阅读说明：本技术 一种基于光催化的无标记电化学生物传感器检测溶菌酶的新方法 (A kind of new method based on light-catalysed unmarked electrochemica biological sensor detection lysozyme ) 是由 许淑霞 唐刚旭 秦晓娇 张信凤 于 2019-09-11 设计创作，主要内容包括：建立了一种无标记、选择性好、特异性强、灵敏高的检测溶菌酶的电化学新方法,通过先把带巯基的捕捉链DNA通过硫金键结合在金电极上,然后将适配体链DNA与捕捉链DNA通过碱基互补配对形成双链,在溶菌酶存在的情况下,溶菌酶与适配体链DNA结合后形成G-四连体,从而使适配体链DNA从双链中解链掉离。电极中余下的单链吸附在GO上,因为GO与碱基都有共同的六边形结构,它们能形成π结构的堆叠反应。最后将PB吸附到GO上,因为PB能通过π-π键吸附到GO的表面,在绿光LED灯照射下,PB吸收能量,继而传递给溶液中的溶解氧产生<Sup>1</Sup>O<Sub>2</Sub>,<Sup>1</Sup>O<Sub>2</Sub>可导致电极表面的DNA单链的断裂,使电极表面对[Fe(CN)<Sub>6</Sub>]<Sup>3-/4-</Sup>的排斥减小,造成传质阻力减少,从而使电化学阻抗信号减小；当没有目标链存在时,[Fe(CN)<Sub>6</Sub>]<Sup>3-/4-</Sup>电化学阻抗信号无明显降低,基于此,可实现对溶菌酶的无标记检测。(Establish the electrochemistry new method of unmarked one kind, selectively good, high specificity, sensitive high detection lysozyme, the capture chain DNA with sulfydryl is incorporated on gold electrode by sulphur gold key by elder generation, then aptamers chain DNA and capture chain DNA are formed into double-strand by base pair complementarity, in the presence of lysozyme, lysozyme in conjunction with aptamers chain DNA after formed G- tetrad, so that the unwinding from double-strand of aptamers chain DNA be made to drop off.In electrode it is remaining it is single-stranded be adsorbed on GO because GO and base have common hexagonal structure, they can be formed π structure stacking reaction.Finally PB is adsorbed on GO, because PB can be adsorbed onto the surface of GO by pi-pi bond, under green light LED light irradiation, PB absorbs energy, and the dissolved oxygen then passed in solution generates 1 O 2 , 1 O 2 The mono- chain break of DNA that can lead to electrode surface, makes electrode surface to [Fe (CN) 6 ] 3‑/4‑ Repulsion reduce, cause resistance to mass tranfer to reduce, thus make electrochemical impedance signal reduce；In the presence of there is no object chain, [Fe (CN) 6 ] 3‑/4‑ Electrochemical impedance signal is without being substantially reduced, based on this, it can be achieved that markless detection to lysozyme.)

1. a kind of new method based on light-catalysed unmarked electrochemica biological sensor detection lysozyme, it is characterised in that including Following four step:

(1) 4 μM of capture chain DNAs of drop coating are incubated for a hour after gold electrode being polished flat and cleaned；

(2) 1 μM of aptamers chain DNA solution of the electrode surface drop coating described in (1) continues to be incubated for two hours；

(3) electrode described in (2) is immersed in 100nM lysozyme soln and continues to be incubated for half an hour；

(4) electrode described in (3) is immersed in 100 μ g/mL GO solution and is incubated for half an hour；

(5) the immersion 100nM PB solution of electrode described in (4) is continued after being incubated for 15 minutes, uses the illumination of LED green light at room temperature 15min, and electrochemical signals are detected with electrochemical impedance spectroscopy (EIS) method with electrochemical workstation, analyze measurement result.

2. method according to claim 1, it is characterised in that selected capture chain DNA sequence is 5 '-CTAAGT AAC TCT GCA CTC TTA TAT ATC ATA GAA TTG GTA GAT-(CH₂)₆-SH-3'；Aptamers chain DNA sequence is 5 '-ATC TAC GAA TTC ATC AGG GCT AAA GAG TGC AGA GTT ACT TAG-3’。

3. method according to claim 1, it is characterised in that Tris-HCl (trishydroxymethylaminomethane-hydrochloric acid) buffer solution Include 10mM trishydroxymethylaminomethane, 100mM NaCl, pH 7.4.

4. method according to claim 1, it is characterised in that the pH of electrolyte solution is 7.4.

Technical field

The present invention relates to a kind of new methods based on the unmarked electrochemica biological sensor detection lysozyme of photocatalysis, belong to In biosensor technique field.

Background technique

Lysozyme is prevalent in animals and plants and microorganism, it is a kind of enzyme of energy dissolution of bacteria cell wall, wide It is general to be applied to bioengineering, in medical diagnosis and food antiseptic.Since lysozyme is in the importance of medical context of detection, therefore develop Many methods measure its content, such as colorimetric method, agarose rocket electrophoresis, Agar diffusion test, ultraviolet spectrophotometry, height Effect liquid phase chromatogram method and electrochemical process etc..But colorimetric method, agarose rocket electrophoresis, Agar diffusion test need to cultivate bacterium, Process is cumbersome；Ultraviolet spectrophotometry does not need culture bacterium, and process is relatively easy, testing result is reliable and stable；Efficient liquid phase Chromatography separation efficiency height, favorable reproducibility, but separate the time length and instrument price valuableness of sample.In contrast, electrochemistry is examined Survey method is due to high sensitivity, detection at low cost and being widely used in lysozyme content.Since the measurement of lysozyme is for facing Bed diagnosis and the research of other aspects have great importance, and therefore, it is good, accurate to design a kind of efficient and sensible, specificity It is most important to spend high detection method.

Summary of the invention

It is an object of the invention to the characteristics using nano material graphene oxide (GO), and the light of phloxine (PB) is urged Change reaction to combine with electrochemical method, devise photocatalysis electrochemical biosensor method to detect the lysozyme in urine, Without additional markers, method, GO has the function of amplified signal to this method simply, simultaneously, can greatly improve sensitivity.

Technical scheme is as follows:

(1) 4 μM of capture chain DNAs of drop coating are incubated for a hour after gold electrode being polished flat and cleaned；

(2) 1 μM of aptamers chain DNA solution of the electrode surface drop coating described in (1) continues to be incubated for two hours；

(3) electrode described in (2) is immersed in 100nM lysozyme soln and continues to be incubated for half an hour；

(4) electrode described in (3) is immersed in 100 μ g/mL GO solution and is incubated for half an hour；

(5) the immersion 100nM PB solution of electrode described in (4) is continued after being incubated for 15 minutes, uses LED green light at room temperature Illumination 15min, and electrochemical signals are detected with electrochemical impedance spectroscopy (EIS) method with electrochemical workstation, analyze measurement result.

Invention effect

Compared with prior art, the present invention has the advantage that

(1) experimentation it is simple, it is at low cost, be not necessarily to labelling technique；

(2) compared with current unmarked electrochemical sensor, the low 1-2 order of magnitude of the detection limit of this method；

(3) high sensitivity, and can specific recognition lysozyme, avoid the generation of false Yangxin number；

(4) identification to variety classes protein or enzyme can be realized by changing the sequence of aptamers chain DNA.

Specific embodiment