CN102288755A - PDMS (Polydimethylsiloxane) multichannel immunoassay chip for rapid field detection of microorganisms - Google Patents
PDMS (Polydimethylsiloxane) multichannel immunoassay chip for rapid field detection of microorganisms Download PDFInfo
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Abstract
The invention discloses a PDMS (Polydimethylsiloxane) micro array immunoassay chip for rapid field detection of a plurality of microorganisms in a food or drinking water sample. In the PDMS micro array immunoassay chip, glass is taken as a substrate, a PDMS chip comprising a plurality of microfluidic channels and the glass are sealed with each other, a plurality of parallel channels are gathered at a gateway, and the quantity of the parallel channels is determined by the quantity of samples which need to be analyzed by one chip and the quantity of the kinds of microorganisms. Due to the adoption of the PDMS micro array immunoassay chip, multicomponent analysis of the samples of different kinds or concentrations can be realized; samples to be detected are introduced by using a negative pressure sample introduction method, so that simultaneous introduction of different solutions in different channels can be realized, the sample introduction time is saved, rapid detection is realized, simultaneous sample introduction of various components is realized, and equipment is simplified; a scattering mark method is introduced into a microfluidic immunoassay chip, so that the sensitivity of microorganism detection is improved, and microorganisms of 2cfu/mL can be detected in minimum; a device has a small size, and has contribution to minimization of the chip; and the channel design has high flexibility, so that high flux detection can be realized.
Description
Technical field
The present invention relates to a kind of hyperchannel PDMS chip, its method for making, and the analytical approach that is used for microbial rapid detection.
Background technology
The detection of pathogenic bacteria is water quality monitoring all the time, the key content of food hygiene and food safety detection, along with constantly advancing of human society and improving constantly of people's quality of life, the new demand new standard is proposed constantly also for the research of pathogenic bacteria detection method.At present, the microorganism detection technology mainly is divided into three major types according to the difference of its principle: the detection method that cultivate based on flat board (1).China still rests on separation and Culture, morphologic observation, biochemical identification and serological typing level mostly for detection, the evaluation of pathogen at present.Although these traditional pathogenic bacteria methods of inspection are highly sensitive, expense is low, can access the qualitative and quantitative result of aspect such as bacterial number and characteristic in water sample or the food samples, but traditional detection method complicated operation, cycle are longer, obtaining the result needs several days time usually; And the bacterial multiplication that requires to detect is visible bacterium colony, can't the pathogen that be difficult to cultivate be detected.Classic method has increased breadboard workload at the everyways such as detection of medium preparation, microbe growth, colony counting and biochemical indicator in addition.Therefore, no matter classic method is the monitoring in environment measuring or food industry production procedure, and still the quality control at finished product is all more and more to show weak point.(2) detection technique of reacting based on the microorganism specific enzyme.Some specific enzyme can be synthesized and discharge to bacterium in its growth and breeding process, the characteristic of pressing enzyme is selected corresponding substrate and indicator for use, and they are formulated in the relevant nutrient culture media.According to the change color that occurs behind the bacterial reaction, determine suspicious bacterial strain to be separated. the qualitative results of reaction helps the quick diagnosis of bacterium.For example. there is active C8 esterase in the salmonella.In nutrient culture media, add chromogenic substrate, detect salmonella by detecting the C8 esterase.In this colour developing differential medium, the salmonella bacterium colony is rendered as pink to purple, the salmonella of separable all serotypes not only, can also identify the bacterium of Serratieae, the vigorous Salmonella of Cray and different enzyme characteristics such as Escherichia coli, mould by the color difference of growth bacterium colony on this nutrient culture media.Though chromogenic culture medium has higher specificity and sensitivity, also some deficiency in testing, as: when detecting the microorganism of mixed infection, a certain proportion of false positive or false negative can appear; May occur different strain on some chromogenic culture medium and present similar color, or same microorganism presents different colours etc., these all remain further to be improved.(3) based on the inspection technology of nucleic acid.Comprising the dna fingerprinting technology that relies on PCR, multiplex PCR detection technique (m-PCR), quantitative fluorescent PCR, biochip technology, quantitative PCR detection technique etc.Polymerase chain reaction (PCR) is nearly most widely used during the last ten years molecular biology method, all be to increase in the detection of food-borne pathogens with the nucleotide sequence of its inhereditary material high conservative design special primer, and then with gel electrophoresis and ultraviolet detection of nucleic acids instrument observation amplification.Other novel round pcrs all grow up on this basis.So far, discovery and synthesizing of correspondent probe to multiple microorganism specific gene have been obtained very big progress, people such as Fillal are separated to the probe that is applicable to that salmonella detects, and this probe can be discerned the different salmonella of 350 strains, and minimum detectable activity is 1 cfu/25 g sample.AOAC approves GENE-TARK salmonella probe analysis method recently, and utilizing this method is 100% to 239 strain salmonella recall rates, and false positive rate is 0.8%.The probe that FDA developed at listeria spp beta hemolysin Disease-causing gene at first in 1987, GENE-TARK develops the DNA probe that commercialization detects listeria spp subsequently, it can specific recognition the 16S rRNA of bacterium, false negative rate is 0.8% ~ 4.7%.Bessesen etc. utilize probe in detecting monocyte hyperplasia listeria spp, and the DNA detection limit is lower than 25 ng (being less than 1000 thalline), method high specificity.But to the requirement of equipment generally higher (as PCR instrument, fingerprint pattern technology etc.), simultaneously, technical level of operators has higher requirements based on the detection method of nucleic acid, is not suitable for on-the-spot fast detecting.(4) based on the detection technique of immunological response, comprising enzyme-linked immuno assay (ELISA) technology, the immune magnetic microsphere isolation technics reaches the nano gold mark immuno analytical method.This technology is aided with immune amplifying technique again and differentiates bacterium by the specificity association reaction of antigen and antibody.Kryinskiand Heimsch etc. were used for ELISA the detection of food salmonella (Salmonella spp.) first in 1977, and in application, constantly developed, the eighties, Paadhye and Park detected Escherichia coli Ol57 with the ELISA of monoclonal or polyclonal antibody respectively: and H7 (Escherichia coli O157:H7).Its second grade of literary composition is used Salmonella monoclonal antibody specific CB8DE and has been set up the direct ELISA method that detects salmonella, and the enzyme mark monoclonal antibody and the Bacterium enteritidis lipopolysaccharides (LPS) of anti-Bacterium enteritidis 09 antigen of application such as Zhang Yanhong have been set up the competitive ELISA method of fast detecting Bacterium enteritidis.Immunological method has high specificity, highly sensitive, plurality of advantages such as the reaction time is short, be beneficial to on-the-spot carrying out of detecting, but traditional immunological technique makes checkout equipment complicated because its label methods such as (fluorescence probe/chemiluminescent labelings/enzyme mark) needs specific high-end devices to detect.In addition, limited based on the sample size that the elisa technique of porous plate allows, the sample less for the unit volume bacterial number is also improper, and because the volume of bacterium is bigger, the dynamic process that combines between the antibody bottom porous plate is slower, thereby prolonged time of elisa assay, reduced sensitivity.
Summary of the invention
The technical problem to be solved in the present invention is that the multi-passage circulating formula immunoassay chip of specified microorganisms content analysis in a kind of potable water or the food is provided.The introducing amount of sample can arbitrarily be controlled in this chip, utilizes the short characteristics of diffusion length in the microchannel, and the immune response speed of microorganism and antibody is obviously accelerated; On chip piece, realize a plurality of samples, the parallel analysis of multiple microorganism simultaneously; This chip can also obtain analytic signal with portable optical detecting instrument scene, realizes the fast detecting of microorganism.
The present invention is achieved by the following technical solutions:
The present invention is a kind of PDMS hyperchannel immunoassay chip that is used for the microorganism field quick detection, with glass as substrate, the PDMS chip and the glass involution that will comprise a plurality of microchannels, parallel a plurality of passages are converged at a gateway, and the quantity of parallel channel depends on the sample quantities of a required analysis of chip and the species number of microorganism.
The present invention is coupled at the microorganism specific antibody in the PDMS passage of amination processing by the method for covalent coupling, form the immunoassay chip of flow type, mouthful place that converges at each passage applies negative pressure, can in each passage, introduce variety classes or contain the sample solution of varying number microorganism, catch objective microbe with making antibody specificity, behind the unconjugated microorganism of flush away, by converging the antibody probe of inlet injection nano-metal particle scattering mark, microorganism is carried out mark, behind the unreacted probe of flush away, nano-metal particle is carried out silver dye the visual signal of generation.
After the present invention adopts the described method of claim (2) to obtain visual signals, its detection mode is: use halogen tungsten lamp, laser or LED are as detection light source, to be radiated at the zone that deposits metallic particles in the passage after the detection light source focusing, CMOS, the CCD picture pick-up device is taken the imaging signal of each passage, and obtain the scattered light intensity of each passage with image processing software, also available fiber spectrometer, or photoelectric conversion device is measured scattered intensity, obtain bacterial number in scattered light intensity and the sample working curve, after detecting the scattered light intensity of actual sample, can obtain the signal of micro organism quantity in the sample according to working curve.
Be connected by unified sample introduction or outlet between many passages of the present invention, changed the complex appts of every independent sample introduction of analysis channel needs of micro-fluidic chip in the past, conversion by malleation and negative pressure, both can realize disposable introducing to solution of the same race in many passages, also can realize disposable introducing to many passage different solutions, reduce the use of pump, saved the time that sample is introduced simultaneously greatly, realized fast detecting.
Beneficial effect of the present invention is as follows:
1, can realize the multicomponent analysis of variety classes or concentration sample.2, utilize negative pressure sampling method to introduce testing sample, introduce when can realize in the different passages different solutions, save sample injection time, realize fast detecting.3, each component while sample introduction, simplified apparatus.4, in micro-fluidic immuno-chip, introduce the method for scattering mark, the sensitivity that has improved microorganism detection, the minimum microorganism that detects 2cfu/mL.5, device volume is little, is beneficial to the microminiaturization of chip.6, the dirigibility of channels designs is strong, can realize high throughput testing.
Description of drawings
Accompanying drawing 1 is that chip side is to structural representation;
The 1st, glass substrate; The 2nd, the PDMS cover plate; The 3rd, injection port; 4a-4d is the microchannel; The 5th, the sample introduction pipe; 6a-6d is a liquid storage tank.
Accompanying drawing 2 is a chip channel design plane synoptic diagram;
The 3rd, injection port; 4a-4d is the microchannel;
Accompanying drawing 3 is the synoptic diagram of chip channel scattered light checkout equipment;
The 7th, light source; The 8th, chip; The 9th, detecting device.
Embodiment
Be described further below in conjunction with the technical scheme of accompanying drawing this law:
(1) design of PDMS chip: design a kind of multichannel PDMS chip, its number of channels is by the sample quantities and the microbe species decision of the each required analysis of chip.Conventional method is adopted in the making of PDMS chip, does not belong to category of the present invention.With PDMS chip and measure-alike glass sheet through sealing mutually after the oxygen plasma treatment and becoming a kind of multichannel chip.The structure of chip such as Fig. 1 are shown in Fig. 2.It comprises: glass substrate 1, the sealing-in PDMS cover plate 2 on glass substrate comprises injection port 3, many microchannel 4a-4d, sample introduction pipe 5, liquid storage tank 6a-6d in the PDMS cover plate 2.Be connected by unified injection port 3 or outlet between many microchannel 4a-4d.When needs solution sample introduction of the same race, inject sample solution by the syringe pump injection pattern by injection port 3, when needs solution difference not of the same race sample introduction, extract so that negative pressure to be provided by injection port 3 by the syringe pump decimation pattern, the solution among the liquid storage tank 6a-6d is entered respectively among each bar microchannel 4a-4d.The design of this chip, changed the complex appts of every independent sample introduction of analysis channel needs of micro-fluidic chip in the past, conversion by malleation and negative pressure, both can realize disposable introducing to solution of the same race in many passages, also can realize disposable introducing to many passage different solutions, reduce the use of pump, saved the time that sample is introduced simultaneously greatly, realized fast detecting.
(2) coupling of microorganism antibody in the passage: the glass table is through after the alkali treatment, clean with distilled water, and with the glass surface in the amination silane agent treated passage, make amination group on the glass surface band, with difunctional coupling reagent (as glutaraldehyde etc.) activation glass surface, converge injection port 3 places at passage and execute negative pressure the solution of microorganism specific antibody is sucked each passage, make antibody coupling in the glazing channel surface.Aforesaid way can be realized the different microorganism antibody of coupling in each passage, detects when carrying out various pathogens.Also can be in each passage the identical microorganism antibody of coupling, carry out the parallel detection of many samples.
(3) utilize hyperchannel immunoassay chip detection microorganism: execute negative pressure at injection port 3 places and in each passage, introduce the sample solution that variety classes or variable concentrations contain microorganism, make antibody specificity catch microorganism, behind the unreacted microorganism of flush away, apply malleation at injection port 3 places and inject the antibody probe of nano-metal particle mark, make it to form sandwich complex with captive microorganism, behind the unreacted probe of flush away, inject silver-colored transfection reagent at injection port 3 places equally, under nano-metal particle catalysis, the silver ion reduction deposition forms the visual signals of immune combination.
(4) observation of immune binding signal in each passage: the silver-colored particle of deposition has extremely strong scattering process to incident light in the passage, can observe corresponding immune binding signal by naked eyes.But visual inspection can only be carried out semi-quantitative analysis to micro organism quantity.If carry out the optical device (as shown in Figure 3) that quantitative test need adopt the present invention to describe the light scattering signal of multi-channel chip is analyzed.The adoptable light source 7 of the present invention comprises: halogen tungsten lamp, laser, or LED, to be radiated at the zone that passage 8 deposits metallic particles after the detection light source focusing, light scattering detector 9 comprises: CMOS, CCD picture pick-up devices etc., the light scattering signal of taking each passage can produce the multi channel imaging signal, and obtains the scattered light intensity of each passage with image processing software.Light scattering detector 9 is the available fiber spectrometer also, or photo-electric conversion element is measured scattered intensity.After obtaining the working curve of bacterial number in scattered light intensity and the sample, the scattered light intensity of actual sample can be detected, micro organism quantity in the sample can be recorded according to working curve.
Below by specific embodiment the present invention is described in detail below:
Embodiment 2: chip finishing specificity Escherichia coli antibody in the passage.To get the precipitation thalline behind 48 hours the Escherichia coli medium centrifugal of 37 ℃ of cultivations, add deionized water 10 doubling dilutions and be 1,10-1,10-2 ..., 10-8 concentration, in liquid storage tank 6a-6d, add the dilution of blank solution (deionized water), 10-2,10-5, three concentration of 10-8 respectively.During detection, with corresponding successively the insertion among the liquid storage tank 6a-6d of sample introduction pipe of microchannel 4a-4d, execute negative pressure to extract the solution in the liquid storage tank at injection port 3, reaction end back is executed malleation at injection port 3 and is injected golden labeling antibody and silver-colored transfection reagent successively.The cleaning of chip is executed malleation at injection port 3 equally and is injected PBS buffer solution irrigation channel.Silver dyes the back and thoroughly cleaned chip 2 ~ 3 minutes with deionized water, dries up the back and pass through 20 * object lens recording channel inscattering spectrum under the metaloscope dark field mode.The scattering spectrum peak strength can be set up linear relationship with bacterial concentration, and can utilize this linear relationship to carry out quantitatively.
Embodiment 3: use this chip that different types of microorganism is detected.Chip finishing specificity Escherichia coli antibody in the passage.Respectively 37 ℃ are cultivated and get the precipitation thalline after 48 hours Escherichia coli and 22 ℃ are cultivated P17 bacterium after 48 hours and spirillum medium centrifugal, add deionized water and be diluted to 10-4 concentration.In liquid storage tank 6a-6d, add blank solution (deionized water), P17 bacterium dilution, spirillum dilution, Escherichia coli dilution respectively.During detection, with corresponding successively the insertion among the liquid storage tank 6a-6d of sample introduction pipe of microchannel 4a-4d, execute negative pressure to extract the solution in the liquid storage tank at injection port 3, reaction end back is executed malleation at injection port 3 and is injected golden labeling antibody and silver-colored transfection reagent successively.The cleaning of chip is executed malleation at injection port 3 equally and is injected PBS buffer solution irrigation channel.Thoroughly cleaned chip 2 ~ 3 minutes with deionized water after silver dyes, dry up the back and under the metaloscope dark field mode, observe the chip channel pictures and write down the scattering spectrum data with CCD with amplifying 20 times of camera lenses.
Claims (4)
1. PDMS hyperchannel immunoassay chip that is used for the microorganism field quick detection, it is characterized in that: with glass as substrate, the PDMS chip and the glass involution that will comprise a plurality of microchannels, parallel a plurality of passages are converged at a gateway, and the quantity of parallel channel depends on the sample quantities of a required analysis of chip and the species number of microorganism.
2. hyperchannel immunoassay chip according to claim 1, it is characterized in that, method by covalent coupling is coupled at the microorganism specific antibody in the PDMS passage of amination processing, form the immunoassay chip of flow type, mouthful place that converges at each passage applies negative pressure, can in each passage, introduce variety classes or contain the sample solution of varying number microorganism, catch objective microbe with making antibody specificity, behind the unconjugated microorganism of flush away, by converging the antibody probe of inlet injection nano-metal particle scattering mark, microorganism is carried out mark, behind the unreacted probe of flush away, nano-metal particle is carried out silver dye the visual signal of generation.
3. hyperchannel immunoassay chip according to claim 2, it is characterized in that, after adopting the described method of claim (2) to obtain visual signals, its detection mode is: use halogen tungsten lamp, laser or LED are as detection light source, to be radiated at the zone that deposits metallic particles in the passage after the detection light source focusing, CMOS, the CCD picture pick-up device is taken the imaging signal of each passage, and obtain the scattered light intensity of each passage with image processing software, also available fiber spectrometer, or photoelectric conversion device is measured scattered intensity, obtain bacterial number in scattered light intensity and the sample working curve, after detecting the scattered light intensity of actual sample, can obtain the signal of micro organism quantity in the sample according to working curve.
4. hyperchannel immunoassay chip according to claim 3, it is characterized in that: be connected by unified sample introduction or outlet between many passages, changed the complex appts of every independent sample introduction of analysis channel needs of micro-fluidic chip in the past, conversion by malleation and negative pressure, both can realize disposable introducing to solution of the same race in many passages, also can realize disposable introducing to many passage different solutions, reduced the use of pump, save simultaneously the time that sample is introduced greatly, realized fast detecting.
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| CN103087899A (en) * | 2013-01-17 | 2013-05-08 | 湖南大学 | Aptamer-based microfluidic chip capable of capturing cancer cells and preparation thereof as well as separation method of cancer cells |
| CN103185802A (en) * | 2011-12-30 | 2013-07-03 | 国家纳米科学中心 | Multiphase microfluidic immunoblotting chip, and preparation method and application thereof |
| CN105510574A (en) * | 2015-11-25 | 2016-04-20 | 深圳出入境检验检疫局食品检验检疫技术中心 | Fluorescent nano probe, preparation method and method for synchronously detecting a plurality of harmful factors in food |
| CN107064493A (en) * | 2017-04-17 | 2017-08-18 | 武汉赛思锐微生物技术有限公司 | The detection method of antigen/antibody in trace sample |
| CN111804356A (en) * | 2020-07-16 | 2020-10-23 | 清华大学 | Microfluidic chip and preparation method thereof, microfluidic device and detection method of pathogenic bacteria |
| CN112570052A (en) * | 2020-12-07 | 2021-03-30 | 厦门大学 | Micro-fluidic device containing probe array and micro-fluidic chip with side channels |
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Cited By (10)
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| CN103185802A (en) * | 2011-12-30 | 2013-07-03 | 国家纳米科学中心 | Multiphase microfluidic immunoblotting chip, and preparation method and application thereof |
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| CN103087899A (en) * | 2013-01-17 | 2013-05-08 | 湖南大学 | Aptamer-based microfluidic chip capable of capturing cancer cells and preparation thereof as well as separation method of cancer cells |
| CN105510574A (en) * | 2015-11-25 | 2016-04-20 | 深圳出入境检验检疫局食品检验检疫技术中心 | Fluorescent nano probe, preparation method and method for synchronously detecting a plurality of harmful factors in food |
| CN105510574B (en) * | 2015-11-25 | 2018-11-20 | 深圳出入境检验检疫局食品检验检疫技术中心 | Fluorescent nano probe and preparation method thereof and the method for the synchronous detection of Hazard factors a variety of in food |
| CN107064493A (en) * | 2017-04-17 | 2017-08-18 | 武汉赛思锐微生物技术有限公司 | The detection method of antigen/antibody in trace sample |
| CN111804356A (en) * | 2020-07-16 | 2020-10-23 | 清华大学 | Microfluidic chip and preparation method thereof, microfluidic device and detection method of pathogenic bacteria |
| CN112570052A (en) * | 2020-12-07 | 2021-03-30 | 厦门大学 | Micro-fluidic device containing probe array and micro-fluidic chip with side channels |
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