CN102435654A - Viral disease diagnosis device and method based on field effect transistor - Google Patents
Viral disease diagnosis device and method based on field effect transistor Download PDFInfo
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- CN102435654A CN102435654A CN201110259970XA CN201110259970A CN102435654A CN 102435654 A CN102435654 A CN 102435654A CN 201110259970X A CN201110259970X A CN 201110259970XA CN 201110259970 A CN201110259970 A CN 201110259970A CN 102435654 A CN102435654 A CN 102435654A
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Abstract
The invention discloses a quick diagnosis device for a viral communicable disease based on a field effect transistor, which is composed of three parts, i.e., a field effect transistor biological chip, a microfluid channel sample introduction encapsulation system and an electronic detection system. A source electrode, a drain electrode and a semiconductor channel between the source electrode and the drain electrode are constructed on a silicon substrate by utilizing a semiconductor manufacturing process; and a covalent modification silane reagent on the channel is taken as a connection molecule for connecting viral antibodies; then a microfluid channel made from polydimethylsiloxane is used to be encapsulated in tight fit with the silicon substrate; finally the source electrode and the drain electrode are connected with a set of a signal detection system, so that a person or an animal can be diagnosed whether to be infected with the viral disease quickly, accurately and sensitively. The quick diagnosis device is a portable epidemic communicable disease detection instrument.
Description
Technical field
The present invention relates to a kind of device and detection method of utilizing field effect set transistor fast diagnosis of human or animal virus sexually transmitted disease.
Background technology
The viral disease that avian influenza virus, SARS virus etc. have an extremely strong infection ability spreads the life and health that has greatly threatened the human and animal on a large scale the whole world.(Avian influenza is the bird infectious disease that is caused by the Influenza Virus A of orthomyxoviridae family type influenza virus AI), is classified as the category-A deadly infectious disease by World Organization for Animal Health and The World Health Organization (WHO) like bird flu.Avian influenza virus is worldwide distribution, and nearly all wild and domestic bird all can infect, it is believed that fowl be influenza virus gene natural with huge depots, be the important substance basis of A type influenza virus new subtype origin.Different strain virulence have very big difference, and the avian influenza virus of had no pathogenicity and low pathogenicity extensively exists in the world and be popular, and wherein the virus of some highly pathogenicity such as H5N1 type can cause a lot of pestilence, are threatening human beings'health.The diagnosis quick and precisely of the disease that infective virus such as bird flu cause has important public hygienics meaning, a kind ofly can detect the mankind or animal real-time and accurately whether by virus infections and be easy to pick-up unit and method integrated, robotization and seem particularly important so develop.Designed a kind of integrated field effect transistor biochip and, whether can be used for detecting people or animal so we invent by the degree of virus infections and infection to the viral detector of the antibody with high specificity of virus.
For the communicable disease that is caused by virus, general biomedical detection method is isolation of virus, immunological detection and molecular Biological Detection method.Wherein, isolation of virus need be done chicken embryo experiment, and the blood of patient suspected or animal is injected into the chicken embryo, cultivates 3 to 5 days, observes whether death of Embryo Gallus domesticus.The method diagnosis speed is slow, is not suitable for fast, delicately extensive epidemic disease is carried out the scene detection.Immunology virus detection method is general detect to as if patient's body in virus or the content of antibody; Because virus is at human or animal's blood, content pettiness extremely in the body fluid; Directly detect very difficulty of virion; Generally need to wait for that the human or animal produces the antibody to virus in vivo, through detecting the detection that corresponding antibody realizes disease.Equally, can not realize early stage, directly detection.For molecular biological detection method generally is to need to extract viral RNA, is reverse transcription-PCR, just can detect the content of virus.More than these classic methods higher to requirement for experiment condition mostly, need particular instrument, and process is loaded down with trivial details, reagent, apparatus expensive, be unfavorable in real time, fast, the on-the-spot virus spread that detects.
Behind the virus infections mammalian cells such as bird flu, in cell, accomplish virus assembling, duplicate, and then, infect other normal cells with new life's the extracellular that is secreted into.Based on these virus rule of life cycles, can realize earlier detection in early days infecting, thereby break through the limitation of traditional detection method disease through the micro-virion that detects in human or animal's body fluid.
Advantage at this diagnostic method is: utilize the field effect transistor biochip that makes up based on nano material or other organic materials etc.; Can direct, overdelicately detect the virion of trace in human or animal's blood, the body fluid; Thereby broken through the constraint of traditional detection method; And device makes up simple, portable, can realize that whether quick, the on-the-spot human or animal of detection is by virus infections.Having real-time sensitivity, characteristics such as portable, high flux, integrated, robotization fast, is whether inspection diagnosis people or animal be by the splendid method of virus infections.
Adopting the portable viral detector of field effect transistor bio-sensing principle development to have in real time, sensitivity, accurately whether detect the human or animal by the effect of virus infections, is efficient detection method and the instrument in prevention from suffering from the diseases, monitoring, detection and the virological investigation.
Summary of the invention
Fundamental purpose of the present invention be make up a cover can quick real-time, the apparatus and method that whether infected of sensitive and accurate field diagnostic human or animal by viral infectious.This method can solve the weak point of existing classic method, more cheap and simple, quick portable diagnosis of viral communicate illness.Utilize biological immune response to catch virus, quick and precisely, have specificity; Carry out disease detection in conjunction with this physics device of field effect transistor; Make this device be easier to integrated, robotization, high flux ground detects sample, be suitable for the sample that carries out enormous amount being carried out fast detecting in the season of the high outburst of epidemic infectious diseases.
The invention provides a kind of detection method of novel viral infection disease; Its principle is: with zooblast or people's blood, humor collecting in a common vessel; Again with lysis or the direct micro-virion that is contained in the body fluid that detects; Through a simple purge process, feed in PDMS (dimethyl silicone polymer) microfluid passage of integrating with the field effect transistor detection chip again.When body fluid flow was crossed the semiconductor channel material of field effect transistor, if contain the target viral particle, specific combination just can take place with virus in nano-modified antiviral antibody on raceway groove, and virus is adsorbed on above the raceway groove.Because the virion surface has in solution electrically; When they are adsorbed on the raceway groove of field effect transistor; Can produce effect of electric field to field effect transistor, weaken or strengthen exhausting of semiconductor channel, thereby influence mobility of charge carrier speed or concentration; Cause having the increase of electric current or weakening, thereby detection system detects the variation of electric signal.The content of virus is high more, can make this electric signal strong more.And the electrical signal of final output is easy to by the computer quick identification.These apparatus and method adopt the sample introduction that flows, the instant detection; Sample flowed out after detection finished; Feeding tens of seconds of a certain amount of buffer solution washing again, can carry out the diagnostic detection of next sample, is quick, accurate, the high-throughout detection method to the virus in the human or animal body.
One of technical scheme of the present invention is, made up the biochip (like Fig. 1) that is used for the field effect transistor that virus detects, and its construction step is:
(1) nano wire or the film with various materials placed SiO through certain method
2On the silicon substrate of insulation course, as the raceway groove of field effect transistor.
(2) through mode such as magnetron sputtering in the both sides of raceway groove, construct the metal electrode about long 1 to 2 centimetre, wide 5 millimeters, thick 200 nanometers, as field effect transistor source electrode, drain electrode.
50 to 100 microns of (3) two electrode distances.
(4) build chip after, drip massfraction at the raceway groove place and be 1% APTES (3-aminopropyl triethoxysilane) or GPTMS (γ (2, the 3-glycidoxy) propyl trimethoxy silicane)) organic connection reagent, modification reaction 3 hours.
(5) in the modification behind organic connection reagent; Drip the antibody protein solution that contains to certain specific virus at the raceway groove place again, reacted 1 hour, feed the bovine serum albumin solution sealing again; The buffer solution washing promptly obtains having the field effect transistor biochip that detects virus capable.
Two of technical scheme of the present invention is, a kind of pick-up unit and method of viral infection disease is provided, its composition and acting as:
(1) be used to adorn receive human or animal's body fluid, blood container bottle and connect flexible pipe.An osculum is opened in the lower end of container, has the plastics or the silica gel hose of machinery valve such as iron clamp in the connection.The effect of this part device is dressing human or animal's body fluid, can open the machinery valve of control flow when next step detects, and lets the body fluid in the container flow into the field effect transistor bio-chip test device of integrating with dimethione through flexible pipe.Devices such as all containers, flexible pipe are all sterilized, are disinfected, and guarantee not allow other pathogenic microorganisms or impurity effect testing result.
(2) the dimethyl silicone polymer microfluid core passage that is connected with flexible pipe.It and the field effect transistor chip close adhesion of having modified antiviral antibody.The purpose that adopts plastic dimethyl silicone polymer macromolecule polymer material and field effect transistor chip to fit tightly be to construct one can sample introduction article solution, flow through transistor channel, flow out the airtight passage of solution again, be easy to control flow direction and circuit like this.Connect a beaker again after flowing out microfluid passage, as waste liquid pool.
(3) field effect transistor biochip.Modify antiviral antibody in the responsive channel part of field effect transistor; Connect outside measurement, checkout equipment again; When the flow of solution that contains virus during through the channel part of field effect transistor, the viruses molecule in the solution will with the combining of antibody specificity on being modified at raceway groove because virion charging property in solution; Can produce effect of electric field to the charge carrier in the semiconductor channel after being combined on the raceway groove; Can strengthen or weaken semi-conductive exhausting, thereby change the electrical signal between field effect transistor source electrode, drain electrode, can detect by measured instrument.
Three of technical scheme of the present invention is the microfluid passage that made up with the field effect transistor integrating biological chip; Step, the method for its structure are: its preparation method does; Utilize the semiconductor light carving technology; On silicon substrate, make up a long number centimetre, the SU-8 photoresist effect template of wide 100 microns, dark 50 microns convexity, then with the dimethyl silicone polymer of liquid state be covered on the template after the hardening agent of doses mixes.Put into vacuum drying chamber subsequently, vacuum state, 90 degree heating down took out after 20 minutes.The dimethyl silicone polymer piece that will solidify separates with template, fits tightly promptly to make up with silicon substrate again and accomplishes.
The feasibility of this viral disease pick-up unit and method and sensitivity derive from the sensitivity response of field effect transistor biochip for the virion of trace in patient's body fluid; The selectivity of this method derives from certain the viral antibody that is directed against that passes through general biological method preparation that is fixed on the fieldistor channel.Through fixing different types of antibody, can realize specific diagnosis to different virus property disease.This method can realize rapidity, portable on-site diagnosis reason be field effect transistor biochip and electronic detection system be easy to miniaturization, integrated, power consumption is low, and can reuse.Be suitable under the condition that lacks conventional viral test experience equipment, carrying out quick diagnosis.The reason that these apparatus and method are easy to robotization, high-throughput diagnostic sample is; The electrical signal of field effect transistor output is easy to discerned by computer; And mobile sample introduction, the instant detection; Sample flowed out, feeds a certain amount of washing buffer solution tens of seconds after detection finished, and can carry out the diagnostic detection of next sample.
The outstanding feature and the marked improvement of the inventive method and device are:
(1) the field effect transistor biochip that the present invention is based on, it is flexible and changeable to make up material, can be organic film, inorganic, metal oxide material, and the method that construction method is connected with antibody is all simple.If the organic material (like Graphene) that uses some good biocompatibility is as raceway groove, antibody protein can need not intermediary and directly be connected on the transistor, reduces Diagnostic Time and cost greatly.
(2) the field effect transistor biochip that the present invention is based on utilizes dimethyl silicone polymer microfluid passage and SiO when detecting
2Passivation layer is protected electrode and chip, effectively improves chip utilization rate and repeatability.
(3) this method directly detects the virus in people or the animal blood, the antibody that not only virus is produced than human body more directly, quicker, and saved the PCR step that detects viral nucleic acid.
(4) this method can realize sensitivity, fast detecting owing to use nano level organic and inorganic semiconductor material to make up the field effect transistor biochip, and the remolding sensitivity classic method improves 10
3To 10
6Doubly, in 1 minute, just can accomplish detection time.In real time, fast, need not fluorescence or other molecular marked compounds.
(5) this method combines the detection of field effect transistor with extensive infectious disease, and the detector of preparing is easy to integrated, robotization, portability, and fast detecting is carried out to great amount of samples in the scene that is adapted at outbreak of communicable diseases.
Description of drawings
Fig. 1 is the method for the invention field effect transistor biochip structural representation.
Fig. 1 mark implication sees the following form:
| Accompanying | Implication | Accompanying drawing 1 | Implication | |
| 1 | | 2 | The | |
| 3 | Semiconductor channel | 4 | | |
| 5 | Antibody to virus | 6 | Coupling agent GPTMS | |
| 7 | | 8 | SiO 2Oxide layer | |
| 9 | Silicon substrate |
Fig. 2 is that the method for the invention ito thin film transistor detects avian influenza virus example signal response diagram.
Fig. 2 mark implication sees the following form:
| Accompanying | Implication | |
| 1 | The sample that does not contain bird flu H 5 | |
| 2 | The sample that contains bird flu H 5 |
Embodiment one
Avian influenza virus diagnostic device based on ITO (Indium Tin Oxides, tin indium oxide) thin film transistor (TFT) biochip:
(1) (Plasma Enhanced Chemical Vapor Deposition, PECVD) method is used SiH through the plasma enhanced chemical vapor deposition method
4And O
2As reacting gas, deposition one deck is based on the porous silica of solid electrolyte on silicon substrate.
(2) method through magnetron sputtering again, under the environment of the pure argon of 0.5Pa, with covering of a special part hollowed-out mask plate, control mask plate above silicon substrate 50 microns, sputtering ITO is made source electrode, drain electrode and raceway groove.The place of not covering is that sputter becomes thicker ITO source electrode, drain electrode, and concealed place is because mask plate has 50 microns apart from silicon substrate, so can form one deck than thin ITO thin layer, as the raceway groove of field effect transistor.Edge at chip scrapes off porous silica layer, exposes silicon substrate and promptly makes grid.
(3) the ito thin film transistor device is cleaned with ethanol, acetone, again channel part was handled 10 minutes with oxydol and ammoniacal liquor mixed solution.Then dripping the GPTMS solution reaction on the raceway groove 3 hours.Drip H 5 N 1 avian influenza type antiviral antibody again, reacted 1 hour, feed the bovine serum albumin(BSA) sealing again, the buffer solution washing has the field effect transistor biochip that detects virus capable.
(4) manufacture the PDMS microfluid passage, passage is long 5 centimetres, and is wide 100 microns, and dark 50 microns, and the field effect transistor biochip that microfluid passage and front are prepared fits tightly, the assurance flow of solution can be from the both sides leakage of passage when passage.
(5) inlet with microfluid passage connects plastics or rubber hose, links to each other with the container that person's sample to be detected is housed again, and the outlet of microfluid passage also connects flexible pipe, leads to waste liquid pool.The source electrode of field effect transistor, drain electrode are connected through lead with electronic detecting device.
Two kinds of samples that (6) will not contain and contain H5N1 virus are successively through flexible pipe inflow microfluid passage; The channel part of the field effect transistor through bio-modification; Can observe the current signal influence and change (like Fig. 2); Be that electronegative avian influenza virus is combined on the raceway groove, cause the ito thin film transistor current to descend.Can whether differentiate this patient or animal quickly and accurately by virus infections based on this signal.
Embodiment two
Based on SnO
2The avian influenza virus diagnostic device of nano-wire transistor biochip:
(1) adopts the synthetic SnO of hydro-thermal method or CVD (Chemical Vapor Deposition) as fieldistor channel
2One-dimensional metal oxide materials such as nano wire.
(2) the exhausted grid layer of field effect transistor is the fine and close silicon dioxide of one deck on the silicon substrate.
(3) the method SnO of employing photoetching and magnetron sputtering
2Metal electrode is prepared at the nano wire two ends, as source electrode, drain electrode, scrapes off silicon dioxide layer at the edge of chip again, exposes silicon substrate and promptly makes grid.
(3) with SnO
2Nano-wire devices is cleaned with ethanol, acetone, channel part is handled 10 minutes with oxydol and ammoniacal liquor mixed solution again.Then dripping the GPTMS solution reaction on the raceway groove 3 hours.Drip H 5 N 1 avian influenza type antiviral antibody again, reacted 1 hour, feed the bovine serum albumin(BSA) sealing again, the buffer solution washing has the field effect transistor biochip that detects virus capable.
(4) manufacture the PDMS microfluid passage, passage is long 5 centimetres, and is wide 100 microns, and dark 50 microns, and the field effect transistor biochip that microfluid passage and front are prepared fits tightly, the assurance flow of solution can be from the both sides leakage of passage when passage.
(5) inlet with microfluid passage connects plastics or rubber hose, links to each other with the container that person's sample to be detected is housed again, and the outlet of microfluid passage also connects flexible pipe, leads to waste liquid pool.The source electrode of field effect transistor, drain electrode are connected through lead with electronic detecting device.
Two kinds of samples that (6) will not contain and contain H5N1 virus are successively through flexible pipe inflow microfluid passage; The channel part of the field effect transistor through bio-modification; Can observe the current signal influence and change, promptly electronegative avian influenza virus is combined on the raceway groove, causes SnO
2The nano wire electric current descends.Can whether differentiate this patient or animal quickly and accurately by virus infections based on this signal.
Claims (9)
1. diagnostic device and method based on a field effect transistor biochip; It is characterized in that the field effect transistor biochip with functional modification is a core; PDMS (dimethyl silicone polymer) microfluid passage and the electronic detection system that add auxiliary feed liquor, fluid are formed, and can detect the virus of the trace in the body fluid of people or animal.
2. like the biochip of right 1 described field effect transistor, it is characterized in that the metallic source electrode, the drain electrode that utilize the semiconductor technology manufacturing on the silicon substrate and be connected source electrode and drain electrode between semiconductor channel.Semiconductor sensitive material raceway groove can be metal oxide nano-material such as SnO
2Nano wire, TiO
2Nano wires etc. also can be other semiconductor materials, like in silicon nanowires, CNT, graphene nanobelt, GaN nano wire, the organic thin film one or more.Passivation one deck SiO on source electrode and the drain electrode
2To adapt to the detection in the WS.
3. like the conducting channel of the biochip of right 1 described field effect transistor, it is characterized in that: transistorized semiconductor channel can be one or by many arrays of forming.
4. like the field effect transistor of right 1 described functional modification; (γ (2 to it is characterized in that passing through on the transistorized semiconductor channel organic coupling agent APTES (3-aminopropyl triethoxysilane) or GPTMS; Propyl trimethoxy silicane) etc. the 3-glycidoxy) intermediation is connected various antibody proteins to virus above the responsive raceway groove of semiconductor.
5. the principle that can detect the content of the specific virus in the body fluid of people or animal like the biochip of right 1 described field effect transistor is to be connected above the responsive raceway groove of semiconductor, when the blood of getting person to be detected or animal to be detected, body fluid to certain viral antibody protein; Through microfluid passage, when flowing through the raceway groove that is modified with antibody, specific combination can take place with virus in antibody; Be adsorbed on above the raceway groove, because the virion surface has in solution electrically; When they are adsorbed on the raceway groove of semiconductor field effect transistor; Can produce effect of electric field to field effect transistor, weaken or strengthen exhausting of semiconductor channel, thereby influence mobility of charge carrier speed or concentration; The increase that finally has electric current perhaps weakens, thereby detection system detects the variation of electric signal.
6. described to certain viral antibody like right 3, the biology of its available routine of originating prepares the monoclonal antibody method preparation, is modified on the raceway groove of field effect transistor again.
7. like the PDMS microfluid passage of right 1 described auxiliary feed liquor, fluid; It is characterized in that making up by PDMS; Controlling liquid flow range and direction, the rectangular parallelepiped block that fits tightly with silicon substrate has long number centimetre on the one side of fitting with silicon substrate; The passage wide 50 microns to 1 millimeter, that dark 50 microns to 100 microns depression is gone down; When fitting tightly with silicon substrate, promptly constructed an airtight passage between silicon substrate and PDMS piece, respectively connect plastics or silica gel hose again at feeder connection and exit.The effect of whole microfluid passage device is that the control sample flows into pick-up unit, and lets flow flow through the channel part of field effect transistor slowly, in an orderly manner, flows out to waste liquid pool again.
8. like the PDMS microfluid passage of right 6 described auxiliary feed liquors, fluid; Its preparation method does, utilizes the semiconductor light carving technology, on silicon substrate, makes up a long number centimetre; The SU-8 photoresist effect template of wide 100 microns, dark 50 microns convexity; Then with the PDMS of liquid state add cover behind the hardening agent of doses with template on, put into vacuum drying chamber subsequently, vacuum state, 90 degree heating down took out after 20 minutes; The PDMS piece that will solidify separates with template, fits tightly promptly to make up with silicon substrate again and accomplishes.
9. like right 1 described electronic detection system, mainly constitute by the supply unit that can give voltage and a reometer at three electrode tips (source electrode, drain electrode, grid) of field effect transistor.
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| CN103424457A (en) * | 2012-05-18 | 2013-12-04 | 中国科学院微电子研究所 | Biosensor and DNA sequencing method thereof |
| CN104655594A (en) * | 2015-01-29 | 2015-05-27 | 山东师范大学 | Nanometer-material-photothermal-effect-based cell detection method and cell detection device |
| CN105301080A (en) * | 2015-11-13 | 2016-02-03 | 南京大学 | Gridless AlGaN/GaN field effect transistor sensor, and preparation method thereof |
| CN105353019A (en) * | 2015-11-13 | 2016-02-24 | 南京大学 | Gridless AlInN/GaN field effect transistor sensor and preparation method thereof |
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| CN104655594A (en) * | 2015-01-29 | 2015-05-27 | 山东师范大学 | Nanometer-material-photothermal-effect-based cell detection method and cell detection device |
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| CN105301080B (en) * | 2015-11-13 | 2018-01-19 | 南京大学 | Without grid AlGaN/GaN field effect transistor sensing devices and preparation method thereof |
| CN105353019A (en) * | 2015-11-13 | 2016-02-24 | 南京大学 | Gridless AlInN/GaN field effect transistor sensor and preparation method thereof |
| CN105301080A (en) * | 2015-11-13 | 2016-02-03 | 南京大学 | Gridless AlGaN/GaN field effect transistor sensor, and preparation method thereof |
| WO2022081330A1 (en) * | 2020-10-14 | 2022-04-21 | Mower Morton M | System, apparatus, and method for viral monitoring in effluent |
| CN112683977A (en) * | 2021-01-05 | 2021-04-20 | 复旦大学 | Novel coronavirus detection module and method based on multi-target-site antibody combination |
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| CN113933365A (en) * | 2021-10-13 | 2022-01-14 | 清华大学 | Renewable field effect transistor biosensor |
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