CN202066861U - Special microfluidic chip used for diagnosing lung cancer rapidly - Google Patents
Special microfluidic chip used for diagnosing lung cancer rapidly Download PDFInfo
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- CN202066861U CN202066861U CN2011200826028U CN201120082602U CN202066861U CN 202066861 U CN202066861 U CN 202066861U CN 2011200826028 U CN2011200826028 U CN 2011200826028U CN 201120082602 U CN201120082602 U CN 201120082602U CN 202066861 U CN202066861 U CN 202066861U
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Abstract
The utility model relates to a rapid lung cancer diagnosis device with composite nanometer particle modified electrodes, in particular to a microfluidic chip, belonging to the field of analyzing and testing. The microfluidic chip is internally provided with pipes, wherein the pipes are in a parallel structure which comprises four branch pipes that are parallel to one another; four working electrodes are respectively arranged in the four branch pipes and are composed of metal electrodes and mixture coated on the surfaces of the metal electrodes, wherein the mixture can be formed by grapheme, nanometer gold, lung cancer diagnosis antibodies and high polymer Nafion solution; the lung cancer diagnosis antibodies on the surfaces of the four working electrodes respectively adopt a specific antibody CEA (Carcinoma Embryonic Antigen) antibody, a CA (Carbonic Anhydrase) 19-9 antibody, an SCC (Squamous Cell Carcinoma) antibody and an NSE (Neuron Specific Enolase) antibody. The chip has a simple and integrated structure; and the chip is beneficial for accelerating the speed of diagnosing the lung cancer, improving the diagnosis accuracy rate and reducing the diagnosis expense.
Description
[technical field]
The utility model relates to a kind of lung cancer quick diagnosis device of composite nano particle modified electrode, and this lung cancer quick diagnosis device is based on the micro-fluidic chip special that the antigen/antibody specific reaction comes diagnosing, belongs to the analytical test field.
[background technology]
Lung cancer (Lung cancer) is modal lung primary malignant tumor, and most lung cancer originate from the tunica mucosa bronchiorum epithelium, so also claim lung bronchogenic carcinoma.Over nearly more than 50 years, countries in the world are industrially developed country particularly, and the incidence of disease of lung cancer and case fatality rate all rise rapidly, and the andropathy philtrum lung cancer of dying from carninomatosis ranks first, the mortality ratio of The World Health Organization's announcement in 2003 is 1,100,000/year, and the incidence of disease is 1,200,000/year.The incidence that the women suffers from lung cancer especially has the trend of rising.Lung cancer is the first place of the whole world cancer cause of the death at present, nineteen ninety-five the whole world have 600,000 people to die from lung cancer, and annual number is all rising.Therefore, exploitation is simple, fast, hypersensitivity and specificity diagnostic method, reagent and device, it is the effective way of preventing and treating lung cancer and reducing case fatality rate that lung cancer is carried out early diagnosis and early treatment.
At present, being used for the method for diagnosing clinically has: X-ray examination, bronchoscopy, ECT inspection, exploratory thoracotomy and methods such as biology and biochemical analysis.Though preceding four kinds of method detection techniques are accurately and reliably, apparatus expensive, pre-treatment is loaded down with trivial details consuming time and need the laboratory and the medical personnel of specialty, is difficult to promote also be popularized for on-the-spot and cheap application of vast patients with lung cancer.Have the distinctive high selectivity of immunoassay itself based on immunologic biology and biochemical analysis method,, judge that aspects such as curative effect and prognosis are all significant the early diagnosis of lung cancer.Yet the detection more complicated of lung cancer, traditional immunoassay only detects at single pulmonary cancer diagnosis antigen usually.Owing to may appear in the different cancers with a kind of diagnostic antigen, or diagnostic antigen not of the same race can occur in same cancer simultaneously, the diagnostic antigen of lung cancer mainly contains: several antigens such as CEA antigen, CA19-9 antigen, SCC antigen, NSE antigen, therefore, carry out multiple efficiently immune analysis method, the kind and the quantity of, quantitative response multiple diagnostic antigen qualitative truly simultaneously from the angle of biological correlativity, aspects such as prevention and detection of lung cancer, judgement lung cancer development degree, O﹠A lung cancer therapy effect all have important medical and are worth in early days.
Electrochemical immunosensor combines the high sensitivity of Electrochemical Detection and the high specific of immunoassay, have outstanding advantages such as amount of samples is little, detection is quick, the result is accurate, be widely used in the cancer diagnosis detection of antigens, unique advantages such as its automaticity height, cheapness, easily preparation, rapid sensitive, cooperate immunoreactive efficiently single-mindedly, be fit to make up very much the analyser that the multiple immunoassay of lung cancer detects.The micro-fluidic chip that the title of " life science integrated circuit " is arranged is the desirable technique that makes up microminiaturized, integrated electrochemical immunosensor.The micro-fluidic chip technology be with sampling, pre-service, add reagent, reaction, separation, detection etc. be integrated in finish on the microchip one in front of the door along technology, have analysis speed fast, contain much information, reagent consumption less, advantage and very suitable suitability for industrialized production such as pollutions is little, operation cost is low, instrument is easy to use.On behalf of the 21 century analytical instrument, microflow control technique move towards microminiaturized, integrated developing direction.The micro-fluidic Amperometric Detection Coupled chip that structure is suitable for the lung cancer quick diagnosis has following advantage: (1) is easy to integrated a plurality of sense channels and detecting electrode on chip by micro-processing technology, accomplishes that multiple pulmonary cancer diagnosis antigen analyzes the technical feasibility height simultaneously.(2) by the micro-flowing injection technology, mobile the guaranteed electrode surface of sample in pipeline upgrades constantly, overcome better that electrode is easily contaminated, false positive rate is high, problem such as collimation difference as a result.(3) microfluidic sensing device upper pipeline diameter has only μ m level, and the reaction tank volume also has only μ L level, and required reagent dosage is few during analysis, and analyte arrives the diffusion length of electrode surface lacks, therefore can significantly reduce the incubation time, realizes the fast joint inspection target.(4) the microfluidic sensing device is many based on the optical detection system at present, and the expensive volume of instrument is big, fully integrated being difficult to, and also it is very high to detect cost, has limited its popularization.And adopting Electrochemical Detection in the ampere immunity sensor, volume is little, automaticity is high, cost is low.
At present, in the micro-fluidic chip technical field, detect the correlation technique of quick diagnosis lung cancer and method not to appear in the newspapers as yet when utilizing the multiple diagnostic antigen of lung cancer.
[utility model content]
Technical problem to be solved in the utility model is, in the so total technological frame of micro-fluidic chip technology, develop a kind of multiple diagnosis antibody that can utilize lung cancer and come that multiple diagnostic antigen to lung cancer detects simultaneously, the micro-fluidic chip special of quick diagnosis.
The utility model solve the technical problem by following scheme, the device that this scheme provides is a kind of micro-fluidic chip of pulmonary cancer diagnosis special use, the structure of this micro-fluidic chip comprises that applying is installed in two plate objects together, described two plate objects are respectively a cover plate and a substrate, on the position of the mutual applying between described two plate objects, be equiped with pipeline, three pond shape things and be installed in the working electrode on the diverse location in the described pipeline in regular turn respectively, to electrode and contrast electrode, one end of pipeline via the manifold shape fluid passage respectively with wherein two pond shape thing UNICOMs, the other end of pipeline and another remaining pond shape thing UNICOM, described working electrode is made up of metal electrode and the film that is coated in surface of metal electrode, and described film is by Graphene, nm of gold, the potpourri of pulmonary cancer diagnosis antibody and macromolecule Nafion solution constitutes.
As further improvement of the utility model, the structure of described pipeline is structure in parallel, the described pipeline that is structure in parallel is made of four lateral parallel connections, and, the quantity of described working electrode is four, the installation position of these four working electrodes lays respectively in described four laterals, and the pulmonary cancer diagnosis antibody materials that these four working electrode top layers apply is respectively CEA antibody, CA19-9 antibody, SCC antibody and the NSE antibody that can combine with pulmonary cancer diagnosis antigen energy specificity.
Film on the described working electrode is the composite nano particle sensitive membrane, wherein embedding pulmonary cancer diagnosis antibody.This film is Graphene, nm of gold, various pulmonary cancer diagnosis antibody are fully mixed with high molecular Nafion solution and to vibrate evenly, drips in surface of metal electrode and its dried overnight film forming is formed by micro syringe.
Pipeline in the described microfluidic chip structure, its internal diameter size all can be selected arbitrarily size, but, for as far as possible less with the consideration of aspects such as liquid sample to be measured and reduction reagent loss, described pipeline is preferably all selected the passage of capillary level for use, the passage meaning of described capillary level is a capillary channel, and the internal diameter capillaceous on its internal diameter and the ordinary meaning is suitable.The shape of cross section of its inner passage of described kapillary can be a shape arbitrarily, described shape of cross section is for example circular, oval, square, rectangle, bar shaped, can certainly be have bending arbitrarily linear, and, described interior shape capillaceous is along with the extension of pipeline, and it is different shapes that the shape of cross section of different parts also can allow.Relate to some kinds of electrodes in the structure of this case micro-fluidic chip, its size is the size that is suitable for being placed in the pipeline of relevant position, but, based on as far as possible less with the consideration of aspects such as liquid sample to be measured and reduction reagent loss, described electrode is the minute sized electrode that is suitable for being placed in the kapillary, the shape of each electrode wherein all can be selected arbitrarily shape, and described selected arbitrarily shape is square shape sheet shape, rectangle sheet, strip or round sheet or the like for example.The material of each electrode wherein all can be selected arbitrarily material, and described selected arbitrarily material is gold, silver, platinum or other metal etc. for example.
Relate to several pond shape things in this case microfluidic chip structure, described pond shape thing is pond shape or the scrotiform structure that is used for actual tested blood serum sample, buffer solution and waste liquid storage, its shape of inner chamber of each pond shape thing wherein all can be selected arbitrarily shape, and described cavity shape is cylindrical cavity shape, square column type cavity-like, oblong cavity shape or spherical hollow space shape or the like for example.The size of described pond shape thing can be arbitrarily selected size, still, for can be as far as possible less with liquid sample to be measured and reduce reagent loss, the hypomegetic pond shape thing that described pond shape thing preferably can mate with kapillary.
This case device can further include some annexes certainly, and described annex is multiple tracks electrochemical workstation and microfluidic pump or the like for example, and the art-recognized meanings of the art-recognized meanings of described multiple tracks electrochemical workstation and microfluidic pump is known.Each working electrode that relates in this case microfluidic chip structure and to electrode and contrast electrode etc., can get lines crossed via corresponding special use is respectively connecting with the corresponding interface of described multiple tracks electrochemical workstation.It is to be used for the private cable that each the corresponding interface with each described electrode and described multiple tracks electrochemical workstation is coupled to each other that described special use is being got lines crossed.Described microfluidic pump is exclusively used in and drives micro liquid and flow, described microfluidic pump can with the described pond of selected any one shape thing UNICOM as required.
Can there be several different methods in the specific implementation path of this case micro-fluidic chip, selects the one detailed annotation as follows:
One, superpolymer (polycarbonate: the PC) preparation of the little gold electrode of electroless plating on the cover plate
1, is photomask with quartz/chromium plate, as light source the selective area on PC cover plate surface carried out radiation, generate carboxyl with the ultraviolet light of selecting wavelength.
2, adopt hexane diamine as aminating agent, 1-[3 ,-(N-N-dimethyl amine) propyl group]-3-hexyl carbodiimide hydrochloride is that coupling agent carries out aminating reaction to the PC cover plate through the selective area irradiation, forms amido link.
3, will immerse in the certain density chlorauric acid solution through the PC cover plate of selective area aminating reaction and react, gold ion is complexed to the amido surface.
4, clean with distilled water after, again it is immersed and reduces 8min in sodium borohydride solution.
5,, with ultrasonic cleaning 30min in the potassium rhodanate solution of its immersion 0.5mol/L, prevented plating again with after the distilled water cleaning.
6, will contain in the plating bath of gold ion, complexing agent and reductive agent formaldehyde through the PC cover plate immersion of cleaning, react 1h down in 45-60 ℃.
7, the cover plate that will be coated with little gold electrode is put into the about 3h of baking oven annealing in process, promptly obtains the many little gold electrode cover plates of superpolymer PC of the present invention.The long 3mm of wherein little gold electrode, wide 3mm, thickness is about 0.1mm, and spacing is about 0.5mm each other.
Two, the superpolymer (polycarbonate: the PC) making of substrate that has the microchannel
1, makes silicon mold
Draw floor layout, the pattern line width is about 25-100 μ m, adopts the output of high-resolution laser photocomposing system on transparent film, promptly obtains mask.
On substrate surface, deposit one deck silicon nitride film as sacrifice layer by chemical vapour deposition technique, on this substrate, cover one deck light-sensitive emulsion, in baking oven, handle 15-20min for 60 ℃ by the rotation film coating technique; Mask is covered on the substrate, the principle by the exposure imaging with the image transfer on the mask to the optical cement layer of substrate surface; Method by dry etching is processed into the spatial structure with certain depth with the planar figure on the optical cement layer.Can obtain the silicon mold of lobed passage.The silicon mold for preparing is used H successively
2O
2: H
2SO
4Solution (volume ratio is 1: 4), acetone and distilled water clean, to remove its surperficial oxide.
2, adopt pressure sintering to duplicate the microchannel
The PC sheet cut into be about 4cm, the size of wide 3cm.Ultrasonic cleaning is dried naturally, in hot-press arrangement the PC sheet is heated to about softening temperature (110 ℃), applies certain pressure then on silicon mold, and continues about 1min, can suppress microchannel with the silicon mold complementation at the PC sheet.Then, formpiston and the PC substrate that is carved with the microchannel are cooled off the back demoulding together, just obtain required microchannel.
3, on cover plate, make three microminiature pond shape things
Adopting electric drill on different positions, to get out three microminiature pond shape things respectively on the cover plate.
Three, little gold electrode cover plate of superpolymer and the superpolymer substrate that has a microchannel are handled in the cryogenic oxygen plasma atmosphere totally.
Four, will contain Graphene, nm of gold, pulmonary cancer diagnosis antibody is coated on corresponding little metal working respectively with the mixture solution of macromolecule Nafion solution and makes on the electrode dried overnight.
Five, chip involution
Aim at little gold electrode cover plate of superpolymer and the superpolymer substrate that has the microchannel at microscopically, with this substrate with cover plate is fixed and clamp with two cover glasses, be placed in the baking oven and under uniform temperature, be incubated 15min, can obtain required four-way micro-fluidic chip.
Six, the PC sheet that step 4 is finished is respectively at working electrode, reference electrode and utilize elargol to connect copper sheet as lead to electrode end, treats that elargol seals up hot melt adhesive after dried fully again with fixing copper sheet, promptly finishes the making of whole four-way micro-fluidic chip.
Its relevant each operating parameter of the above-mentioned specific implementation path of this case micro-fluidic chip of detailed annotation can be adjusted according to actual needs.
The using method of this case micro-fluidic chip:
Employing adds Micropump and drives the steady flow in the capillary channel of four-way micro-fluidic chip of liquid stream, utilizes the four-way electrochemical analytical instrument respectively four kinds of pulmonary cancer diagnosis antigens to be detected.
The concrete detection of this case micro-fluidic chip uses step as follows:
1, adopts the four-way electrochemical analyser, in the microtubule road, feed certain density potassium ferricyanide solution, adding under the Micropump driving, adopt the ampere method to detect the size of current of said process as electron mediator.
2, add actual blood serum sample liquid in the microtubule road, adding under the Micropump driving, various pulmonary cancer diagnosis antigen molecules are caught by corresponding pulmonary cancer diagnosis antibody molecule on the working electrode surface in each passage.
3, the pulmonary cancer diagnosis antigen molecule in pulmonary cancer diagnosis antibody molecule and the blood serum sample forms immune complex, and certain inhibition is played in the electron transport of the electron mediator potassium ferricyanide, and the electric current that the ampere method detects said process reduces degree.
4, the result who electric current is reduced degree carries out analysis-by-synthesis, obtains the kind and the content of various pulmonary cancer diagnosis antigen molecules thus, and the lung cancer situation is carried out comprehensive diagnos.
The utility model has the advantages that, at a physical device is integrated four working electrodes that are coated with four kinds of pulmonary cancer diagnosis antibody materials respectively on the micro-fluidic chip, at four kinds of feature diagnostic antigens of detection of lung cancer, the design feature of the integrated structure of this case micro-fluidic chip has determined the use of this chip to help to accelerate pulmonary cancer diagnosis speed, has improved accuracy rate of diagnosis, has reduced diagnostic fees usefulness these four working electrodes respectively.
[description of drawings]
Fig. 1 is this case micro-fluidic chip embodiment organigram, and what showed is the form of the perspective under the depression angle of this example structure, does not depict described annex among the figure.
Among the figure, 1,2,11 is respectively three pond shape things that installation position is different, the 3rd, the manifold shape fluid passage, 4 represent the installation position difference but the various pipelines and the lateral of formation parallel with one another UNICOM in parallel structure, the 5th, its top layer that is installed in the pipeline is graphene coated, nm of gold, the working electrode of the potpourri of pulmonary cancer diagnosis CEA antibody and macromolecule Nafion solution, the 6th, its top layer that is installed in the pipeline is graphene coated, nm of gold, the working electrode of the potpourri of pulmonary cancer diagnosis CA19-9 antibody and macromolecule Nafion solution, the 7th, its top layer that is installed in the pipeline is graphene coated, nm of gold, the working electrode of the potpourri of pulmonary cancer diagnosis SCC antibody and macromolecule Nafion solution, the 8th, its top layer that is installed in the pipeline is graphene coated, nm of gold, the working electrode of the potpourri of pulmonary cancer diagnosis NSE antibody and macromolecule Nafion solution, the 9th, to electrode, the 10th, contrast electrode.
[embodiment]
In this case embodiment that Fig. 1 showed, this device also is the structure of micro-fluidic chip, comprising that applying is installed in two plate objects together, and two plate objects are respectively the cover plate of micro-fluidic chip and the substrate of micro-fluidic chip.On the position of the mutual applying between two plate objects, be equiped with pipeline, and, three pond shape things.These three pond shape things are respectively the pond shape thing 1 of actual blood serum sample storage, the pond shape thing 2 of buffer solution storage and the pond shape thing 11 of waste liquid storage.One end of described pipeline via manifold shape fluid passage 3 respectively with pond shape thing 1 and pond shape thing 2 UNICOMs, the other end of described pipeline and remaining pond shape thing 11 UNICOMs.This micro-fluidic film-making also is provided with and is installed in the working electrode on the diverse location in the described pipeline in regular turn respectively, to electrode 9 and contrast electrode 10.Working electrode comprises metal electrode and applies the sensitive membrane that the potpourri by Graphene, nm of gold, pulmonary cancer diagnosis antibody and macromolecule Nafion solution constitutes at metal electrode surface layer.The structure of described pipeline is structure in parallel, and this pipeline that is structure in parallel is made of four lateral parallel connections, and the quantity of working electrode is four, and these four working electrodes are respectively working electrodes 5,6, and 7,8.Wherein, working electrode 5 is that its top layer that is installed in the lateral is graphene coated, nm of gold, the working electrode of the potpourri of pulmonary cancer diagnosis CEA antibody and macromolecule Nafion solution, working electrode 6 is that its top layer that is installed in the lateral is graphene coated, nm of gold, the working electrode of the potpourri of pulmonary cancer diagnosis CA19-9 antibody and macromolecule Nafion solution, working electrode 7 is that its top layer that is installed in the lateral is graphene coated, nm of gold, the working electrode of the potpourri of pulmonary cancer diagnosis SCC antibody and macromolecule Nafion solution, working electrode 8 are that its top layer that is installed in the lateral is graphene coated, nm of gold, the working electrode of the potpourri of pulmonary cancer diagnosis NSE antibody and macromolecule Nafion solution.
The annex of the utility model micro-fluidic chip also has annexes (not shown) such as microfluidic pump and multiple tracks electrochemical workstation.Each pond shape thing in this routine structure can be as required with as the microfluidic pump of annex UNICOM by any means.Each working electrode in this routine structure and to electrode and contrast electrode can be respectively via special-purpose separately cable or say get lines crossed respectively with as the corresponding cable interface of the multiple tracks electrochemical workstation of annex or say the interface of getting lines crossed and connect.
Claims (3)
1. the micro-fluidic chip of lung cancer quick diagnosis special use, it is characterized in that: the structure of this micro-fluidic chip comprises that applying is installed in two plate objects together, described two plate objects are respectively a cover plate and a substrate, on the position of the mutual applying between described two plate objects, be equiped with pipeline, three pond shape things and be installed in the working electrode on the diverse location in the described pipeline in regular turn respectively, to electrode and contrast electrode, one end of pipeline via the manifold shape fluid passage respectively with wherein two pond shape thing UNICOMs, the other end of pipeline and another remaining pond shape thing UNICOM, described working electrode is made of metal electrode and the sensitive membrane that contains pulmonary cancer diagnosis antibody that is coated in surface of metal electrode.
2. the micro-fluidic chip of lung cancer quick diagnosis according to claim 1 special use, it is characterized in that: the structure of described pipeline is structure in parallel, the described pipeline that is structure in parallel is made of four lateral parallel connections, and, the quantity of described working electrode is four, and the installation position of these four working electrodes lays respectively in described four laterals.
3. according to the micro-fluidic chip of any described lung cancer quick diagnosis special use among the claim 1-2, it is characterized in that: described pipeline is capillary channel.
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CN107199058A (en) * | 2016-03-18 | 2017-09-26 | 葛宇杰 | The general type Diagnostic Value of Several Serum Tumor Markers joint inspection chip apparatus conveniently disassembled |
CN107199059A (en) * | 2016-03-18 | 2017-09-26 | 葛宇杰 | For the classifiable tumor mark joint inspection chip apparatus of women physical examination examination |
CN107199056A (en) * | 2016-03-18 | 2017-09-26 | 葛宇杰 | Easily-disassembled male's classifiable tumor markers in detecting chip apparatus |
CN107199057A (en) * | 2016-03-18 | 2017-09-26 | 葛宇杰 | Dismounting brief classifiable tumor mark joint inspection chip apparatus convenient to both |
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CN107225002A (en) * | 2016-03-25 | 2017-10-03 | 葛宇杰 | The classifiable tumor markers in detecting micro flow control chip device that women is applicable |
CN107225005A (en) * | 2016-03-25 | 2017-10-03 | 葛宇杰 | Six kinds of classifiable tumor mark multichannels are while detection micro flow control chip device |
CN107225004A (en) * | 2016-03-25 | 2017-10-03 | 葛宇杰 | General type is used for the micro flow control chip device for detecting Diagnostic Value of Several Serum Tumor Markers simultaneously |
CN107282147A (en) * | 2016-04-01 | 2017-10-24 | 葛宇杰 | Six kinds of classifiable tumor mark joint inspection chip apparatus of dual drive coupling running |
CN107282148A (en) * | 2016-04-01 | 2017-10-24 | 葛宇杰 | Using the chip apparatus for joint inspection Diagnostic Value of Several Serum Tumor Markers of hydrophobic substrate |
CN107282145A (en) * | 2016-04-01 | 2017-10-24 | 葛宇杰 | The female tumor mark joint inspection chip apparatus of many type of drive coupling runnings |
CN107282146A (en) * | 2016-04-01 | 2017-10-24 | 葛宇杰 | The male tumor mark joint inspection chip apparatus of two kinds of type of drive coupling runnings |
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