CN102072931A - Method for preparing biosensor based on silicon nanowires and application of biosensor to detecting DNA - Google Patents

Abstract

Translated fromChinese

本发明公开了一种基于硅纳米线的生物传感器制备方法及在DNA检测中的应用，通过湿化学法制备硅纳米线，将金纳米粒子通过硅烷偶联剂修饰于硅纳米线上；再通过金纳米粒子与DNA之间的化学键的结合，将探针DNA嫁接到硅纳米线上，制备成传感器探头，用于探测待测靶溶液中未知DNA序列；检测结果主要由循环伏安法测得的数据分析得到。本发明的特点：①通过简单的微加工工艺能够进行大批量生产，成本低且与大规模集成电路工艺相兼容；②主要利用了硅纳米线、金纳米粒子和DNA相互之间的特异性和生物相容性，易于实现，具有广泛的适用性；③所得生物传感器，制作简便、重现性好、灵敏度高、易于实现微型化并且能够实现实时监测。The invention discloses a method for preparing a biosensor based on silicon nanowires and its application in DNA detection. The silicon nanowires are prepared by a wet chemical method, and the gold nanoparticles are modified on the silicon nanowires through a silane coupling agent; The combination of chemical bonds between gold nanoparticles and DNA, the probe DNA is grafted onto silicon nanowires, and a sensor probe is prepared to detect unknown DNA sequences in the target solution; the detection results are mainly measured by cyclic voltammetry data analysis obtained. The characteristics of the present invention: ① mass production can be carried out through simple micro-processing technology, low cost and compatible with large-scale integrated circuit technology; ② mainly utilize the specificity and Biocompatibility, easy to implement, and wide applicability; ③The obtained biosensor is easy to manufacture, has good reproducibility, high sensitivity, is easy to realize miniaturization, and can realize real-time monitoring.

Description

A kind of based on the biology sensor preparation method of silicon nanowires and the application in DNA detection

Technical field

The present invention relates to micro-nano sensor, electrochemica biological sensor technical field, specifically a kind of biology sensor preparation method and the application of this sensor in DNA detection based on silicon nanowires.

Background technology

Along with the enforcement of the Human Genome Project,, make genetic test particularly important in molecular biology and biomedical research for the diagnosis and treatment of genopathy provide scientific basis.In recent years, the biotechnology fast development, many biological new technologies are arisen at the historic moment, gene tester highly sensitive for developing, high specific has injected new vitality, the various DNA biosensor techniques that wherein utilize the base complementrity pair principle of dna double chain to grow up have been subjected to numerous researchers' great attention.

The DNA biology sensor, combines with detection techniques such as fluorescent technique, QCM (Quartz Crystal Microbalance) technology, surface plasma body resonant vibration technology, electrochemical techniques as sensitive element with dna molecular, can make the sensor of the multiple DNA of being used for fast measuring.Although all kinds of DNA sensors have been obtained significant progress, obtain attracting attention of common people, but still exist many insoluble problems, for example technical costs and checkout equipment costliness, make problems such as complicated, that detection sensitivity is lower, poor repeatability, analyst coverage are narrower.And these problems show that mainly preparation, the probe of sample synthesize and fixing, the mark of molecule, several aspects such as reading and analyze of data.Signal obtain with analytically, current most methods use fluorescence methods to detect and analyze, repeatability better, but detection sensitivity is still not high.Therefore, the exploration cost is cheaper, making is simpler, the check and analysis method of the more convenient sensitivity of detection is numerous scientific research personnel's the task of top priority.

Summary of the invention

The purpose of this invention is to provide a kind of biology sensor preparation method and the application of this sensor in detecting DNA based on silicon nanowires, solving and to improve the deficiencies in the prior art, for the making of following DNA electrochemica biological sensor provide a kind of new, be applicable to large-scale production and feasible program with low cost.

Purpose of the present invention can be achieved through the following technical solutions.

A kind of biology sensor preparation method based on silicon nanowires comprises following concrete steps:

A) preparation of silicon nanowires

Use H₂SO₄: H₂O₂=1:1 solution cleaning silicon chip is rinsed it well with deionized water again; Prepare 70 mMs/liter AgNO₃Solution, ultrasonic vibration 15～20 minutes, compound concentration is 20% HF solution again; Two kinds of solution 1:1 by volume mixes, and the silicon chip of cleaning is put in the above-mentioned mixed solution reacted, and the reaction time is 50～60 minutes; Use HNO then₃Solution and deionized water clean up this silicon chip, obtain being carved with the silicon chip of silicon nanowires after the oven dry;

B) preparation of golden nanometer particle

Prepare the chlorauric acid solution of 0.001 mol; Simultaneously, prepare 38.8 mMs/liter sodium citrate solution; Then the chlorauric acid solution for preparing is heated to boiling, again sodium citrate solution is added to wherein, chlorauric acid solution and sodium citrate solution volume ratio are 10:1, make mixed solution leavestandstill reaction 10～15 minutes, the color of cooling back solution is become dark red brown by yellow, i.e. reaction finishes, and obtains the golden nanometer particle aqueous solution ofparticle diameter 8～10 nanometers;

C) modify silicon nanowires with golden nanometer particle

Use ethanol and deionized water rinsing clean respectively the silicon chip of step a preparation; Put into then in the ethanolic solution that is added with silane coupling agent 3-aminopropyl trimethoxysilane (APTMS), the volume ratio of silane coupling agent and ethanol is 1:1000, and the ethanolic solution that is added with APTMS is heated to boiling, 40～45 minutes time; Take out silicon chip, fall unnecessary APTMS with alcohol flushing earlier, wash ethanol off with deionized water again; This silicon chip being put into solution of gold nanoparticles soaked 12 hours again; Take out, use deionized water rinsing, the drying box of putting into 100 ℃ is dried; With conductive silver paste and epoxy resin lead is connected on the silicon chip at last, makes biology sensor based on silicon nanowires.

The application of described biology sensor in DNA detection, be with the grafting of single-stranded probe dna sequence dna to this sensor; Then this sensor is put into the DNA target solution of unknown nucleotide sequence,, scanned detection with cyclic voltammetry by electrochemical workstation.

The DNA electrochemica biological sensor is an emerging cross discipline that relates to fields such as biology, chemistry, galvanochemistry, medical science and electronics.The prepared biology sensor of the present invention is based on the silicon nanowires that golden nanometer particle is modified, silicon nanowires has higher specific surface area, add the electric conductivity and the bio-compatibility of golden nanometer particle excellence, the two perfect adaptation, a kind of brand-new DNA detection technology can be provided, have simple, reliable, inexpensive, sensitivity and good selective, will have broad application prospects at the aspects such as screening of clinical gene diagnosis, cancer therapy drug.

Description of drawings

Fig. 1 is a process flow diagram of the present invention;

Fig. 2 is a silicon nanowires sectional view of the present invention;

The surface of silicon nanowires EDS figure that Fig. 3 modifies for golden nanometer particle of the present invention;

Fig. 4 is the cyclic voltammogram of sensor exploring electrode of the present invention in damping fluid;

Fig. 5 is the cyclic voltammogram of sensor exploring electrode of the present invention in target solution to be measured;

Fig. 6 for sensor exploring electrode of the present invention at voltage is-during the 1.1V place, and target solution concentration and reduction current graph of a relation.

Embodiment

Further set forth technical characterstic of the present invention below in conjunction with the drawings and specific embodiments.

Embodiment

By the present invention is further set forth in one 0.5 * 0.5 square centimeter silicon chip design, the description of making and testing.

The preparation of A, described biology sensor

1) preparation of silicon nanowires

Silicon nanowires is made by chemical etching method; At first use H₂SO₄: H₂O₂=1:1 solution cleaning silicon chip, scavenging period is 10 minutes, with deionized water it is rinsed well again; Prepare 70 mMs/liter AgNO₃20 milliliters of solution, ultrasonic vibration 20 minutes, compound concentration is 20 milliliters of 20% HF solution again; The silicon chip of cleaning is put into above-mentioned AgNO₃With react in the mixed solution of HF, the reaction time is 60 minutes; Use HNO at last respectively₃Solution and deionized water clean up the silicon chip that this is carved with silicon nanowires, dry stand-by; Prepared silicon nanowires as shown in Figure 2.

2) preparation of golden nanometer particle

Golden nanometer particle is by the preparation of sodium citrate reduction gold chloride method; At first, prepare the chlorauric acid solution of 300 milliliters 0.001 mol; Simultaneously, the preparation 30 milliliters 38.8 mMs/liter sodium citrate solution; The chlorauric acid solution that will prepare is heated to boiling then, again sodium citrate solution is added to wherein, make mixed solution leavestandstill reaction 10 minutes, can see after the cooling that the color of solution is become dark red brown by yellow, i.e. reaction finishes, and obtains the golden nanometer particle that particle diameter is about 10 nanometers.

3) modify silicon nanowires with golden nanometer particle

Use silane coupling A PTMS,, golden nanometer particle is self-assembled on the silicon nanowires by chemical bond.At first, the silicon nanowires that preparation is finished uses ethanol and deionized water rinsing clean respectively; Put into then in the ethanolic solution that is added with APTMS and be heated to boiling, totally 45 minutes; Take out sample, fall unnecessary APTMS with alcohol flushing earlier, wash ethanol off with deionized water again; This sample being put into solution of gold nanoparticles soaked 12 hours again; Take out, use deionized water rinsing, the drying box oven dry of putting into 100 ℃ is stand-by; With conductive silver paste and epoxy resin lead is connected on the sample at last, the sensor preparation finishes.The silicon nanowires EDS figure that Fig. 3 modifies for golden nanometer particle, as seen from the figure, gold content is 7.4%, promptly golden nanometer particle is successfully grafted on the silicon nanowires.

B, the application of described sensor in DNA detection, concrete steps are as follows:

1) grafting of single-stranded probe DNA

Chosen following dna fragmentation as example:

Dna probe sequence: 5 '-TTTTTTTTTTTCTCGGAATTCGTTGGTGGG-3 '

The target dna sequence of coupling: 5 '-TTTTTTTTTTCCCACCAACGAATTCCGAGA-3 '

The target dna sequence of non-coupling: 5 '-TTTTTTTTTTAGGAATGTGCTTCTCACGTA-3 '

At first be the single-stranded probe DNA(Capture DNA of 2 micromoles per liter with concentration) grafting to the biology sensor that has made-the silicon nanowires electrode promptly modified by golden nanometer particle on, grafting time is 3 hours; Form the Au-S key between the dna probe molecule of utilization band sulfydryl and the gold nano grain, can make the dna molecular self assembly to electrode surface.Grafting finishes, and rinses out unnecessary single-stranded probe dna solution with deionized water, uses N again₂Electrode is dried up.

2) hybridization reaction

The sensor that is fixed with single-stranded probe DNA is placed target solution to be measured, and target solution is the target dna sequence that an end is modified through golden nanometer particle, and modifying purpose is in order to strengthen detection signal.If sub-thread dna probe and target sequence carry out recognition reaction. coupling, then hybridization forms double-stranded DNA.This course of reaction will be noted in real time and intactly by electrochemical workstation.By the test data of cyclic voltammetry, can analyze learn target dna sequence whether with the dna probe sequences match, if coupling then can be passed through known probe DNA base sequence, the base sequence on the target dna that pushes away unknownly.

3) test

By electrochemical workstation LK3200A (Tianjin, China), scan with the DNA target solution of cyclic voltammetry (CV method) unknown nucleotide sequence, the DNA biology sensor result of detection prepared to the present invention carries out analytical test.

ⅰ, damping fluid test

In order to judge whether damping fluid can disturb the sensor among the present invention, the CV test of the advanced line sensor of the first step in damping fluid.As shown in Figure 4, though the CV curve presents a pair of redox peak, peak value is very little, and the peak is very wide, and reversibility is poor, and it is little to judge that back of the body bottom buffering liquid disturbs the detection of this sensor, can continue test.

ⅱ, DNA survey

Withsensor place 1 nanomole/liter the target solution (tDNA) of coupling, carry out the cyclic voltammetry test.By Fig. 5 (a) curve as can be known, the CV curve presents a pair of redox peak, and the peak is more sharp-pointed, and peak value is very big, and at the reduction potential place of-1.1V, electric current can reach 700 microamperes.Andsensor place 1 nanomole/liter the target solution (tDNA) of non-coupling, the CV curve that records (as Fig. 5 (b) curve) does not have tangible redox peak, and hybridization reaction does not promptly take place.Contrast can get thus, and this sensor can successfully be distinguished coupling and non-coupling tDNA.

According to the result of detection of (10 nanomoles/rise to, 1 picomole/liter) under the variable concentrations, sum up the current value curve that obtains as shown in Figure 6 at the reduction potential place of-1.1V.(a) curve is coupling tDNA measurement result, and this curve is linear substantially, and matched curve is Y=2534+212X, and Y is a current value, and X is the logarithm of tDNA concentration value.And (b) curve is non-coupling tDNA test result, and this curve is substantially near 0.Can get thus, the DNA biology sensor of made can be successfully applied to DNA detection among the present invention.

The present invention has following purposes and advantage:

Purposes: prepared biology sensor, can be studied widely and be used in fields such as clinical medical inspection, genetic engineering, drug mechanism, new medicament screen, environmental monitoring and food engineerings.

Advantage: 1. can produce in enormous quantities by simple micro fabrication, cost is low and mutually compatible with lsi technology.

2. the biology sensor that makes has utilized silicon nanowires, golden nanometer particle and DNA specificity and biocompatibility each other dexterously, and the idea novelty is easy to realize having widely applicability.

3. easy, the favorable reproducibility, highly sensitive of preparation method is easy to realize microminiaturization and can realize Real-Time Monitoring.

Claims (2)

Translated fromChinese

1.一种基于硅纳米线的生物传感器制备方法，其特征在于该方法包括以下具体步骤：1. A method for preparing a biosensor based on silicon nanowires, characterized in that the method comprises the following steps:a）硅纳米线的制备a) Preparation of silicon nanowires用H₂SO₄:H₂O₂=1:1溶液清洗硅片，再用去离子水将其冲洗干净；配制70毫摩尔/升的AgNO₃溶液，超声振动15～20分钟，再配制浓度为20%的HF 溶液；两种溶液按体积比1:1混合，将洗净的硅片投入到上述混合溶液中进行反应，反应时间为50～60分钟；然后用HNO₃溶液和去离子水将该硅片清洗干净，烘干后得到刻有硅纳米线的硅片；Clean the silicon wafer with H₂ SO₄ :H₂ O₂ =1:1 solution, and then rinse it with deionized water; prepare a 70 mmol/L AgNO₃ solution, ultrasonically vibrate for 15-20 minutes, and then prepare the concentration It is 20% HF solution; the two solutions are mixed at a volume ratio of 1:1, and the cleaned silicon wafer is put into the above mixed solution for reaction, and the reaction time is 50-60 minutes; then use HNO₃ solution and deionized water The silicon wafer is cleaned and dried to obtain a silicon wafer engraved with silicon nanowires;b）金纳米粒子的制备b) Preparation of gold nanoparticles配制0.001摩尔/升的氯金酸溶液；同时，配制38.8毫摩尔/升的柠檬酸钠溶液；然后将配制好的氯金酸溶液加热至沸腾，再将柠檬酸钠溶液加至其中，氯金酸溶液与柠檬酸钠溶液体积比为10:1，使混合溶液静置反应10～15分钟，冷却后溶液的颜色由黄色变成深红棕色，即反应结束，得到粒径8～10纳米的金纳米粒子水溶液；Prepare 0.001 mol/liter of chloroauric acid solution; at the same time, prepare 38.8 mmol/liter of sodium citrate solution; then heat the prepared chloroauric acid solution to boiling, then add sodium citrate solution to it, and The volume ratio of the acid solution to the sodium citrate solution is 10:1, and the mixed solution is left to react for 10 to 15 minutes. After cooling, the color of the solution changes from yellow to dark reddish brown. Aqueous solution of gold nanoparticles;c）用金纳米粒子修饰硅纳米线c) Modification of silicon nanowires with gold nanoparticles将步骤a制备的硅纳米线分别用乙醇和去离子水冲洗干净；然后投入到加有硅烷偶联剂3-氨丙基三甲氧基硅烷的乙醇溶液中, 硅烷偶联剂与乙醇的体积比为1:1000，将乙醇溶液加热至沸腾，时间40～45分钟；取出硅片，先用乙醇冲洗掉多余的APTMS，再用去离子水洗掉乙醇；再将该硅片放入金纳米粒子溶液中浸泡12小时；取出，用去离子水冲洗，放入100℃的干燥箱中上烘干；最后用导电银浆和环氧树脂将导线连接于硅片上，制得基于硅纳米线的生物传感器。The silicon nanowires prepared in step a are rinsed with ethanol and deionized water respectively; then put into the ethanol solution added with silane coupling agent 3-aminopropyltrimethoxysilane, the volume ratio of silane coupling agent to ethanol 1:1000, heat the ethanol solution to boiling for 40-45 minutes; take out the silicon chip, first wash off the excess APTMS with ethanol, and then wash off the ethanol with deionized water; then put the silicon chip into gold nanoparticles Soak in the solution for 12 hours; take it out, rinse it with deionized water, put it in a drying oven at 100°C and dry it; finally use conductive silver paste and epoxy resin to connect the wire to the silicon chip to make a silicon nanowire-based biological sensor.2.一种权利要求1所述生物传感器在DNA检测中的应用，其特征在于：将单链探针DNA序列嫁接到该传感器上；然后将该传感器放入未知序列的DNA靶溶液中，通过电化学工作站，用循环伏安法进行扫描检测。2. The application of a biosensor according to claim 1 in DNA detection, characterized in that: the single-stranded probe DNA sequence is grafted onto the sensor; then the sensor is put into the DNA target solution of unknown sequence, and passed Electrochemical workstation, scanning detection by cyclic voltammetry.

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