CN102435653A - Antibiotic drug screening device and method based on field effect transistor - Google Patents
Antibiotic drug screening device and method based on field effect transistor Download PDFInfo
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
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Abstract
A method for screening antibiotic drugs based on a field effect transistor comprises a field effect transistor biochip, a micro liquid storage tank and an electronic detection system. A pair of source and drain electrodes and a semiconductor channel between the source and the drain electrodes are constructed on a silicon substrate by utilizing a semiconductor manufacturing process, and the channel is subjected to biological functional modification. Then a micro liquid storage tank is constructed on a channel of the chip by using polydimethylsiloxane for culturing bacteria, and finally a source electrode and a drain electrode are connected with a set of signal detection system, so that the antibiotic drugs can be screened at high flux, and the method is an integrated, automatic, rapid and cheap screening method for the antibiotic drugs.
Description
Technical field
The present invention relates to a kind of apparatus and method of utilizing field effect set transistor fast high-flux screening antibiotics.
Background technology
Microbiotic is called antibiotic again, and it can kill bacteria, again other pathogenic microorganismss such as mould, mycoplasma, Chlamydia is also had good inhibition and killing action.Microbiotic is a kind of material that produces in the certain micro-organisms growth and breeding process, and the microbiotic that is used to cure the disease also has fully with synthetic or part synthetic except that directly extracting thus.Antibiotic main effect is the medicine that is used to treat various bacterial infections or suppresses pathogenic microorganism infection.
China is that microbiotic uses big country, also is production of antibiotics big country, produces about 210,000 tons of microbiotic raw material per year, exports 30,000 tons, per capita (U.S. is 13 grams only) about year consumption figure 138 grams.Show according to 2006 to 2007 annual Ministry of Public Health whole nation bacterial resistance monitoring results; Whole nation hospital antibacterials year utilization rate is up to 74%; In developed countries such as the U.S. and Britains, the microbiotic utilization rate of hospital is merely 22%-25%, and China is one of the most serious country of abuse of antibiotics problem in the world.Scientifically using antibiotic method is to do bacterium to cultivate and do drug sensitive test, selects extremely sensitive medicaments for use according to the result of drug sensitive test, has so just avoided blindness, and can receive excellent curative.
The principle of antibiotic antibacterial or sterilization; Mainly be to kill and wound to the mechanism of " bacterium have and people's animals and plants such as (or) other height do not have "; Following 4 big types of mechanisms of action are arranged: 1, hinder the synthetic of bacteria cell wall; Cause bacterium swelling fracture under the Hyposmolality environment dead, the microbiotic of effect mainly is a beta-lactam antibiotic by this way.2, with the bacterial cell membrane interaction, strengthen bacterial cell membrane permeability, open the ion channel on the film, let the inner utility of bacterium spill thalline or electrolyte balance imbalance and dead.The antibiosis of effect have polymyxins and gramicidins etc. by this way.3, used each other with bacterial ribosome or its reaction substrate (like tRNA, mRNA), CKIs matter synthetic.The microbiotic of effect comprises TCs, macrolide antibiotics, aminoglycoside antibiotics, chloromycetin etc. by this way.4, hinder duplicating and transcribing of DNA of bacteria.Effect mainly is the antimicrobial quinolonyl class (like Ofloxacin) of synthetic by this way.No matter be which kind of above-mentioned mechanism of action; Bacterium receives under the influence of antibiotic medicine all can suppress its normal growth; Different when causing its nutrient culture media and normal cultured; And this change can be detected by these apparatus and method, thereby can monitor the growth conditions of bacterium, further judges the bactericidal effect of antibiotic medicine then.
The drug sensitive test that utilizes microbe growth to do on the conventional biomedical sector need be done agar and move piece method, liquid diffusion method etc., need pave plate, cultivates, screens bacterium.Higher to requirement for experiment condition, need some specific experimental facilitiess, and the loaded down with trivial details reagent of process is expensive, be unfavorable for high flux, fast, the validity of cheap, screening drug-resistance of bacteria and antibiotics.Receive existence situation, metabolic capability under the drug effect and utilize this method can judge bacterium directly, obviously, fast.
In order to satisfy current screening to a large amount of antibiotics; Solve antibiotics abuse, bacterial drug resistance problem; We develop a kind of can be fast, the method for high flux, trace (being less than 1 milliliter) screening antibiotics; Gathered field effect transistor the output of highly sensitive, real-time electric signal, be easy to characteristics such as integrated, can be used for the high flux screening antibiotics.
Summary of the invention
Fundamental purpose of the present invention be make up a cover can be fast, high flux, be easy to the method for the screening antibiotics of integrated, robotization.This method can solve the weak point of existing classic method, more cheap and simple, fast a large amount of evaluation antibacterials are to the influence of bacterium.Utilize bacterial reproduction, metabolism to change the character of solution composition on every side; Carry out input in conjunction with this physics device of field effect transistor; Make this device be easier to integrated, robotization, high flux ground screening of medicaments; Be suitable for current social microbiotic and produce in a large number, bacterium more and more has the resistance to the action of a drug, so medication needs large-scale rapid screening before, estimates the inhibiting effect of various antibacterials to bacterial growth.
The invention provides a kind of method of novel screening antibiotics, its principle is: on fieldistor channel, make up a miniature liquid storage tank culture of bacteria with dimethyl silicone polymer.Bacterium utilizes the glucose in the nutrient solution to carry out the metabolic while; Can in solution, discharge some metabolic products; Like lactic acid, citric acid; They can change the pH value of solution on every side, and the change of pH also can cause the variation of liquid storage tank bottom field effect transistor electric signal in this solution.And after in liquid storage tank, adding some antibiotics (like penicillin); Antibiotic medicine can interfere with bacterial carry out metabolism; Suppress the generation of metabolic product, thereby suppress the change of the pH value of solution, cause the change in electric of field effect transistor also to weaken.And the antibacterials of adding are strong more to the bacterial metabolism inhibiting effect, so the change in electric of field effect transistor also just more a little less than, thereby can come to judge fast a kind of ability of inhibition bacterial metabolism of antibiotic medicine through the intensity of variation of electric signal.In addition on the one hand; Because the lipopolysaccharides on bacterium such as Escherichia coli surface makes its meeting in solution electronegative; So bacterium can be adsorbed on the semiconductor channel of the polylysine that is modified with positively charged, 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, produce the variation of electric signal.If certain antibiotic medicine antibacterial action is not obvious; Bacterium can breed in a large number so; Increasing electronegative bacterium is adsorbed on the raceway groove of field effect transistor; Can produce strong change in electric, thereby can come the growing state of reacting bacteria under drug effect from the degree of change in electric.
One of technical scheme of the present invention is, made up the biochip (like Fig. 1) of the field effect transistor that is used to screen antibiotics, 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 polylysine solution again, reacted 1 hour at the raceway groove place.Promptly obtain the field effect transistor biochip of functional modification.
Two of technical scheme of the present invention is, a kind of apparatus and method of screening antibiotic medicine are provided, its composition and acting as:
(1) the miniature liquid storage tank that constitutes by PDMS (dimethyl silicone polymer) on the semiconductor channel.It is characterized in that being made up by PDMS, dissolving long-pending is about 500 microlitres, and the Xiao Chi of circular or square control storaging liquid fits tightly with silicon substrate, guarantee that the liquid in the liquid storage tank does not outflow.The effect of this liquid storage tank is the liquid storage culture of bacteria, owing on raceway groove, make up, so the subtle change of the SOLUTION PROPERTIES in the liquid storage tank all can be required by pmos sense transistor.So devices such as container all sterilize, sterilization, clean, guarantee not bring into other pathogenic microorganisms or impurity effect testing result.
(2) field effect transistor biochip.Modify the polylysine of positively charged in the responsive channel part of field effect transistor; Connect outside measurement, checkout equipment again; When the bacterial reproduction in the liquid storage tank was grown, the bacterium in the solution can be because total usefulness that positive negative electricity attracts each other be combined on the semiconductor channel; Because bacterium is electronegative in solution; Be combined on the raceway groove or bacterial metabolism produces the pH value that meeting such as lactic acid, citric acid change solution and can the charge carrier in the semiconductor channel be exerted an influence, thereby change the field effect transistor source electrode, the signal of the electricity between draining, measured instrument detects.
(3) signal 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.Act as and detect the electrical signal variation.
The feasibility of this antibiotics screening technique derives from the field effect transistor biochip and changes the sensitivity response of SOLUTION PROPERTIES on every side for bacterial reproduction, metabolism; The ubiquity of this method derives from and can be used to cultivate the bacterium that most laboratory can be cultivated, breed; Can screen various antibiotics; This method can realize fast, the reason of high flux screening is that field effect transistor biochip and electronic detection system are easy to miniaturization, power consumption is low, and simple, can reuse, produce in enormous quantities; Be suitable under the condition that does not have general Biochemistry Experiment chamber cheap fast screening antibiotic medicine.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 and by the computer quick identification; And bacterial reproduction speed is fast, the signal in real time monitoring and detection, only needs several hrs just can draw the inhibition degree of a kind of antibiotic medicine to bacterial metabolism.Outwell the liquid in the liquid storage tank, the simple cleaning can be carried out the screening of next medicine, makes that this device easily is automated, the high flux screening medicine.
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; The structure material is flexible and changeable; Can be organic film, inorganic, metal oxide material, construction method be simple, if use the organic material (like Graphene) of some good biocompatibility; Be more conducive to bacterial growth, improve the confidence level and the accuracy of screening.
(2) the field effect transistor biochip that the present invention is based on utilizes PDMS liquid storage tank and SiO when detecting
2Passivation layer is protected electrode and chip, effectively improves chip utilization rate and repeatability.
(3) this method can realize sensitivity, apace medicine screened owing to use nano level organic and inorganic material construction field effect transistor biochip, and screening sensitivity improves 10 than classic method
3To 10
6, just can accomplish detection time in several hours, and efficient improves about 10 times.Need not the other biological instrument and equipment.
(4) this method combines field effect transistor and extensive antibiotic-screening; The screening instrument of preparing, the output electric signal is easy to integrated, robotization, portability; Be fit to carry out quick, high flux screening instantly to a large amount of, miscellaneous antibiotics.。
Description of drawings
Fig. 1 is the method for the invention field effect transistor screening plant structural representation.
Fig. 1 mark implication sees the following form:
| Accompanying drawing 2 marks | Implication | Accompanying drawing 2 marks | Implication |
| 1 | Bacterium | 2 | The electrode passivation layer |
| 3 | Source electrode | 4 | The PDMS liquid storage tank |
| 5 | Semiconductor channel | 6 | Drain electrode |
| 7 | Silicon dioxide layer | 8 | Silicon substrate |
Embodiment one
Screening technique based on the antibiotics of 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.The polylysine protein solution reacted 1 hour again.
(4) making volume is 500 microlitre PDMS liquid storage tanks; Promptly at first on field effect transistor chip, construct a liquid storage tank template; 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, guarantees that the liquid storage tank no leakage gets final product.
(5) adding bacterium in the liquid storage tank cultivates.Source electrode, drain electrode with field effect transistor simultaneously is connected through lead with electronic detecting device.When cultivating certain hour, stage by stage, concentration adds glucose solution from low to high in the solution; Do not have the stage adding to need the microbiotic of screening simultaneously, make the concentration of glucose be ladder and rise, monitoring in real time each in stage bacterium to the degree of utilizing of glucose sugar; Whether receive the inhibition of antibiotic medicine; The promptly electric variation of leading is more little, and it is strong more to say that medicine suppresses ability to bacterial metabolism, explains that the antibiotic property of this type of antibiotic medicine is better.
Embodiment two
Based on SnO
2The screening technique of the antibiotics 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
2The nano-wire transistor device 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.The polylysine protein solution reacted 1 hour again.
(4) making volume is 500 microlitre PDMS liquid storage tanks; Promptly at first on field effect transistor chip, construct a liquid storage tank template; 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, guarantees that the liquid storage tank no leakage gets final product.
(5) adding bacterium in the liquid storage tank cultivates.Source electrode, drain electrode with field effect transistor simultaneously is connected through lead with electronic detecting device.When cultivating certain hour, stage by stage, concentration adds glucose solution from low to high in the solution; Do not have the stage adding to need the microbiotic of screening simultaneously, make the concentration of glucose be ladder and rise, monitoring in real time each in stage bacterium to the degree of utilizing of glucose sugar; Whether receive the inhibition of antibiotic medicine; The promptly electric variation of leading is more little, and it is strong more to say that medicine suppresses ability to bacterial metabolism, explains that the antibiotic property of this type of antibiotic medicine is better.
Claims (9)
1. high flux antibacterials screening technique based on the field effect transistor biochip; It is characterized in that the field effect transistor chip with the functionalization bio-modification is a core; Add that miniature PDMS (dimethyl silicone polymer) liquid storage tank, electronic detection system three parts form, can be fast, the high flux screening antibiotics.
2. like the biochip of right 1 described field effect transistor, it is characterized in that on silicon substrate, utilizing metallic source electrode, the drain electrode of semiconductor technology manufacturing, and be connected the semiconductor channel between source electrode and drain electrode.Semiconductor sensitive material raceway groove can be silicon nanowires, CNT, SnO
2Nano wire, TiO
2In nano wire, GaN nano wire, graphene nanobelt, 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 biochip of right 1 described field effect transistor, it is characterized in that: described 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 polylysine above the responsive raceway groove of semiconductor.
5. the principle that can detect the bacterial growth situation like the biochip of right 1 described field effect transistor is; On fieldistor channel, make up a miniature liquid storage tank culture of bacteria with PDMS; Bacterium such as Escherichia coli are because the lipopolysaccharides of surface detail after birth can be electronegative in solution; So bacterium can be adsorbed on the semiconductor channel of the polylysine that is modified with positively charged, semiconductor channel is produced effect of electric field, weaken or strengthen exhausting of raceway groove; Thereby influence the migration rate or the concentration of charge carrier in the raceway groove, finally cause electric current to change.If bacterial reproduction quantity increases, so just have increasing electronegative bacterium and be adsorbed on the raceway groove of field effect transistor, cause electric current that bigger variation takes place, thus the growth change of coming reacting bacteria through the variation that detects electric signal.
As the biochip of right 1 described field effect transistor can the rapid screening antibiotics principle be; On fieldistor channel, make up a miniature liquid storage tank culture of bacteria with PDMS; Bacterium can utilize the glucose in the nutrient solution to carry out metabolism, can in solution, get rid of some metabolic products, like lactic acid, citric acid; They can change the pH value of solution on every side, and the change of pH also can cause the field effect transistor change in current in this solution.And after in liquid storage tank, adding some antibiotics (like penicillin); Antibiotic medicine can interfere with bacterial carry out metabolism, suppresses the generation of metabolic product, thereby suppresses the change of the pH value of solution; Cause the electric current variation of field effect transistor also to weaken; The antibacterials of this adding are strong more to the bacterial metabolism inhibiting effect, a little less than the electric current of field effect transistor changes more, thus the ability that can come to judge fast a kind of inhibition bacterial metabolism of antibiotic medicine like this.
7. like the right 1 described miniature liquid storage tank that on semiconductor channel, constitutes by PDMS; It is characterized in that being made up by PDMS, dissolving long-pending is about 500 microlitres, the Xiao Chi of circular or square control storaging liquid; Fit tightly with silicon substrate, guarantee that the liquid in the liquid storage tank does not outflow.The effect of this liquid storage tank is the liquid storage culture of bacteria.Owing on raceway groove, make up, so the subtle change of the SOLUTION PROPERTIES in the liquid storage tank all can be required by pmos sense transistor.
8. like the right 6 described miniature liquid storage tanks that on semiconductor channel, constitute by dimethyl silicone polymer; Its preparation method is for constructing a liquid storage tank template on the effect transistor chip at first on the scene; 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, and the dimethyl silicone polymer liquid storage tank that will solidify separates with template promptly to make up to be accomplished.
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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