CN104878075A - An electrochemical biochip sensor array for rapidly detecting mycobacterium tuberculosis and a preparing method - Google Patents
An electrochemical biochip sensor array for rapidly detecting mycobacterium tuberculosis and a preparing method Download PDFInfo
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- CN104878075A CN104878075A CN201410068230.1A CN201410068230A CN104878075A CN 104878075 A CN104878075 A CN 104878075A CN 201410068230 A CN201410068230 A CN 201410068230A CN 104878075 A CN104878075 A CN 104878075A
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Abstract
The invention belongs to the technical fields of bioelectrochemistry and biochip sensors and relates to an electrochemical biochip sensor array for mycobacterium tuberculosis 16SrDNA and for detecting mycobacterium tuberculosis and a preparing method thereof. Starting with molecular level detection of the mycobacterium tuberculosis 16SrDNA, Fe3O4@SiO2 composite nanometer particles and a nanometer Au structure material are adopted as mediators, a novel and functionalized nanometer electrochemical biochip superparamagnetism nano-detection microsphere sensor interface is designed, the mycobacterium tuberculosis is subjected to double enrichment extraction and purification detection, and the electrochemical biochip sensor array for rapidly detecting the mycobacterium tuberculosis is constructed. A clinical sample containing the mycobacterium tuberculosis is directly added into a magnetic reaction micropore. A mycobacterium tuberculosis 16SrDNA specific sequence can be subjected to double enrichment and biological stabilization through utilizing a capture probe labeled by the Fe3O4@SiO2 composite nanometer particles and a nanometer Au labeled detecting probe respectively. A high mycobacterium tuberculosis target molecule separating efficiency and high detection sensitivity are expected to be achieved by utilization of nano-detection microsphere superparamagnetism. In addition, target molecule "on-chip" separation is expected to be achieved through characteristics of the electrochemical biochip sensor array without the need of extra separating steps, thus largely simplifying operation procedures and shortening the detection time.
Description
Technical field
The invention belongs to bioelectrochemistry and biochip field of sensing technologies, relate to a kind of mycobacterium tuberculosis 16S
relectrochemica biological sensing chip array of DNA (rDNA) and preparation method thereof, for detecting mycobacterium tuberculosis.
Background technology
Mycobacterium tuberculosis causes phthisical pathogenic agent, cause about 2,000,000 people dead every year, also be the important factor of bringing out respiratory infection diseases simultaneously, significant damage is brought to human health, whole world tuberculosis number of patients increases fast with the speed of annual 2%, and China's number of the infected lungy occupies the second in the world, infection rate reaches 50%, is one of 22 tuberculosis high burden countries.Tuberculosis is higher at the sickness rate of low per capita income country, and one of most important reason lacks accurate, reliable, the convenient diagnostic method low with cost and technology.Existing a variety of TB detection method now: (1), as mycobacterium tuberculosis culture method, although have very high specificity and accuracy, although separation and Culture accuracy and susceptibility are all higher, the time in minimum needs a few week can obtain result; (2) mycobacterium tuberculosis quick Ziehi-Neelsen stain susceptibility is lower, and recall rate fluctuates larger in difference infection background crowd.(3) feature of immuno-chromatographic assay technology is simple to operate, and detection speed is very fast; But it is single that it detects mark kind, and sensitivity and specificity are all lower.
Along with m tuberculosis infection problem is day by day serious, global problem is become to the control of this bacterium, set up quick, easy, accurate, sensitive and accurately mycobacterium tuberculosis detection method be the important foundation of quick diagnosis.Electrochemical biosensor detection technique has the advantage that detection speed is fast, highly sensitive and reliability is strong, receives much concern in tuberculosis pathogenic microbes detect new technology.
The present invention detects from mycobacterium tuberculosis 16S rDNA molecular level and starts with, with Z 250 silicon-dioxide (Fe3O4SiO2) composite nanoparticle and nanometer Au structured material for mediator, novel in design, the nano electrochemical chip biological sensing interface of functionalization, simultaneously with electrochemica biological sensing chip array for relying on, utilize nano DNA probe biomolecule system, build super-paramagnetism nano and detect microballoon, carry out dual enrichment extraction and purification to mycobacterium tuberculosis to detect, build a kind of electrochemica biological sensing chip array for mycobacterium tuberculosis rapid detection.
Summary of the invention
The object of the invention is to provide one can sensitive, mycobacterium tuberculosis 16S fast and in detection by quantitative clinical samples
relectrochemica biological sensing chip array of DNA and preparation method thereof, its concrete preparation process is as follows:
Step one: electrochemica biological sensing chip array Fe
3o
4siO
2the preparation of the DNA probe bio-sensing layer of magnetic nano-composite particle and nanometer Au mark;
Step 2: electrochemica biological sensing chip array horseradish peroxidase (HRP), hydrogen peroxide (H
2o
2) and the preparation of cascade detection signal amplification system that forms of the DNA detection probe of nanometer Au mark.
Electrochemica biological sensing chip array Fe described in step one
3o
4siO
2the preparation of the DNA probe bio-sensing layer of magnetic nano-composite particle mark, is characterized in that:
(1) Fe is got
3o
4magnetic fluid, with washes of absolute alcohol twice, then uses ultrasonic cell disruption instrument ultrasonic disperse even, moves in three-necked flask, add ammoniacal liquor, regulate pH at room temperature to stir with average rate value;
(2) to Fe
3o
4dropwise add appropriate tetraethoxy (TEOS) in magnetic fluid mixed solution and react certain hour, again use washes of absolute alcohol twice after reaction, then clean twice with ultrapure water, the brown solution obtained, this magnetic fluid week age is soaked, by not wrapped Fe again with concentrated hydrochloric acid
3o
4magnetic ball removes, then with ultrapure water cleaning and Fe
3o
4siO
2magnetic nano-composite particle;
(3) electrochemica biological chip is cleaned twice in distilled water, dry; After liquid cell being covered electrochemica biological chip surface, put into electrochemica biological chip reaction tank together;
(4) Fe is added in 16 reaction members of corresponding electrochemica biological chip respectively
3o
4siO
2the mycobacterium tuberculosis that magnetic nano-composite particle is modified detects DNA probe, then the reaction tank containing electrochemica biological chip is put into after the electrochemica biological chip being full of nitrogen hatches and hatch in box, taking-up dehydrated alcohol cleans gently, and dries in nitrogen;
(5) put into electrochemica biological chip after adding mycobacterium tuberculosis Pretreated mixed solution in 16 reaction members of corresponding electrochemica biological chip respectively and hatch box.
Chemical-biological chip sensor array horseradish peroxidase (HRP) described in step 2, hydrogen peroxide (H
2o
2), the preparation of cascade detection signal amplification system that forms of the Mycobacterium tuberculosis DNA detection probes of tetramethyl biphenyl peace (TMB) and nanometer Au mark, it is characterized in that:
(1) add the Mycobacterium tuberculosis DNA detection probes of nanometer Au mark in 16 reaction members of corresponding electrochemica biological chip respectively, hatch rear taking-up PBS and rinse gently;
(2) horseradish peroxidase (HRP), hydrogen peroxide (H is added in 16 reaction members of corresponding electrochemica biological chip respectively
2o
2), tetramethyl biphenyl peace (TMB), by color reaction naked-eye observation colour developing result.
Step 3: by differential pulse map of current and current-time curvel figure statistic data, according to the mycobacterium tuberculosis drawing standard curve of detected different concns.
The present invention detects the principle of mycobacterium tuberculosis:
Clinical samples containing mycobacterium tuberculosis is directly joined magnetic reaction micropore, respectively by capture probe and the nanometer Au marker detection probe of Fe3O4SiO2 magnetic nano-composite particle mark, " dual enrichment " and biologically stable effect can be played to mycobacterium tuberculosis 16S rDNA specific sequence, utilize nanometer detection microballoon superparamagnetism to be expected to obtain very high mycobacterium tuberculosis target molecule separation efficiency and detection sensitivity; To be expected to realize target molecule " on-chip " by electrochemica biological sensing chip array characteristics be separated, do not need extra separating step, simplify the operation flow process greatly, shortens detection time simultaneously.
By horseradish peroxidase (HRP), hydrogen peroxide (H
2o
2) and nanometer Au form double cascade detection signal amplification system, with tetramethyl benzidine (TMB) for response matrix, make mycobacterium tuberculosis target nucleic acid molecule detected result visual, by electrochemica biological chip, mycobacterium tuberculosis bacillus 16S rDNA target sequence hybridization information is converted into readable electrical signal to export simultaneously, this signal is directly proportional to the mycobacterium tuberculosis 16S rDNA target sequence concentration in liquid to be checked, by drawing standard curve, can detect the mycobacterium tuberculosis in unknown clinical samples and quantitative analysis, result can carry out the visual and dual judgement of machine examination result.
The Rapid Detection of Mycobacterium Tuberculosis that the present invention sets up electrochemica biological sensing chip array and preparation method thereof, its feature embodied is:
(1) detect mycobacterium tuberculosis without the need to microbial culture from nucleic acid molecule, the detection speed that causes because of long-time microbial culture can be avoided slow;
(2) HRP-TMB-H is adopted
2o
2biological signals amplification system, good stability is highly sensitive, without the need to the amplification of PCR to target nucleic acid molecule, can carry out Visual Outcomes judgement, and for scene tuberculosis sample in enormous quantities examination provides possibility, and testing cost is lower;
(3) mycobacterium tuberculosis 16S rDNA " dual enrichment " isolation technique-electrochemica biological sensing chip array " on-chip " technology: namely directly complete the separation of mycobacterium tuberculosis 16SrDNA molecule in knot clinical samples, purification and detection on electrochemica biological sensing chip array, obviate traditional separation method centrifugation step time and effort consuming, the complicated troublesome drawback of tuberculosis sample preparation link, make whole experimentation operating process become more simple, rapid.
Embodiment:
Detect the electrochemica biological sensing chip array preparation method of mycobacterium tuberculosis bar:
(1) getting solid content is 25mg/mL magnetic fluid 50mL, with washes of absolute alcohol twice, then uses ultrasonic cell disruption instrument ultrasonic disperse even, move in 250mL three-necked flask, add ammoniacal liquor, pH value is adjusted to 11, at room temperature stir with 50rlmin ~ 500r/min speed.Then dropwise add appropriate TEOS by dropping funnel and react 5h ~ 24h.Again use washes of absolute alcohol twice after reaction, then clean twice with ultrapure water, the brown solution obtained.Fe
3o
4magnetic ball is by SiO
2first time wraps up, and soaks this magnetic fluid week age, by not wrapped Fe with 1mol/L concentrated hydrochloric acid
3o
4magnetic ball removes, and then with ultrapure water cleaning, is 25mg/mL by its constant volume to solid content;
(2) electrochemica biological chip is cleaned twice in distilled water, after liquid cell is covered electrochemica biological chip surface by oven dry 1min, put into electrochemica biological chip reaction tank together;
(3) Fe is added in 16 reaction members of corresponding electrochemica biological chip respectively
3o
4siO
2the mycobacterium tuberculosis that magnetic nano-composite particle is modified detects DNA probe solution 50 μ L, then the reaction tank containing electrochemica biological chip is put into after the electrochemica biological chip being full of nitrogen hatches and hatch 20min in box, taking-up dehydrated alcohol cleans gently, and dries 1min in nitrogen;
(4) put into electrochemica biological chip after adding mycobacterium tuberculosis Pretreated mixed solution in 16 reaction members of corresponding electrochemica biological chip respectively and hatch box.
(5) add the Mycobacterium tuberculosis DNA detection probes 50 μ L of nanometer Au mark in 16 reaction members of corresponding electrochemica biological chip respectively again, take out after hatching 20min and rinse 1-2 time gently with PBS;
(6) horseradish peroxidase (HRP), hydrogen peroxide (H is added in 16 reaction members of corresponding electrochemica biological chip respectively
2o
2), tetramethyl biphenyl peace (TMB) mixed solution 20 μ L, by color reaction naked-eye observation colour developing result.
(7) again by differential pulse map of current and current-time curvel map analysis data, according to the mycobacterium tuberculosis drawing standard curve of detected different concns.
Claims (4)
1. the electrochemica biological sensing chip array and preparation method thereof of a mycobacterium tuberculosis rapid detection, is characterized in that comprising the following steps:
(1) electrochemica biological sensing chip array Fe
3o
4siO
2the preparation of the DNA probe bio-sensing layer of magnetic nano-composite particle and nanometer Au mark;
(2) electrochemica biological sensing chip array horseradish peroxidase (HRP), hydrogen peroxide (H
2o
2) and the preparation of cascade detection signal amplification system that forms of the DNA detection probe of nanometer Au mark.
2. electrochemica biological sensing chip array Fe according to claim 1
3o
4siO
2the preparation of the DNA probe bio-sensing layer of magnetic nano-composite particle mark, is characterized in that:
(1) Fe is got
3o
4magnetic fluid, with washes of absolute alcohol twice, then uses ultrasonic cell disruption instrument ultrasonic disperse even, moves in three-necked flask, add ammoniacal liquor, at room temperature stir with average rate;
(2) to Fe
3o
4dropwise add appropriate TEOS in magnetic fluid mixed solution and react certain hour, after reaction, again use washes of absolute alcohol twice, then clean twice with ultrapure water, the brown solution obtained, then soak this magnetic fluid week age, by not wrapped Fe with concentrated hydrochloric acid
3o
4magnetic ball removes, then with ultrapure water cleaning and Fe
3o
4siO
2magnetic nano-composite particle;
(3) electrochemica biological chip is cleaned twice in distilled water, dry; After liquid cell being covered electrochemica biological chip surface, put into electrochemica biological chip reaction tank together;
(4) Fe is added in 16 reaction members of corresponding electrochemica biological chip respectively
3o
4siO
2the mycobacterium tuberculosis that magnetic nano-composite particle is modified detects DNA probe, then the reaction tank containing electrochemica biological chip is put into after the electrochemica biological chip being full of nitrogen hatches and hatch in box, taking-up dehydrated alcohol cleans gently, and dries in nitrogen;
(5) put into electrochemica biological chip after adding mycobacterium tuberculosis Pretreated mixed solution in 16 reaction members of corresponding electrochemica biological chip respectively and hatch box.
3. chemical-biological chip sensor array horseradish peroxidase (HRP) according to claim 1, hydrogen peroxide (H
2o
2), the preparation of cascade detection signal amplification system that forms of the Mycobacterium tuberculosis DNA detection probes of tetramethyl biphenyl peace (TMB) and nanometer Au mark, it is characterized in that:
(1) add the Mycobacterium tuberculosis DNA detection probes of nanometer Au mark in 16 reaction members of corresponding electrochemica biological chip respectively, hatch rear taking-up PBS and rinse gently;
(2) horseradish peroxidase (HRP), hydrogen peroxide (H is added in 16 reaction members of corresponding electrochemica biological chip respectively
2o
2), tetramethyl biphenyl peace (TMB), by color reaction naked-eye observation colour developing result.
4. the drafting of electrochemica biological chip detection intestinal bacteria typical curve according to claim 1, it is characterized in that: by differential pulse map of current and current-time curvel figure statistic data, according to the mycobacterium tuberculosis drawing standard curve of detected different concns.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017137192A1 (en) * | 2016-02-09 | 2017-08-17 | Mirnax Biosens, S.L. | Magnetic beads-based electrochemical biosensor |
| CN109321668A (en) * | 2018-10-24 | 2019-02-12 | 安徽大千生物工程有限公司 | Detect the preparation application method of the condenser type biochip of bacillus tuberculosis |
| CN110231479A (en) * | 2017-06-14 | 2019-09-13 | 杨华卫 | A kind of biochip |
-
2014
- 2014-02-27 CN CN201410068230.1A patent/CN104878075A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017137192A1 (en) * | 2016-02-09 | 2017-08-17 | Mirnax Biosens, S.L. | Magnetic beads-based electrochemical biosensor |
| CN110231479A (en) * | 2017-06-14 | 2019-09-13 | 杨华卫 | A kind of biochip |
| CN109321668A (en) * | 2018-10-24 | 2019-02-12 | 安徽大千生物工程有限公司 | Detect the preparation application method of the condenser type biochip of bacillus tuberculosis |
| CN109321668B (en) * | 2018-10-24 | 2022-05-17 | 梁鑫 | Preparation and use method of capacitive biochip for detecting human mycobacterium tuberculosis |
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Application publication date: 20150902 |