CN102288755B - 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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- CN102288755B CN102288755B CN2011102014024A CN201110201402A CN102288755B CN 102288755 B CN102288755 B CN 102288755B CN 2011102014024 A CN2011102014024 A CN 2011102014024A CN 201110201402 A CN201110201402 A CN 201110201402A CN 102288755 B CN102288755 B CN 102288755B
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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 microbes detect method.At present, the microorganism detection technology, according to the difference of its principle, mainly is divided into three major types: the detection method that cultivate based on flat board (1).China still rests on separation cultivation, 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 the aspect such as bacterial number and characteristic in water sample or food samples, but traditional detection method complicated operation, cycle are longer, obtaining result needs the time of several days 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 the workload in laboratory at the everyways such as detection of nutrient culture media 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, or in the quality control of finished product, is all more and more to show weak point.The detection technique of (2) based on the microorganism specific enzyme, reacting.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, and they are formulated in relevant nutrient culture media., according to the change color that occurs after 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 salmonella.Add chromogenic substrate in nutrient culture media, by detecting the C8 esterase, detect salmonella.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 be different by the color of growth bacterium colony on this nutrient culture media, identify the bacterium of Serratieae, the vigorous Salmonella of Cray enzyme characteristics different from Escherichia coli, mould etc.Although chromogenic culture medium has higher specificity and sensitivity, also some deficiency in testing, as: while detecting the microorganism of mixed infection, there will be a certain proportion of false positive or false negative; May occur on some chromogenic culture medium that different strain presents similar color, or same microorganism presents different colours etc., these all remain further to be improved.(3) take the inspection technology of nucleic acid as basis.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 most widely used closely during the last ten years molecular biology method, all to increase with the nucleotide sequence of its inhereditary material high conservative design special primer in the detection of food-borne pathogens, 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 the synthetic of correspondent probe to the multiple-microorganism specific gene make great progress, the people such as Fillal are separated to and are applicable to the probe that salmonella detects, and this probe can be identified 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 for listeria spp beta hemolysin Disease-causing gene at first in 1987, GENE-TARK develops commercialization and detects the DNA probe of listeria spp subsequently, it can specific recognition the 16S rRNA of bacterium, false negative rate is 0.8% ~ 4.7%.Bessesen etc. utilize the probe in detecting Listeria Monocytogenes, and the DNA detection limit is lower than 25 ng (being less than 1000 thalline), method high specificity.But the detection method take nucleic acid as basis is to the requirement of equipment generally higher (as PCR instrument, fingerprint pattern technology etc.), and simultaneously, operating personnel's technical merit has higher requirements, and is not suitable for on-the-spot fast detecting.(4) take the detection technique of immunological response as basis, comprising enzyme-linked immuno assay (ELISA) technology, immune magnetic microsphere isolation technics, and nano gold mark immuno analytical method.The specific binding reaction of this technology by antigen and antibody, then be aided with immune amplifying technique and carry out discriminating bacteria.Kryinskiand Heimsch etc. were used for ELISA the detection of salmonella in food (Salmonella spp.) first in 1977, and constantly developed in application, 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 application Salmonella monoclonal antibody specific of literary composition CB8DE has set up the Salmonella method that detects salmonella, and enzyme mark monoclonal antibody and the Bacterium enteritidis lipopolysaccharides (LPS) of anti-Bacterium enteritidis 09 antigen of the 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, the plurality of advantages such as the reaction time is short, be beneficial to carrying out of Site Detection, but traditional immunological technique detects because the methods such as its label (fluorescence probe/chemiluminescent labeling/enzyme mark) need specific high-end devices, makes checkout equipment complicated.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 larger, and between the antibody of porous plate bottom, the dynamic process of combination is slower, thereby extended 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 food is provided.In this chip, the introducing amount of sample can arbitrarily be controlled, and utilizes the short characteristics of diffusion length in microchannel, and the immune response speed of microorganism and antibody is obviously accelerated; Realize simultaneously a plurality of samples, the parallel analysis of multiple-microorganism on chip piece; 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 hyperchannel of PDMS for microorganism field fast detecting Immunoassay Chip, use glass as substrate, 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 introduce variety classes in each passage or contain the sample solution of varying number microorganism, make antibody target acquisition microorganism specifically, after washing away unconjugated microorganism, inject the antibody probe of nano-metal particle scattering mark by converging entrance, microorganism is carried out mark, after washing away unreacted probe, 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, after being focused on, detection light source is radiated in passage the zone that deposits metallic particles, 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 the scattering light intensity, obtain bacterial number in scattered light intensity and sample working curve, after detecting the scattered light intensity of actual sample, can obtain according to working curve the signal of micro organism quantity in sample.
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 the disposable introducing to solution of the same race in many passages, also can realize the disposable introducing to many passage different solutions, reduce the use of pump, the time of greatly having saved sample introduction simultaneously, 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 the interior different solutions of different passages, save sample injection time, realize fast detecting.3, each component while sample introduction, simplified apparatus.4, introduce the method for scattering mark in micro-fluidic immuno-chip, the sensitivity that has improved microorganism detection, the minimum microorganism that 2cfu/mL detected.5, device volume is little, is beneficial to the microminiaturization of chip.6, the dirigibility of channels designs is strong, can realize that high flux detects.
Description of drawings
Accompanying drawing 1 is that chip side is to structural representation;
The 1st, glass substrate; The 2nd, PDMS cover plate; The 3rd, injection port; 4a-4d is microchannel; The 5th, sample feeding pipe; 6a-6d is liquid storage tank.
Accompanying drawing 2 is chip channel design plane schematic diagram;
The 3rd, injection port; 4a-4d is microchannel;
Accompanying drawing 3 is the schematic 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 to this law:
(1) design of PDMS chip: design a kind of multichannel PDMS chip, its number of channels is determined by sample quantities and the microbe species 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.PDMS chip and measure-alike glass sheet are sealed mutually and become a kind of multichannel chip after oxygen plasma treatment.The structure of chip such as Fig. 1, 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 feeding pipe 5, liquid storage tank 6a-6d in 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 ejection of syringe pump pattern by injection port 3, when needs solution difference not of the same race sample introduction, extract to provide negative pressure by the syringe pump decimation pattern by injection port 3, the solution in liquid storage tank 6a-6d is entered respectively in the 4a-4d of each microchannel.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 the disposable introducing to solution of the same race in many passages, also can realize the disposable introducing to many passage different solutions, reduce the use of pump, the time of greatly having saved sample introduction simultaneously, realized fast detecting.
(2) coupling of microorganism antibody in passage: the glass table is after alkali treatment, clean with distilled water, and with the glass surface in 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 testing of many samples.
(3) utilize the hyperchannel Immunoassay Chip to detect microorganism: to execute negative pressure at injection port 3 places and introduce variety classes or variable concentrations contain the sample solution of microorganism in each passage, make the antibody specificity catch microorganism, after washing away unreacted microorganism, 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, after washing away unreacted probe, 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: in passage, the silver-colored particle of deposition has extremely strong scattering process to incident light, by naked eyes, can observe corresponding immune binding signal.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 to 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, be radiated at passage 8 after detection light source is focused on and deposit the zone of metallic particles, 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 the scattering light intensity.After obtaining the working curve of bacterial number in scattered light intensity and sample, the scattered light intensity of actual sample can be detected, according to working curve, micro organism quantity in sample can be recorded.
Below by specific embodiment, the present invention is described in detail below:
Embodiment 1, uses this chip variable concentrations Escherichia coli sample to be analyzed and obtained the typical curve of scattered light data and bacterial concentration.Chip finishing specificity Escherichia coli antibody in passage.To get the precipitation thalline after 37 ℃ of cultivations Escherichia coli medium centrifugal of 48 hours, add deionized water 10 doubling dilutions and be 1,10-1,10-2 ..., 10-8 concentration, add respectively the dilution of blank solution (deionized water), 10-2,10-5, three concentration of 10-8 in liquid storage tank 6a-6d.During detection,, with the sample feeding pipe of microchannel 4a-4d corresponding the insertion in liquid storage tank 6a-6d successively, at injection port 3, execute negative pressure with the solution in extracting liquid storage tank, execute malleation at injection port 3 after reaction finishes and inject successively golden labeling antibody and silver-colored transfection reagent.The cleaning of chip is executed malleation at injection port 3 equally and is injected PBS buffer solution irrigation channel.Silver dyes and with deionized water, thoroughly cleaned chip 2 ~ 3 minutes afterwards, takes image in chip channel by 20 * object lens after drying up under the metaloscope dark field mode, can judge that by naked eyes the brightness of image increases with bacterial concentration, can do quantitative test.
Embodiment 2: chip finishing specificity Escherichia coli antibody in passage.To get the precipitation thalline after 37 ℃ of cultivations Escherichia coli medium centrifugal of 48 hours, add deionized water 10 doubling dilutions and be 1,10-1,10-2 ..., 10-8 concentration, add respectively the dilution of blank solution (deionized water), 10-2,10-5, three concentration of 10-8 in liquid storage tank 6a-6d.During detection,, with the sample feeding pipe of microchannel 4a-4d corresponding the insertion in liquid storage tank 6a-6d successively, at injection port 3, execute negative pressure with the solution in extracting liquid storage tank, execute malleation at injection port 3 after reaction finishes and inject successively golden labeling antibody and silver-colored transfection reagent.The cleaning of chip is executed malleation at injection port 3 equally and is injected PBS buffer solution irrigation channel.Silver dyes and with deionized water, thoroughly cleaned chip 2 ~ 3 minutes afterwards, after drying up under the metaloscope dark field mode by 20 * object lens recording channel inscattering spectrum.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 to detect different types of microorganism.Chip finishing specificity Escherichia coli antibody in passage.Respectively 37 ℃ are cultivated after the Escherichia coli of 48 hours and 22 ℃ are cultivated P17 bacterium after 48 hours and spirillum medium centrifugal and get the precipitation thalline, add deionized water and be diluted to 10-4 concentration.Add respectively blank solution (deionized water), P17 bacterium dilution, spirillum dilution, Escherichia coli dilution in liquid storage tank 6a-6d.During detection,, with the sample feeding pipe of microchannel 4a-4d corresponding the insertion in liquid storage tank 6a-6d successively, at injection port 3, execute negative pressure with the solution in extracting liquid storage tank, execute malleation at injection port 3 after reaction finishes and inject successively golden labeling antibody and silver-colored transfection reagent.The cleaning of chip is executed malleation at injection port 3 equally and is injected PBS buffer solution irrigation channel.Silver dyes and with deionized water, thoroughly cleaned chip 2 ~ 3 minutes afterwards, after drying up under the metaloscope dark field mode with magnify 20 camera lens observation chip channel picture and with CCD, record the scattering spectrum data.
Claims (3)
1. PDMS hyperchannel Immunoassay Chip that is used for the microorganism field fast detecting, it is characterized in that: use glass as substrate, PDMS chip and the glass involution that will comprise a plurality of microchannels, parallel a plurality of passages are converged at a gateway, the quantity of parallel channel depends on the sample quantities of a required analysis of chip and the species number of microorganism, 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 introduce variety classes in each passage or contain the sample solution of varying number microorganism, make antibody target acquisition microorganism specifically, after washing away unconjugated microorganism, inject the antibody probe of nano-metal particle scattering mark by converging entrance, microorganism is carried out mark, after washing away unreacted probe, nano-metal particle is carried out silver dye the visual signal of generation.
2. hyperchannel Immunoassay Chip according to claim 1, it is characterized in that, after adopting method claimed in claim 1 to obtain visual signals, its detection mode is: use halogen tungsten lamp, laser or LED are as detection light source, after being focused on, detection light source is radiated in passage the zone that deposits metallic particles, 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 the scattering light intensity, obtain the working curve of bacterial number in scattered light intensity and sample, after detecting the scattered light intensity of actual sample, can obtain according to working curve the signal of micro organism quantity in sample.
3. hyperchannel Immunoassay Chip according to claim 2, 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 the disposable introducing to solution of the same race in many passages, also can realize the disposable introducing to many passage different solutions, reduced the use of pump, the time of greatly having saved sample introduction simultaneously, realize fast detecting.
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| CN103185802B (en) * | 2011-12-30 | 2016-03-02 | 北京纳迅科技股份有限公司 | Heterogeneous micro-fluidic Western blotting chip and its production and use |
| CN102879565B (en) * | 2012-09-26 | 2014-12-03 | 四川大学 | Microorganism sample rapid detection method and detection device thereof |
| CN103087899B (en) * | 2013-01-17 | 2014-08-20 | 湖南大学 | Aptamer-based microfluidic chip capable of capturing cancer cells and preparation thereof as well as separation method of cancer cells |
| 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 |
| CN111804356B (en) * | 2020-07-16 | 2021-11-09 | 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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