H,et al.Angewandte Chemie International Edition, 2013,52 (45): 11912-11915.], ruthenium and its derivative [Deng C, Chen J, Nie L, et al.Analytical Chemistry, 2009,81 (24): 9972-9978.] etc..Especially quanta point material, Because its preferable biocompatibility by favor [Rohrbach F, F,Fichte M A H,et al.Angewandte Chemie International Edition,2013,52(45):11912-11915.]。
Since in short distance ECL Resonance energy transfer [Liu J, Cao Z, Lu occur for CdS:Mn in 2009 and silver nanoclusters Y.Chemical Reviews, 2009,109 (5): 1948-1998.], the quenching effect based on ECL Resonance energy transfer is It is proved to be a kind of effective method for improving bio-sensing, for example CdS:Mn excites CdTe NCs [Feng C, Dai S, Wang L.Biosensors&Bioelectronics, 2014,59:64-74.], CdS NCs-Ru (bpy)3 2+[Zhao W W,Xu J J, Chen H Y.Trac Trends in Analytical Chemistry, 2016,82:307-315.], pass through what is adjusted the distance The enhancing to ECL signal also may be implemented in control.
Silver nanoclusters (Ag NCs) because of its own unique optics, electricity, that chemical characteristic has caused related fields is wide General research interest [Shan Y, Xu J J, Chen H Y.Chem.Commun.2010,46 (28), 5079-5081.], silver from Son can be incorporated into silver ion on the ssDNA of rich C the efficient affinity of cytimidine, and DNA becomes preferable support Ag The skeleton of NCs and be widely used.Nearest report discovery, using ssDNA as the ultraviolet-visible spectrum of the Ag NCs of templated synthesis Have at 492nm absorption peak [Wu M S, Shi H W, Xu J J, et al.Chemical Communications, 2011,47 (27): 7752-7754.], Ag NCs may be one of energy acceptor of CdS quantum dot.This nanocluster usually by it is several to Dozens of atom composition, size are less than 2nm, and the discrete energy level with size-dependent, this may make it have and be similar to Molecule catalytic performance [Cheng Y, Lei J, Chen Y, et al.Biosensors&Bioelectronics, 2014,51 (1):431-436.].Based on the above principle, a kind of ECL-RET based on Ag NCs and electronics transfer ECL sensing body can be designed System.
Isothermal exponential amplification reacts (EXPAR) as one of most important amplification strategy of nucleic acid, expands because itself is simple Increasing form and relatively high amplification efficiency and become a kind of up-and-coming method [Li J, Zhu J J, Xu K.Trac Trends in Analytical Chemistry,2014,58:90-98.].By dexterously devising two stage amplification, Open neck ring chain and ATP molecule can be quickly converted into the new DNA of no ATP molecule by the template, and then when generating product Next efficient EXPAR can be induced again, to further improve the sensitivity of detection.
The present invention devises a kind of electrochemical luminescence based on Ag NCs dual quenching effect and Multiple Cycle amplifying technique Biosensor realizes the super sensitivity detection to ATP using the online reduction of ECL Resonance energy transfer and Ag NCs.Pass through The specific recognition of aptamer acts on, and opens neck ring after target ATP is in conjunction with its aptamer, exposing can be in conjunction with template DNA Sequence, polymerase and shearing enzyme under the action of realize Multiple Cycle iodine, generate a large amount of DNA product chains.Meanwhile The Ag NCs of DNA profiling fabricated in situ is connected on electrode by circulation products DNA chain, due to CdS quantum dot and Ag on electrode NCs and between the consumption to the coreagent near electrode surface of ECL Resonance energy transfer and Ag NCs, realize to electricity The dual quenching of extremely upper ECL signal carries out super sensitivity detection to ATP according to the variation of ECL signal.
Summary of the invention:
An object of the present invention is to provide a kind of preferable CdS quantum dot of ECL luminescent properties as probe, mentions for detection For more stable signal source.Specifically includes the following steps:
By the CdCl of 5mmol in three-necked flask2·2.5H2O is dissolved in 30mL secondary water, and 20 μ L MPA are added, and is generated The precipitating of white is later 10~11 with the pH that the NaOH of 1.0M adjusts solution, and precipitating of solution gradually dissolves during this, connects Logical nitrogen deoxygenation 30min, then by the Na of the 0.02M newly prepared2Above-mentioned reaction solution is added dropwise in S solution 20mL solution In, orange-yellow precipitating is obtained, continues logical nitrogen, keeps reaction temperature at 70 DEG C, stops heating after reaction 3h to get carboxyl The CdS quantum dot of modification, to collect after reaction solution cooled to room temperature be placed in 4 DEG C it is spare.
It is a kind of based on the dual quenching effect of Ag NCs and Multiple Cycle amplifying technique the second object of the present invention is to provide Electrochemical luminescence biosensor realizes the super sensitivity detection to ATP.It is comprised the steps of:
The preparation of biosensor:
The building of step 1. sensor: the CdS quantum dot solution for taking 10 μ L to prepare equably is dripped in electrode surface, at room temperature The gold electrode that naturally dry is modified to get CdS；Contain EDC (20mg/mL) and NHS (10mg/mL) next, electrode is immersed Solution in, carry out quantum dot surface carboxyl activation processing 1h；Electrode surface is gently rinsed with PBS later, to remove remnants EDC and NHS, the surface that the HP2 (1 μM) of 10 μ L is then dripped to electrode is protected from light 4h under wet environment；Use PBS Electrode surface is carefully rinsed, the DNA not connected is removed and then carries out sealing plate processing 1h with MCH, then rinses electrode.
Next electrode is immersed in the pipe of reacted each concentration, and the Mg (Ac) of 10 μ L is added2(1mM), 100 μ L PBS (0.1M) and 5 μ L silver nanoclusters and DNA template chain reaction 1h；Finally, electrode washing is detected ECL signal.
ECL detection liquid is to contain coreagent K2S2O8The PBS solution of (0.1M) and KCl (0.1M), the voltage range of detection ﹣ 1.5V, sweep speed 0.1V/s are arrived for 0, photomultiplier tube (PMT) is ﹣ 600V to ﹣ 800V.
Simultaneously use unified photomultiplier transit condition, on optical window cover different wave length optical filter (400nm, 425nm, 440nm, 460nm, 490nm, 535nm, 555nm, 575nm, 620nm), detect change of the ECL luminous intensity in different wavelength Change.
The present invention uses Ag using the preferable CdS quantum dot of ECL luminescent properties as electrochemical luminescence signals probe The electrochemical luminescence biosensor of the dual quenching effect of NCs and Multiple Cycle amplifying technique, is successfully realized to ATP Gao Ling Quick, highly selective detection.The research is expected to be used in the early diagnosis of disease.
Compared with prior art, the present invention major advantage is: the present invention is using the CdS quantum dot of preparation as electrochemistry Luminous signal probe has stronger electrochemical luminescence signals, greatly improves detection sensitivity；The present invention is by preparation CdS quantum dot signal probe is combined with Multiple Cycle amplifying technique, is greatly exaggerated ECL signal；The present invention utilizes CdS amount Son point and Ag NCs and between the consumption to the coreagent near electrode surface of ECL Resonance energy transfer and Ag NCs, The dual quenching to ECL signal on electrode is realized, highly sensitive, the highly selective detection to ATP is realized.
Electrochemiluminescsensor sensor of the invention shows excellent accuracy, high sensitivity, highly selective, stable Property and reproducibility, analysis detection rapidly, conveniently, the biosensor bio-medical analysis detection and early clinical diagnosis in have There is huge application potential, can be used for the detection of actual sample.
Detailed description of the invention:
The TEM figure (grain size distribution that illustration is CdS quantum dot) of Fig. 1 (A) CdS quantum dot, (B) DNA- silver nanoclusters TEM figure, the UV absorption figure of (C) CdS quantum dot, illustration be CdS quantum dot luminous intensity figure and wavelength relation curve, (D) UV absorption figure of silver nanoclusters.
Fig. 2 is based on the dual quenching effect of silver nanoclusters and Multiple Cycle amplifying technique electrochemical luminescence super sensitivity detection ATP Schematic diagram.
Fig. 3 electrophoresis characterization: (electrophoresis characterization: (a) marker, (b) HP1, (c) template DNA, (d) HP1+ Template DNA, (e) HP1+template DNA+ATP, (f) HP1+template DNA+ATP+phi29, (g) HP1+ Template DNA+ATP+phi29+Nt.BbvCI, (h) HP2, (i) template of Ag, (j) HP2+template of Ag, (k) HP1+template+DNA+ATP+phi29+Nt.BbvCI+HP2+template of Ag.
The electrochemical luminescence performance of Fig. 4 CdS quantum dot: the ECL intensity-potential curve of (A) CdS quantum dot, illustration are to follow Ring voltammetric scan curve, the ECL strength-duration curve of (B) CdS quantum dot, (C) continuous scanning 16 enclose after ECL intensity-when Half interval contour, (D) continuous scanning 16 enclose after cyclic voltammetry scan curve.
The ECL of Fig. 5 (A) different phase electrode is responded, the cyclic voltammetry curve in (B) each stage: (a) bare electrode, (b) CdS quantum dot modification is to gold electrode surfaces, and (c) HP2 is modified to the surface of electrode, (d) MCH sealing plate, after (e) opening by SI After being hybridized with the DNA with silver nanoclusters.
The ECL signal of Fig. 6 (A) various concentration object responds, (B) corresponding ECL signal intensity value: (a) 1aM, (b) 10aM, (c) 100aM, (d) 1fM, (e) 10fM, (f) 100fM, (g) 1pM.
The graph of relation of Fig. 7 ECL signal intensity value and ATP concentration, illustration are the standards calibration curve of ATP detection.
Selection Journal of Sex Research of Fig. 8 sensor to target ATP.
Specific embodiment:
The preparation of 1. electroluminescent chemiluminescence biosensor of embodiment and detection to ATP
Target primase auxiliary circulation iodine: first prepare a certain concentration gradient ATP solution, be 10pM, 1pM, The object solution of 100fM, 10fM, 1fM, 100aM, 10aM, 1aM, 0.1aM, be placed in 4 DEG C it is spare.Then, respectively several anti- 10 μ L (1 μM) HP1 is added in Ying Guanzhong, then is separately added into the ATP of the 10 each concentration of μ L, and what is be uniformly mixed is placed on the isothermal vibration phase After middle reaction 30min, 1 μM of template DNA (10 μ L), the dNTPs of 10 μ L, 5U phi29 polymerization are added in reaction tube Enzyme and 5U Nt.BbvCI restriction endonuclease and corresponding buffer, react 90min at a temperature of 37 DEG C after mixing, connect The inactivation for getting off for reaction tube to be placed on to carry out enzyme in 60 DEG C of water bath, be finally placed in 4 DEG C it is spare.
The preparation of silver nanoclusters: the AgNO of 0.02M is first configured3Solution, according to DNA and Ag+The ratio that ratio is 1:10 will The AgNO of 10 μM of the DNA and 1 μ L of 200 μ L3Mixing, is vigorously mixed 90s, then by reaction tube in 4 DEG C of items on turbine mixer 30min is reacted under part.Then the NaBH for the 0.01M for taking 1 μ L newly to prepare4It is added in reaction tube, continues acute with turbine mixer Reaction tube finally is continued to be placed in 4 DEG C of environment by strong mixing 90s, and reaction overnight, can be obtained on template DNA The silver nanoclusters of generation.
The pretreatment of electrode: successively carrying out surface polishing treatment with 1.0 μm, 0.3 μm and 0.05 μm of aluminium powder for electrode, uses Secondary water rinses surface well.Then electrode is placed in dehydrated alcohol and is ultrasonically treated 10min, finally rinsed again with secondary water dry It is net spare.
The CdS quantum dot solution of preparation is taken, is added after ethyl alcohol, 5min is centrifuged under 6000rpm, later collects precipitating, Supernatant liquid is removed, precipitating is redissolved in the secondary water of equivalent；The quantum dot solution of 10 μ L is taken equably to drip with liquid-transfering gun On the surface of electrode, it is protected from light the gold electrode that naturally dry is modified to get CdS at room temperature；Next, electrode immersion is contained In the solution of EDC (20mg/mL) and NHS (10mg/mL), the activation processing of quantum dot surface carboxyl, reaction time 1h are carried out；Instead After answering, electrode surface is gently rinsed with PBS, to remove remaining EDC and NHS, the HP2 (1 μM) of 10 μ L is then dripped into electricity The surface of pole is protected from light 4h so that the amino of HP chain end and the carboxyl of quantum dot surface combine under wet environment；Instead After answering, electrode surface is carefully rinsed with PBS, the DNA not connected is removed and then carries out sealing plate processing 1h with MCH, most Carry out electrode washing again afterwards.
Next electrode is immersed in the pipe of reacted each concentration, and the Mg (Ac) of 10 μ L is added2(1mM), 100 μ L PBS (0.1M) and 5 μ L silver nanoclusters and DNA template chain reaction 1h；Electrode is finally carried out to careful flushing, is detected ECL signal.
The preparation of 2. electroluminescent chemiluminescence biosensor of embodiment and detection to ATP
Will " it is then, several respectively to be added in reaction tube 10 μ L (1 μM) HP1, then it is separately added into the 10 each concentration of μ L ATP, what is be uniformly mixed is placed on the interim reaction 30min of isothermal vibration." be changed to it is " then, several respectively to be added in reaction tube 10 μ L (1 μM) HP1, then it is separately added into the ATP of the 10 each concentration of μ L, what is be uniformly mixed is placed on the interim reaction of isothermal vibration 40min." preparation other conditions with embodiment 1, obtain pattern and property be similar to the biosensor of embodiment 1.To CEA The result of detection is the same as embodiment 1.
The preparation of 3. electroluminescent chemiluminescence biosensor of embodiment and detection to CEA
Will " be added 1 μM of 10 μ L of template DNA chain in reaction tube, the dNTPs of 10 μ L, 5U phi29 polymerase and 5U Nt.BbvCI restriction endonuclease and corresponding buffer react at a temperature of 37 DEG C in shaking table after being uniformly mixed 90min." be changed to " 1 μM of template DNA chain 10 μ L, the dNTPs of 10 μ L, 7U phi29 polymerase be added in reaction tube With 5U Nt.BbvCI restriction endonuclease and corresponding buffer, reacted at a temperature of 37 DEG C in shaking table after being uniformly mixed 90min." preparation other conditions with embodiment 1, obtain pattern and property be similar to the biosensor of embodiment 1.To CEA The result of detection is the same as embodiment 1.
The preparation of 4. electroluminescent chemiluminescence biosensor of embodiment and detection to CEA
Will " be added 1 μM of 10 μ L of template DNA chain in reaction tube, the dNTPs of 10 μ L, 5U phi29 polymerase and 5U Nt.BbvCI restriction endonuclease and corresponding buffer react at a temperature of 37 DEG C in shaking table after being uniformly mixed 90min." be changed to " 1 μM of template DNA chain 10 μ L, the dNTPs of 10 μ L, 5U phi29 polymerase be added in reaction tube With 7U Nt.BbvCI restriction endonuclease and corresponding buffer, reacted at a temperature of 37 DEG C in shaking table after being uniformly mixed 90min." preparation other conditions with embodiment 1, obtain pattern and property be similar to the biosensor of embodiment 1.To CEA The result of detection is the same as embodiment 1.
The preparation of 5. electroluminescent chemiluminescence biosensor of embodiment and detection to CEA
Will " be added 1 μM of 10 μ L of template DNA chain in reaction tube, the dNTPs of 10 μ L, 5U phi29 polymerase and 5U Nt.BbvCI restriction endonuclease and corresponding buffer react at a temperature of 37 DEG C in shaking table after being uniformly mixed 90min." be changed to " 1 μM of template DNA chain 10 μ L, the dNTPs of 10 μ L, 5U phi29 polymerase be added in reaction tube With 5U Nt.BbvCI restriction endonuclease and corresponding buffer, reacted at a temperature of 37 DEG C in shaking table after being uniformly mixed 100min." preparation other conditions with embodiment 1, obtain pattern and property be similar to the biosensor of embodiment 1.To CEA The result of detection is the same as embodiment 1.
The preparation of 6. electroluminescent chemiluminescence biosensor of embodiment and detection to CEA
" next electrode will be immersed in the pipe of reacted each concentration, and the Mg (Ac) of 10 μ L is added2(1mM), The silver nanoclusters of the PBS (0.1M) and 5 μ L of 100 μ L and the template chain reaction 1h of DNA." be changed to " next immerse electrode anti- In the pipe for each concentration answered, and the Mg (Ac) of 10 μ L is added2(1mM), the silver nanoparticle of the PBS (0.1M) and 5 μ L of 100 μ L The template chain reaction 70min of cluster and DNA." preparation other conditions with embodiment 1, obtain pattern and property be similar to embodiment 1 Biosensor.To CEA detection result with embodiment 1.

Claims (3)

1.一种基于银纳米簇双重猝灭效应和多重循环放大技术的电化学发光生物传感器，其特征是：通过目标ATP与其适体结合以及酶聚合剪切作用实现多重循环放大反应，生成大量DNA产物链，将原位合成Ag NCs的DNA接到电极上，实现电极上CdS量子点与Ag NCs与之间的ECL能量转移以及Ag NCs对电极表面附近的共反应剂的消耗，从而构建电化学发光生物传感器。1. An electrochemiluminescence biosensor based on the double quenching effect of silver nanoclusters and multiple cyclic amplification technology, characterized in that: multiple cyclic amplification reactions are realized through the combination of target ATP and its aptamer and enzymatic polymerization and shearing, and a large amount of DNA is generated. The product chain connects the DNA of the in situ synthesized Ag NCs to the electrode, realizes the ECL energy transfer between the CdS quantum dots and the Ag NCs on the electrode and the consumption of the co-reactant near the electrode surface by the Ag NCs, thereby constructing the electrochemical structure. Luminescent biosensors. 2.一种制备权利要求1所述的基于银纳米簇双重猝灭效应和多重循环放大技术的电化学发光生物传感器的方法和应用，其特征方法由下列步骤组成：2. a method and application of the electrochemiluminescence biosensor based on silver nano-cluster double quenching effect and multiple cycle amplification technology according to claim 1 are prepared, and its characteristic method is made up of the following steps: 步骤1.CdS量子点的制备：采用水热合成方法合成了CdS量子点作为信号材料，在三口烧瓶中将5mmol的CdCl2·2.5H2O溶解于30mL二次水中，加入20μL MPA，生成白色的沉淀，之后用1.0M的NaOH调节溶液的pH为10～11，此过程中溶液的沉淀逐渐溶解，接着通氮气除氧30min，然后将新配制的0.02M的Na2S溶液20mL溶液逐滴地加入上述反应溶液中，得到橙黄色的沉淀，继续通氮气，保持反应温度在70℃，反应3h之后停止加热，即得羧基修饰的CdS量子点，待反应液自然冷却至室温之后收集置于4℃备用。Step 1. Preparation of CdS quantum dots: CdS quantum dots were synthesized by hydrothermal synthesis method as a signal material. In a three-necked flask, 5 mmol of CdCl 2 ·2.5H 2 O was dissolved in 30 mL of secondary water, and 20 μL of MPA was added to form a white Then, the pH of the solution was adjusted to 10-11 with 1.0M NaOH. During this process, the precipitation of the solution was gradually dissolved. Then, nitrogen was used to deoxygenate for 30min, and then 20mL of the newly prepared 0.02M Na 2 S solution was added dropwise. Add it to the above reaction solution to obtain orange-yellow precipitate, continue to pass nitrogen gas, keep the reaction temperature at 70 °C, stop heating after 3 h of reaction, to obtain carboxyl-modified CdS quantum dots, after the reaction solution is naturally cooled to room temperature, collected and placed in Reserve at 4°C. 步骤2.目标引发酶辅助循环放大反应：首先配制一定浓度梯度的ATP溶液，为10pM，1pM，100fM，10fM，1fM，100aM，10aM，1aM，0.1aM的目标物溶液，置于4℃备用。Step 2. Target priming enzyme-assisted cyclic amplification reaction: First, prepare a certain concentration gradient of ATP solution, which is the target solution of 10pM, 1pM, 100fM, 10fM, 1fM, 100aM, 10aM, 1aM, 0.1aM, and put it at 4 °C for use. 然后，分别几个在反应管中加入10μL(1μM)HP1，再分别加入10μL各个浓度的ATP，混合均匀之后置于恒温震荡期中反应30min，之后，在反应管内加入1μM的template DNA链10μL，10μL的dNTPs，5U phi29聚合酶和5U Nt.BbvCI内切酶以及各自对应的缓冲液，混合均匀之后在在摇床里37℃温度下反应90min，接下来将反应管放在60℃的水浴环境中进行酶的灭活，最后置于4℃备用。Then, add 10 μL (1 μM) of HP1 to the reaction tubes respectively, and then add 10 μL of ATP of each concentration, mix well and place them in a constant temperature shaking period to react for 30 min. After that, add 10 μL, 10 μL of 1 μM template DNA chain to the reaction tubes. dNTPs, 5U phi29 polymerase and 5U Nt.BbvCI endonuclease and their corresponding buffers, mixed well and reacted in a shaker at 37°C for 90min, then placed the reaction tube in a 60°C water bath environment Enzyme inactivation was carried out, and finally placed at 4 °C for later use. 步骤3 银纳米簇的制备：先配置0.02M的AgNO3的溶液，按照DNA与Ag+比例为1:10的比例将200μL的10μM的银序列与1μL的AgNO3混合，在涡旋混合器上剧烈混合90s，再将反应管在4℃条件下反应30min。然后将新配制的0.01M的NaBH4取1μL加入到反应管内，继续用涡旋混合器剧烈混合90s，最后将反应管继续放置于4℃的环境中，反应过夜，即可得到在templateDNA上生成的银纳米簇。Step 3 Preparation of silver nanoclusters: First prepare a solution of 0.02M AgNO 3 , mix 200 μL of 10 μM silver sequence with 1 μL of AgNO 3 according to the ratio of DNA to Ag + 1:10, on a vortex mixer Mix vigorously for 90s, and then react the reaction tube at 4°C for 30min. Then add 1 μL of the freshly prepared 0.01M NaBH 4 into the reaction tube, continue to mix vigorously with a vortex mixer for 90s, and finally place the reaction tube in a 4°C environment for overnight reaction. of silver nanoclusters. 步骤4 生物传感器的制备：传感器制备之前，首先进行电极的处理。将金电极用0.3μm的氧化铝粉进行表面的抛光处理，之后用二次水将电极表面残留的氧化铝粉冲洗干净，最后在二次水浴中超声10min，最后吹干备用。Step 4 Preparation of biosensor: Before the preparation of the sensor, the electrode is first processed. The surface of the gold electrode was polished with 0.3 μm alumina powder, then the residual alumina powder on the electrode surface was rinsed with secondary water, and finally ultrasonicated in a secondary water bath for 10 min, and finally dried for use. 取制备的CdS量子点溶液，加入乙醇之后，6000rpm下离心5min，之后将沉淀收集，除去上层液体，将沉淀重新溶于等量的二次水中；用移液枪取10μL的量子点溶液均匀地滴在电极的表面，在室温下避光自然晾干，在电极表面形成一层薄膜，即得CdS修饰的金电极；接下来，将电极浸入含有EDC(20mg/mL)和NHS(10mg/mL)的溶液中，进行量子点表面羧基的活化处理，反应时间1h；反应之后，用PBS轻轻冲洗电极表面，以除去残余的EDC和NHS，然后将10μL的HP2(1μM)滴到电极的表面，在湿润的环境下避光反应4h以使HP链末端的氨基和量子点表面的羧基结合；反应之后，用PBS小心地冲洗电极表面，将没有连接的DNA除去然后用MCH进行封板处理1h，最后再进行电极冲洗。Take the prepared CdS quantum dot solution, add ethanol, centrifuge at 6000 rpm for 5 min, then collect the precipitate, remove the upper liquid, and redissolve the precipitate in an equal amount of secondary water; use a pipette to take 10 μL of the quantum dot solution evenly. Drop it on the surface of the electrode, dry it naturally in the dark at room temperature, and form a thin film on the surface of the electrode to obtain a CdS-modified gold electrode; ), the carboxyl groups on the surface of the quantum dots were activated, and the reaction time was 1 h; after the reaction, the electrode surface was gently rinsed with PBS to remove residual EDC and NHS, and then 10 μL of HP2 (1 μM) was dropped on the surface of the electrode. , react in a humid environment for 4 h in the dark to allow the amino group at the end of the HP chain to combine with the carboxyl group on the surface of the quantum dots; after the reaction, carefully rinse the electrode surface with PBS, remove the unconnected DNA, and then seal the plate with MCH for 1 h , and finally wash the electrode. 3.根据权利要求2所述的ATP检测方法，其特征是：所述的电化学发光测试是将表面进行反应完成的电极作为工作电极，三电极体系中检测ECL信号。ECL检测液为pH为7.4的PBS溶液(0.1M)，含有K2S2O8(0.1M)和KCl(0.1M),其中K2S2O8作为共反应剂与量子点发生反应，检测电压范围为0～-1.5V，扫描速率为0.1V/s，光电倍增管为-600V到-800V。同时采用统一的光电倍增条件，在光窗上加盖不同波长的滤光片(400nm，425nm，440nm，460nm，490nm，535nm，555nm，575nm，620nm)，检测ECL发光强度在不同的波长内的变化。3. ATP detection method according to claim 2, is characterized in that: described electrochemiluminescence test is to use the electrode whose surface carries out the reaction to complete as working electrode, and detects ECL signal in three-electrode system. The ECL detection solution is a PBS solution (0.1M) with a pH of 7.4, containing K 2 S 2 O 8 (0.1M) and KCl (0.1M), wherein K 2 S 2 O 8 acts as a co-reactant to react with the quantum dots, The detection voltage range is 0 to -1.5V, the scan rate is 0.1V/s, and the photomultiplier tube is -600V to -800V. At the same time, the uniform photomultiplier conditions are used, and filters of different wavelengths (400nm, 425nm, 440nm, 460nm, 490nm, 535nm, 555nm, 575nm, 620nm) are placed on the light window to detect the luminous intensity of ECL in different wavelengths. Variety.
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