CN103645229B - Array for bacterial detection multiple galvanochemistry constant-temperature amplification chip and preparation method thereof - Google Patents
Array for bacterial detection multiple galvanochemistry constant-temperature amplification chip and preparation method thereof Download PDFInfo
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- CN103645229B CN103645229B CN201310676222.0A CN201310676222A CN103645229B CN 103645229 B CN103645229 B CN 103645229B CN 201310676222 A CN201310676222 A CN 201310676222A CN 103645229 B CN103645229 B CN 103645229B
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Abstract
The invention belongs to technical field of electrochemical detection, be specially multiple electrochemical constant-temperature amplification chip of a kind of array for Bacteria Detection and preparation method thereof.This chip is made up of the ito glass electrode basement of laser ablation and dimethyl silicone polymer microchip.With ito glass and dimethyl silicone polymer for material, be prepared from by laser ablation and micro-processing technology.This chip space array ordered arrangement, detects while utilizing the space region of amplified signal to assign to realize multiple nucleic acid object; Respectively containing a set of three-electrode system in each amplification pond: working electrode, to electrode, contrast electrode, by being connected with extraneous multi-channel electrochemical workstation, and carrying out LAMP isothermal reaction, detecting in real time; Quantitative analysis results is obtained by carrying out data processing to real-time curve chart.This chip manufacturing is simple, easy to operate, is easy to extensive preparation, provides effective scheme for LAMP method realizes the detection of clinical multiple pathogens simultaneous quantitative.
Description
Technical field
The invention belongs to technical field of electrochemical detection, be specifically related to a kind of array many galvanochemistry constant-temperature amplification chip and preparation method thereof.
Background technology
The infection of the upper respiratory tract, refers to the acute infection occurring in respiratory tract, and comprise common cold, influenza, nasopharyngitis, neck gland inflammation and laryngitis etc., how in self limiting, but incidence is higher.The pathogen 80% infected is virus, is secondly bacterium.And the patient of 78% is taken the microbiotic invalid to virus by diagnosis, this will seriously cause the generation of bacterial drug resistance.Diagnosis is generally diagnosed by the method for serology and microbe growth clinically, and sensitivity is lower, length consuming time.Therefore, timely qualitative and quantitative detection causes the bacterium of flu, for suiting the remedy to the case clinically and judging that disease degree is of great importance.
Ring mediated isothermal nucleic acid amplification (Loop-mediated isothermal amplification, LAMP) invented by Japanese Scientists the earliest, this has the advantages such as highly sensitive, specificity good, reaction velocity is fast, reaction conditions is simple in time, through the academic research of nearly 10 years, now come into the market with the form of various detection kit.But the quantitative test of LAMP reaction needs expensive accurate instrument, the laboratory that equipment is superior and specially trained technician.This is for timely diagnosis, is especially inapplicable in the diagnosis in the area of economics of underdevelopment.Galvanochemistry microarray technology, has gathered microflow control technique and electrochemical method, make use of the significantly enhancing of microflow control technique in micro-meter scale underflow effect, surface tension effect, capillary effect, and electrochemical apparatus economy is easy to get, the advantage of convenient operation.Traditional electrode preparation method comprises electrode print, plating, electro-deposition techniques, but plating and electro-deposition techniques comparatively loaded down with trivial details, be not easy to commercialization, the electric conductivity that prints electrode and reappearance general.And laser ablation indium tin oxide (indium tin oxide, ITO) glass electrode, laser is utilized to etch electrode accurately, both print electrode cheap had been remained, be easy to the extensive advantage made, improve its shortcoming simultaneously, accomplish electric conductivity, favorable reproducibility, can be intended to according to individual the object designing any pattern.This technology will exert far reaching influence to fields such as galvanochemistry, life science and clinical medicine.
Summary of the invention
The object of the invention is for the deficiencies in the prior art, a kind of array for Bacteria Detection multiple galvanochemistry constant-temperature amplification chip and preparation method thereof is provided.
Array for Bacteria Detection provided by the invention multiple galvanochemistry constant-temperature amplification chip, it is made up of the ito glass electrode basement of laser ablation and dimethyl silicone polymer (Polydimethylsiloxane, PDMS) microchip.See shown in accompanying drawing 1.Wherein, ito glass electrode, be independently made up of multiple independent array, each array forms by three-electrode system, and array quantity can according to the Demand Design of template detection thing, as triple, five weights, eightfold etc., unrestricted.Electrochemical signals detects by adding electrochemical activity molecule methylene blue, the double-stranded DNA constantly generated during itself and LAMP react has interaction, there is not same-action in the differential responses stage, and have multi-channel electrochemical workstation to detect in real time, thus reach the object of multiplex real-time identification.
The present invention also provides the preparation method of above-mentioned amplification chip, and its concrete steps are:
The preparation of a.ITO glass electrode substrate: electrode is can any shape, is etched on a certain size ito glass, as long as the place of etching pattern is conduction accurately by laser.It can comprise several independent working electrodes, and several are to electrode phase, and connect formation large ring, and draw electrochemical cell, to be connected with outer lead; All contrast electrodes are pooled to a middle formation circle, and deposit Ag/AgCl;
The pre-service of b.ITO glass electrode: used by the ito glass electrode etched distilled water, absolute ethyl alcohol and redistilled water ultrasonic cleaning clean, stand-by successively;
C. the structure of PDMS chip: include several circular amplification ponds, each passage has well, outage respectively, and all amplification ponds isolate all mutually, without interruption;
D. the making of chip template: utilize micro-processing technology (as MEMS, photoetching etc.), make chip template, formwork structure is corresponding with above-mentioned PDMS chip structure, and mould material can be silicon materials;
E. chip cast, degassed and solidification: after dimethyl siloxane and hardening agent are mixed, be poured in chip template, vacuum outgas, solidification, opens several wells, outage respectively, chip is cut into corresponding shape;
F. by plasma treatment, by the ito glass electrode handled well and PDMS chip bonding;
G. the chip of bonding is connected wire with conductive silver glue, after drying, use multi-purpose adhesive again to fix.
Above-mentioned amplification chip provided by the invention can be directly used in the Electrochemical Detection of DNA of bacteria; Its detection method is as follows:
By the working electrode of described chip, electrode is connected with multi-channel electrochemical workstation with contrast electrode, forms multiple three-electrode system; The appropriate LAMP reaction mixture configured is added from well, containing a certain amount of methylene blue solution in LAMP reaction mixture, a certain amount of bacterial lysate and LAMP reaction reagent; Chip is placed on constant temperature heating plate and heats, when temperature reaches 63 DEG C, start electrochemical measurement; Within the scope of certain potentials, carry out square wave voltammetry scanning, measured once every 1 minute, obtain real-time detector data; Process acquired results, obtains the linear relationship curve of the time of variable concentrations bacterium and the logarithm of concentration, utilizes calibration curve method to carry out analysis to clinical sample and detects.
Advantage and disadvantage of the present invention is as follows:
(1) present invention utilizes the advantage that ITO etches electrode, can precisely etch any electrode pattern by laser, not only avoid the technological process that complicated electro-deposition, plating etc. are complicated, also retains production in enormous quantities of printing electrode, the advantage that cost is low, its electric conductivity and reappearance excellence.
(2) the present invention utilizes the advantage of LAMP technology constant-temperature amplification, and combined with electrochemical workstation cost is low, and simply, the advantage such as easy to operate, avoids and use expensive experiment equipment, be very beneficial for the promotion and application of LAMP method in basic unit.
(3) utilize the advantage of electrochemical workstation multi-channel detection, realize Multiple detection, so that the multiple pathogen of Rapid identification.
(4) obtaining quantitative testing result from being loaded onto, being no more than 1h, simple and quick.
Accompanying drawing explanation
Fig. 1 is array multiple galvanochemistry constant-temperature amplification chip diagram.Wherein, top is divided into amplification chip pictorial diagram, and bottom is divided into ITO etching glass electrode basement structural representation.
Fig. 2 is the pulmonary bacillus (a-e, 2.8 × 10 that cause respiratory tract infection
5, 2.8 × 10
4..., 2.8 × 10
1copies/ μ L) time m-current differential curve map.Wherein, right figure is time m-log (concentration) curve map of the standard solution of bacterium.
Fig. 3 is the haemophilus influenzae (a-e, 1.7 × 10 that cause respiratory tract infection
5, 1.7 × 10
4..., 1.7 × 10
1copies/ μ L) time m-current differential curve map.Wherein, right figure is time m-log (concentration) curve map of the standard solution of bacterium.
Fig. 4 is the Klebsiella Pneumoniae (a-e, 1.6 × 10 that cause respiratory tract infection
5, 1.6 × 10
4..., 1.6 × 10
1copies/ μ L) time m-current differential curve map.Wherein, right figure is time m-log (concentration) curve map of the standard solution of bacterium.
Number in the figure: 1 is amplification pond, and 2 is well, and 3 is outage, and 4 is working electrode, and 5 is to electrode, and 6 is contrast electrode.
Embodiment
Below in conjunction with instantiation, the present invention is described further, but instantiation does not do any restriction to the present invention.
Embodiment
Utilize this array multiple galvanochemistry isothermal amplification technology platform, develop a eight passages, pulmonary bacillus, haemophilus influenzae, the array multiple galvanochemistry constant-temperature amplification chip of Klebsiella Pneumoniae somatotype.
The preparation process of the multiple galvanochemistry of the array for the Bacteria Detection constant-temperature amplification chip in the present embodiment is as follows:
(1) preparation of ito glass electrode basement: be etched in 4cm accurately by laser wide, the ito glass that 1mm is thick etches the array-type electrode of Eight-Diagram tactics shape, only has the place of etching pattern to be conduction.It comprises eight independent working electrodes, and one is interconnected to form a large ring to electrode, and draws electrochemical cell to be connected with outer lead.All contrast electrodes are pooled to a middle formation circle, and deposit Ag/AgCl; Used by the ito glass electrode etched distilled water, absolute ethyl alcohol and redistilled water ultrasonic cleaning clean, stand-by successively;
(2) preparation of PDMS chip: if it comprises eight circular amplification ponds, each passage has well, outage respectively.All amplification ponds isolate all mutually, without interruption; Utilize the method for micro-processing technology (MEMS, photoetching etc.) to make chip template, formwork structure is corresponding with above-mentioned PDMS chip structure, and mould material can be silicon materials; After by dimethyl siloxane and hardening agent alkoxy silane, 5-12:1 mixes in mass ratio, be carefully poured in chip template, after vacuum outgas 30min, 80 DEG C of solidification 2h, open several wells respectively, outage and; Chip is cut into octagon;
(3) for the preparation of the array multiple galvanochemistry constant-temperature amplification chip of Bacteria Detection: by plasma treatment, by the above-mentioned ito glass electrode handled well and the permanent bonding of PDMS chip; The chip of bonding is connected wire with conductive silver glue, spends the night after drying, use multi-purpose adhesive again to fix.
The electrochemical monitoring of the present embodiment
The LAMP reactant liquor 20 μ L of three kinds of bacterium master samples containing 1.6 μ L and 10 μMs of methylene blues is added between two respectively, wherein containing 10 μMs of methylene blue solutions in 6 amplification ponds; Not containing bacteria sample in two other amplification pond, as blank.Capping hole, respective electrode is connected with the wire of eight passage electrochemical workstations, and is placed on constant temperature heating plate by chip and heats, and when temperature reaches 63 DEG C, starts electrochemical measurement; In the potential range of-0.1 V, carry out square wave voltammetry scanning from-0.6 V, measured once every 1 minute, obtain real-time detector data; Process acquired results, obtains the linear relationship curve of the time of three groups of variable concentrations bacteriums and the logarithm of concentration, utilizes calibration curve method to carry out analysis to clinical sample and detects, as shown in Figure 2, Figure 3 and Figure 4.Pulmonary bacillus (a-e, 2.8 × 10
5, 2.8 × 10
4..., 2.8 × 10
1copies/ μ L), haemophilus influenzae (a-e, 1.7 × 10
5, 1.7 × 10
4..., 1.7 × 10
1copies/ μ L), Klebsiella Pneumoniae (a-e, 1.6 × 10
5, 1.6 × 10
4..., 1.6 × 10
1copies/ μ L) linear equation be respectively y1=-4.62x+45.930; Y1=-6.95962x+51.928; Y3=-6.26595x+44.8908, linearly dependent coefficient is respectively, r1=0.9917; R2=0.9944; R3=0.9943; Detectability is respectively 28 copies/ μ L, 17 copies/ μ L, 16 copies/ μ L.
The present invention is combined with electrochemical method by array micro-fluidic chip, and the LAMP signal realizing various bacteria detects in real time, improves the present situation of the accurate expensive instrument of LAMP reaction needed, is very beneficial for the promotion and application of LAMP method in basic unit.Prepared the method for electrode by laser ablation ito glass, avoid the technological process that complicated electro-deposition, plating etc. are complicated, any spatial multiplex array arrangement in the pond that simultaneously can realize as required chip increases.This chip structure pattern theory has unlimited extensibility, LAMP multiple arbitrarily can be completed according to the actual requirements and effectively increase, realize the Quantitative detection of Mutiple Targets, thus meet the quick diagnosis of clinical any multiple pathogens or genetic disease.
Claims (2)
1., for a preparation method for the array multiple galvanochemistry constant-temperature amplification chip of Bacteria Detection, this chip is made up of the ito glass electrode basement of laser ablation and polydimethylsiloxane microchip; Wherein, ito glass electrode is independently made up of multiple independent array, and each array forms by three-electrode system, the Demand Design of array quantity basis template detection thing; Described electrode is any shape, is etched on a certain size ito glass by laser accurately, the place conduction of etching pattern; It comprises several independent working electrodes, and several are to electrode phase, and connect formation large ring, and draw electrochemical cell, to be connected with outer lead; All contrast electrodes are pooled to a middle formation circle, and deposit Ag/AgCl;
It is characterized in that concrete steps are:
The preparation of a.ITO glass electrode substrate: electrode is any shape, is etched on a certain size ito glass accurately by laser, the place conduction of etching pattern; It comprises several independent working electrodes, and several are to electrode phase, and connect formation large ring, and draw electrochemical cell, to be connected with outer lead; All contrast electrodes are pooled to a middle formation circle, and deposit Ag/AgCl;
The pre-service of b.ITO glass electrode: used by the ito glass electrode etched distilled water, absolute ethyl alcohol and redistilled water ultrasonic cleaning clean, stand-by successively;
The preparation of c.PDMS chip: PDMS chip includes several circular amplification ponds, and each passage has well, outage respectively, and all amplification ponds isolate all mutually, without interruption;
D. the making of chip template: utilize micro-processing technology to make chip template, formwork structure is corresponding with above-mentioned PDMS chip structure, and mould material is silicon materials;
E. chip cast, degassed and solidification: after 5-12:1 mixes in mass ratio by dimethyl siloxane and hardening agent alkoxy silane, be poured in chip template, vacuum outgas, solidification, opens several wells, outage respectively, chip is cut into corresponding shape;
F. by plasma treatment, by the ito glass electrode handled well and PDMS chip bonding;
G. the chip of bonding is connected wire with conductive silver glue, after drying, use multi-purpose adhesive again to fix.
2. the application of array multiple galvanochemistry constant-temperature amplification chip in DNA of bacteria Electrochemical Detection that prepare of preparation method as claimed in claim 1, is characterized in that concrete steps are as follows:
By the working electrode of described chip, electrode is connected with multi-channel electrochemical workstation with contrast electrode, forms multiple three-electrode system; The LAMP reaction mixture configured is added from well, containing a certain amount of methylene blue solution, bacterial lysate and LAMP reaction reagent in LAMP reaction mixture; Chip is placed on constant temperature heating plate and heats, when temperature reaches 63 DEG C, start electrochemical measurement; Within the scope of certain potentials, carry out square wave voltammetry scanning, measured once every 1 minute, obtain real-time detector data; Process acquired results, obtains the linear relationship curve of the time of variable concentrations bacterium and the logarithm of concentration, utilizes calibration curve method to carry out analysis to clinical sample and detects.
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| SG10201808874XA (en) * | 2014-04-09 | 2018-11-29 | Agency Science Tech & Res | Microfluidic device |
| CN103913486B (en) * | 2014-04-12 | 2016-06-08 | 太原理工大学 | The preparation method of AuNPs-PDMS compound mems thin film biosensor |
| CN104407036B (en) * | 2014-11-06 | 2017-12-08 | 上海慧观贸易有限公司 | Preparation and its application for the electrochemical microfluidic control device of nucleic acid isothermal amplification |
| CN105891192A (en) * | 2016-06-24 | 2016-08-24 | 大连理工大学 | Electrochemical and electrochemical luminescence detection method based on integrated three-electrode system microchip |
| CN106124596A (en) * | 2016-06-30 | 2016-11-16 | 英太格电子科技（苏州）有限公司 | A kind of preparation technology of biochemical test sheet |
| CN106198675A (en) * | 2016-09-28 | 2016-12-07 | 扬州大学 | The manufacture method of the integrated chip sensor of biological immune electrochemical impedance |
| CN106483288B (en) * | 2016-10-14 | 2018-01-30 | 哈尔滨工业大学 | A kind of enterorrhagia Bacillus coil 0157:H7 detection platforms and preparation method thereof |
| EP3668650A4 (en) * | 2017-10-03 | 2021-06-02 | Avails Medical, Inc. | Apparatus, systems, and methods for determining the concentration of microorganisms and the susceptibility of microorganisms to anti-infectives based on redox reactions |
| CN112175785B (en) * | 2019-07-03 | 2023-10-13 | 中国科学院苏州生物医学工程技术研究所 | Dual-channel isothermal amplification device and method |
| CN114720537B (en) * | 2022-03-21 | 2024-03-22 | 上海科技大学 | Polyelectrolyte hydrogel ion diode, preparation method thereof and application thereof in nucleic acid detection |
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