CN1228635C - Biosensor and quantified detection method using said biosensor - Google Patents
Biosensor and quantified detection method using said biosensor Download PDFInfo
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- CN1228635C CN1228635C CN 01104109 CN01104109A CN1228635C CN 1228635 C CN1228635 C CN 1228635C CN 01104109 CN01104109 CN 01104109 CN 01104109 A CN01104109 A CN 01104109A CN 1228635 C CN1228635 C CN 1228635C
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Abstract
The present invention relates to a biosensor which utilizes an electrochemical method to quantitatively detect and measure protein and nucleic acid of liquid samples in real time, and a method which utilizes the biosensor to carry out quantization detection on protein and nucleic acid of liquid samples. The biosensor comprises a base sensor, a capillary film and a compound pad, wherein the base sensor is provided with a working electrode, a reference electrode and at least one third electrode which are coated on a plastic base. First conjugate which can be combined with analyte is fixed on the working electrode. The electrodes of the base sensor are connected with a readable instrument to carry out electrochemical measurement. The capillary film is arranged on the base sensor, and is used for detecting the sample transportation in the horizontal direction. The compound pad comprises second conjugate, wherein the second conjugate can be combined with the analyte in a different way.
Description
Technical field
The present invention relates to a kind of can be to being present in the sensor device that protein in the fluid sample and nucleic acid carry out detection by quantitative, relate in particular to a kind of biology sensor that utilizes protein and nucleic acid in electrochemical method detection by quantitative and the The real time measure fluid sample.
The invention still further relates to the method that this biology sensor of utilization quantizes to detect to the protein in the fluid sample and nucleic acid.
Background technology
Along with the development of modern medicine, medical test also more and more higher for the requirement of the modern medicine inspection technology that is used for medical diagnosis on disease, quick, efficient, pinpoint accuracy can provide strong support and help for the early detection and the diagnosis of disease.For doctor and patient, the idealized model of medical test should be at any time, everywhere, test sample check system that need not to pass on, full-automatic.People more and more recognize, when carrying out medical diagnosis on disease, can determine for example content of the various compositions of blood, seminal fluid etc. of human body fluid, and the importance that can detect the pick-up unit of quantized result rapidly that designs for the layman.In the various detection indexs that are used for medical diagnosis on disease, the detection of protein and nucleic acid is the important indicator of the preventative examination of various angiocardiopathies, prostatic disorders, cancer, genetic disease and various diseases in the body fluid.
Immunoassay has been widely used in detecting antigen and antibody.Commonly used is exactly enzyme immunoassay (EIA) (EIAs).EIAs is specially adapted to clinical analysis, medical diagnosis, medicine analysis, environmental monitoring, food quality control and bioprocess analysis.Its importance is high sensitivity and accuracy, and it can detect the various check compositions that derive from multiple sample matrix.According to the disposal route of sample, EIAs is divided into heterogeneous enzyme immunoassay (EIA) (needing free antigen is separated) usually in the antigen of those and antibodies, with homogeneous EIA (need not to separate or cleaning step).According to the existence condition of antibody combining site, EIAs also can be divided into competitive or noncompetitive.Usually, for the conventional analysis of great amount of samples, EIAs is easy and is suitable for the analysis of various detection things.Yet in analytic process, these methods need repeatedly cleaning and incubation step to finish.And for most situation, these methods must realize by means of complicated and expensive analytical equipment.In addition, repeatedly clean and incubation step has also limited the development and the exploitation of the portable check and analysis equipment of the common medical center that is applicable to dispersion.
In the last few years,, can overcome the limitation of heterogeneous EIAs by people's effort, and found homogeneous phase, fast with the immunoassay method that need not to separate cleaning step, this method is convenient to use at general curative point.And developed a kind of simply, EIAs detects fast, can carry out single analyte by visual change color and detect.Based on the technology that antigen or antibody is solidificated on the solid phase object, just by using some can obtain clinical required analysis result simply qualitatively apace as surveying liquid meter, test tube and these routine test utensils of permeable membrane testing cassete.This membrane type detection method is solidificated in antigen, antibody on the solid phase object, shifts utensil by using some conventional liquid, just can carry out simple qualitative analysis apace.This method not only extensively is applied in home test, and just more and more manyly is adopted by the laboratory of doctor and hospital.For example be used for the inspection of pregnancy, the infection of throat strep, bacterium and ovulation prediction.Relevant this class detects at United States Patent (USP) Pat.Nos.5, and is on the books in 622,871,4703,017,5,468,647 and 5,798,273..
Said method has overcome a difficult problem that needs separation, cleaning step in the conventional sense method, for clinical detection provides quick, easy new way.But the defective of this method for quick is that it does not have high sensitivity and quantitative detection performance.Thereby some need targetedly that the medical diagnosis of detection by quantitative can only carry out in the special laboratory that has such complicated immunoassay analytical instrument.A main tendency of tachysynthesis detection method in recent years, develops towards the detection by quantitative direction exactly.People have proposed the scheme of a kind of membrane type immunologic detection method as quantitative test, and instantiation is seen United States Patent (USP) No. 5753517, have wherein put down in writing and have utilized antibody conjugates, object of reference and membrane type liquid transfer method to carry out quantitative immunity-stratographic analysis.But this method need not sample separation, the purpose that detects of step to also existing certain difficulty, reason to be that this method can't reach in the diagnostic detection of common medical center.Perhaps use the existing outstanding problem of membrane type immunologic detection method to be to have contradiction for the aspect that requires of film.For example, the employed film of the solidifying requirements of protein has very strong protein adhesion characteristic in detection, but requires aspect the analyte film can not adhesion protein matter again transmitting.And, be generally used for improving between each key element of sensitivity of this membrane type detection method and but have exclusiveness.For example, passivator has also reduced the amount of specific reaction signal when reducing the nonspecific reaction signal.Owing to usually run into the contradiction to the film demand in the performance history of membrane type immunologic detection method, the use of conventional membrane type detection system in quantitative immunologic detection method is greatly limited.
Therefore, this just requires immunoassay device and the method that can providing of a kind of improvement is quick, accurate, quantize testing result can be provided.The high sensitivity of Electrochemical Detection, in addition antibody-antigen-reactive intrinsic characteristic an advanced person's technology----electro-chemistry immunity is detected be achieved.This technology is except succinct, the sensitive advantage that possesses Electrochemical Detection, and its salient point is to measure the undressed sample that has other interfering material to exist.Adopting the immunoassay technology of amp gauge Electrochemical Detection to be applied aspect the assay determination of liquid sample.United States Patent (USP) No.5 has recorded and narrated the patent that the immunoassay device that adopts amp gauge to detect carries out the body fluid analysis deagnostic test for 830, No. 680.This device comprises the electrochemical detection system of a sandwich type immunologic detection method that need not to separate, and protein analyte wherein as HCG, is sandwiched in and solidifies between absorption antibody and a kind of alkaline phosphate antibody-like on the microporous membrane gold metal electrode.Though this class device has the advantage that need not separating sample, when handling and hatching sample, but need spend on the long time, this has just limited the application of this detection method in quick diagnosis test.
United States Patent (USP) No.5756362 number carries out to a kind of liposome that adopts that signal produces and the method for Electrochemical Detection is described in immune detection.In this method, the liposome and the analyte of parcel being gone into electric responsive thing are compound.The sample that at first will contain analyte is put into a proving installation and is hatched, this sample contain simultaneously a kind of can with the material of analyte and analyte-liposome complex specific bond.After hatching, this mixed solution transfers to an electrochemical measurement part through adsorption bar, and here liposome is dissolved by cell lytic agent and discharges electric responsive thing.The responsive thing burst size of electricity can detect with electrochemical method, and its amount what with sample in the content of analyte corresponding relation is arranged.
In United States Patent (USP) No.5391272 number, recorded and narrated and on colloidal metal, wrapped quilt, then the method for bag quilt on sensor with biological active matter.The detection of analyte is to be solidificated in the electric current that the electric sensitive kinds material on the sensor produced by measurement to realize that this class material is analyte/enzymic catalytic reaction part.Detect some analytes (as hormone or herbicide) though this method is applicable to, it need be through reaching desirable sensitivity than complex separation process and the process of hatching.
The method that other Applied Electrochemistry detection method is carried out immune detection all need rely on some conventional methods in heterogeneous immune detection, such as, need hatch for a long time and cleaning step repeatedly, free antigen and detecting reactant could be separated in antigen that is cured and reactant.Separate the experiment report that galvanometer carries out immunologic detection method though existing several research institutions have issued about using not have, a kind of simple, quick, successful report that sample need not the galvanometer immunoassay of separating step is not arranged so far yet.
The gene diagnosis technology is developed rapidly at the twentieth century end, adopts the gene diagnosis technology to find that disease takes place and the trend of development in the very early time of various diseases.The development of gene diagnosis technology has been experienced the pcr amplification technology of traditional molecular hybridization, gene and comprehensive above-mentioned technology and to the biochip technology of microcosmic and integrated development.The DNA chip is that known gene order is synthesized the surface of solidifying in medium by printing or original position, and the latter can accurately analyze out by molecular hyridization with gene element in the sample.Though biochip technology is expected to obtain huge development in 21st century, final marketization still has some key issues to need to be resolved hurrily.Wherein very important problem is the robotization and the universalness of genetic chip checkout equipment.At present, the testing result of DNA chip generally all is to adopt the scanner scanning chip, and this equipment price costliness needs special equipment and professional and technical personnel operation, has limited the application of biochip technology in clinical diagnosis greatly.
Therefore, a kind of can provide simple, quantitatively and the detecting instrument measured of real-time diagnosis and the technology of method be still the task of top priority.
Summary of the invention
At the defective of prior art, the object of the present invention is to provide a kind of utilize electrochemical method directly, fast, the protein in the detection by quantitative sample and the biosensor arrangement of nucleic acid.
Another object of the present invention provides the method for utilizing protein and DNA in this biology sensor detection by quantitative sample.
This invention has solved many technical matterss in this field, a kind of simple, quick and reliable method successfully is provided, and the mode of utilizing immunoassay, molecular hyridization and Electrochemical Detection to combine comes direct quantitative to detect the analyte that is present in the liquid sample.
The present invention has used combined electrical chemical biosensor (being sensor diaphragm).This sensor diaphragm comprises a substrate sensor, and it is printed on conductor material on the one corresponding matrix by serigraphy and processes.A kind of specific reaction thing corresponding to analyte (first bond) is solidificated in working electrode surface, and this reactant can be specific antibody, the nucleic acid corresponding to target analytes, also can be target analytes itself.
Sensor device of the present invention is made up of a sample region and a detection zone.Wherein, detection zone comprises the electrode surface area that is used to solidify the specific reaction thing.Sample region then comprises a composite pad, wherein include predetermined fixed can with second bond of analyte specific bond.This second bond can be a kind of through the label mark can with nucleic acid, protein or the high score subclass material of analyte specific bond, when when reacting, it can produce a kind of Electrochemical Detection signal with substrate (or detect thing).
Sensor device of the present invention can also comprise a capillary film, by it sample region and detection zone is coupled together.This capillary film is delivered to detection zone as a kind of carrier by the liquid sample that capillary action will contain analyte and detecting reactant, makes it be cured in electrode surface by the molecular hyridization between antibody antigen reaction or the nucleic acid.The material that can be used as the capillary film includes but not limited to nylon, cellulose, paper and materials similar.Preferred film is the reticulate texture film.The advantage of reticulate texture film is that its planar structure is suitable for liquid sample and flows to the detection zone plane from sample region, and then directly contacts with the specific reaction thing that solidifies in electrode surface.
The composite pad that sensor device of the present invention is subsidiary, be used for absorbing and the on purpose release of the detecting reactant of control mark, also can be simultaneously/or subsidiary separating and filtering device, be used for blood plasma is separated from blood, blood plasma is combined with second bond on the composite pad.
Sensor device of the present invention also is provided with a waste liquid absorption pad, and this absorption pad should have enough spaces and capacity to receive the liquid sample that is used to detect.
The present invention has also further introduced the method for using protein and nucleic acid content in biology sensor detection by quantitative one analyte of the present invention.When using this biology sensor and carrying out enzyme immune detection or detection of nucleic acids, the sample that will contain target analytes is put into sample region.This sample will be along the capillary film flow that is installed on the sensor diaphragm, reacts with second bond of mark, and then reacts with first bond that is solidificated in sensor surface (antibody or nucleic acid).When adopting the sandwich immunologic detection method, analyte will be sandwiched between second bond of antibody that solidifies on the sensor and mark.In the liquid sample content of analyte with in this process, exist certain proportionate relationship by the antibody antigen amount be solidificated in the sensor surface analyte that interacts, and the content of analyte can record by the antibody-multienzyme complex that is fixed on sensor surface.The amount of analyte just can be determined according to the typical curve of target analytes.Combine with the specificity of solidifying between the detecting reactant on the electrode by analyte, utilize the label that is marked on the specific reaction thing to carry out detection by quantitative as indicator.
Method of the present invention combines the principle and the method for immune response (molecular hyridization) and electrochemical reaction, and utilization can detect as indicator with the label of analyte generation specific reaction.Electric current and the proportional relation of the concentration of analyte in liquid sample that produces by sensor device under certain conditions, and this electric current can be measured by reometer.No matter be the detection that to finish analyte in damping fluid and the body fluid sample with competitive mode of the present invention or sandwich biology sensor.
When biology sensor of the present invention and an electrochemical appliance, as a kind of hand-held detector, can carry out Electrochemical Detection after being connected, directly testing sample is dropped in and carry out Electrochemical Detection on the disposable sensor diaphragm, can obtain testing result rapidly.Even the layman also can obtain highly sensitive quantitative test result in the common again medical center within the short time (tens of seconds to 30 minutes).In addition, method of the present invention almost can be common to the immunocompetence class material in any liquid sample and the detection by quantitative of nucleic acid, and therefore, it is with a wide range of applications in medical diagnosis, agricultural and environmental monitoring field.
In addition, biosensor arrangement of the present invention also can be designed to box-packed pattern, is suitable for family, clinic and common medical center and uses.
Description of drawings
Below in conjunction with accompanying drawing,, describe in detail but do not limit the present invention by description to preferred embodiment of the present invention.
Figure 1A is the diagrammatic top view of sensor of the present invention.
Figure 1B is the diagrammatic top view of based sensor in the sensor of the present invention.
Fig. 2 is the exploded view of electronics immunosensor.
Fig. 3 represents the corresponding relation of the current response that prostate class antigen is produced in the content of damping fluid (■-) and serum (▲-) sample and electronics immunosensor detection zone.
The content of AFP in Fig. 4 express liquid sample is with the corresponding relation that uses the sensor diaphragm form in the current response of detection zone.
Fig. 5 represents the electrochemical reaction of the liquid sample cardiac Troponin I measured by the electronics immunosensor.
Embodiment
Biosensor arrangement of the present invention comprises a substrate sensor and film, forms a sensor device (being sensor diaphragm) jointly, and example is seen Fig. 1 and Fig. 2.
Shown in Figure 1B, substrate sensor 4, have on it by serigraphy and be coated on the working electrode 2 on the plastic-substrates, contrast electrode 8 and at least one third electrode 10, substrate sensor has multiple method for making, for example be printed in the suitable substrate, for example Mylar plastic pvc or the like by a kind of electric conduction coating liquid (as the carbon element masking liquid) barrier film.Substrate can be cut apart with a knife or scissors then and make a plurality of substrate sensor diaphragms.The all available carbon element masking liquid of working electrode and third electrode is printed, and the then the most handy silver masking liquid of contrast electrode is printed.Want to make working electrode and third electrode that better electric conductivity is arranged, can be chosen in the following silver masking liquid of last layer that is coated with earlier of carbonaceous coatings.Can also on conductive coating, be coated with one deck insulating material again with corresponding position, sample collection district.The substrate sensor of making of said method need not further processing when solidifying absorption first bond.Third electrode can be a correcting electrode, is used for the error of correct detection system, also can be one second working electrode, is used for the blank as working electrode 2.
Corresponding to first bond of analyte, can be protein or nucleic acid, for example in the sandwich immune detection, first antibody is solidificated in electrode surface by suitable mode, an available spraying or a mode that is coated with.This first bond can be a strain specific antibodies, nucleic acid or the target analytes itself corresponding to target analytes, and preferred curing antibody should be a monoclonal antibody, and its concentration should meet or exceed 10
9Liter/mole.
On substrate sensor 4, be furnished with respectively: a sample region 14 ', its top is provided with a composite pad 20, is used to gather the liquid sample; One detection zone 16 ', the liquid sample of collection comes in contact reaction at this and described electrode; An and waste liquid absorption pad 18; Sample region is connected by a capillary film 22 with detection zone, and the liquid sample is sent to detection zone from sample region, and analyte promptly is sandwiched in and solidifies in advance or be deposited on second bond in the composite pad and be cured between first bond of electrode surface.
Figure 1A is depicted as the capping of sensor diaphragm 12.This capping 12 has two openings 14 and 16.When being contained in this capping 12 on the sensor diaphragm 4, opening 14 and 16 corresponds respectively to sample region 14 ' and the detection zone 16 ' on the sensor diaphragm, and they are exposed to working electrode surface.
Three electrodes shown in Figure 1B provide being connected between sensor diaphragm of the present invention and the potential measurement instrument, can carry out current measurement with the detected material of electrochemical method to working electrode surface.When detection zone 16 ' had liquid sample, therefore this biology sensor produced continuous potential difference (PD) just as same electrochemical cell between working electrode and third electrode, just formed electric current between them.The size of the electric current in the circuit directly is directly proportional detailed being described below with the amount that is solidificated in the detecting reactant of working electrode surface by absorption antibody.The supporting potential measurement instrument of commercially available and the present invention comprise (West Lafayette, IN), Cypress System ElectrochemicalAnalyer (Laerence, KS), and AndCare ElectrochemicalMonitor (Durham, NC).
Sensor device of the present invention also can be loaded onto a separator-filter as required in the composite pad position, when the liquid vertical direction was passed through, this separator-filter can be separated blood plasma from blood.
Shown in the exploded view of Fig. 2, sensor device of the present invention has also designed the device of levels of reagent direction mobile (from left to right), in the present invention, absorption pad 18 and 22 terminal linking to each other of capillary film, composite pad 20 links to each other with the other end of capillary film 22, like this, absorption pad 18, capillary film 22 and composite pad 20 have covered sample region 14 ' and detection zone 16 '.Second bond, for example sign has (compound with it) can produce the antibody of the enzyme of Electrochemical Detection signal with the reaction of substrate and electronic transmission medium, is cured on the composite pad 20, and composite pad 20 is as the absorption of compound and sustained release effectively.The sample region 14 ' below capillary film 22 connects and the detection zone 16 ' of electrode surface, here analyte will be transported on the detection zone of electrode surface.
More particularly, after the sample that will be contained analyte by opening 14 adds sensor device, this sample composite pad 20 of at first flowing through, in composite pad, the contact of sample causes the release of the antibody-multienzyme complex (or nucleic acid) that is deposited on or is solidificated in advance on the composite pad, then, this sample and d/d antibody-recombination reaction thing (or nucleic acid) is brought to the detection zone 16 ' of sensor surface by capillary action through capillary film 22, and here analyte is adsorbed on the first antibody (or nucleic acid) that electrode surface solidifies and contains in the formed interlayer of second antibody (or nucleic acid) compound of label.
Electrochemical enzymatic immune detection (EEIA)
Compare with most other methods, EEIA combines the sensitivity of electro-chemical test and the selectivity and the specificity of immunoassay, can carry out the high detection of sensitivity, and can enlarge the scope that detects, electrochemical detection method is specially adapted to the immunosensor that antigen-antibody response is arranged of electrode surface, also can be used for the nucleic acid sensor that electrode surface carries out making nucleic acid molecular hybridization.(Ngo.T.T.Ed.Electrochemical Sensors in Immunological Analysis Plenum Press.ew York, 1987; And Monroe, D.Amperometric Immunoassays in CriticalReviews in clinical Laboratory Sciences 23 (1): 1-18,1990) the most frequently used enzyme sign of carrying out the EEIA detection comprises alkaline phosphatase (AP) and horseradish peroxidase (HRP).Usually a kind of desirable enzyme should be able to this zymolyte of efficient catalytic in the electron transfer reaction of respective media.
In one embodiment of the invention, used a kind of sandwich immune detection mode, the second antibody that horseradish peroxidase (HSRP) is used with forming sandwich is carried out compound.Can determine the amount (electric current that electrochemical reaction produces in also therefore having determined to detect) of electrode surface enzyme antibody compound with the amount of the analyte of the antibody generation specific reaction of curing on the working electrode, thereby can further measure the amount of target analytes.In addition, a kind of immune detection of state of conflict can be used for horseradish peroxidase (HRP) situation mutually compound with analyte.Analyte and analyte HRP compound are at the binding site of the electrode surface competition limited quantity of curing antibody.Because the competition essence of detection method, the concentration of analyte is inversely proportional in analyte of electrode surface-multienzyme complex quantity (electric current that electrochemical reaction produces in also promptly detecting) and the sample.
The activity of enzyme is that the electrochemical reduction character by electronic transmission medium is determined.The electronic transmission medium that may use among the present invention comprises ferrocene and derivant thereof, benzoquinones, as ascorbic acid or 3,3 ', 5,5 '-tetramethyl benzidine (TMB).The preferred medium of carrying out the active ammeter detection of HRP is TMB, ELISA reported once that TMB was adapted at using in the spectrophotometer measurement, and being used as that a kind of electrochemical mediators is used for HRP is the immune detection (G.Volpe et al, Analyst 123:1303-130T7,1998) of enzyme.Find that in the test of the sandwich method of inspection TMB is the good substrates of low content HRP being carried out Electrochemical Detection.
Therefore, in the methods of the invention, add preferred medium TMB and hydrogen peroxide HPR compound with the exploring electrode surface.The concentration that abundant TMB and hydrogen peroxide will be arranged in the experiment is to guarantee effective enzymatic reaction.Because the existence of the hydrogen peroxide that reacts with the HRP compound is arranged, and TMB is oxidized, reduced by substrate under the low potential relatively then.Owing to use low potential, 0mv-200mv (vs.Ag/AgCl) for example, many bioprocess of Electrochemical Detection that can disturb usually can not produce undesired signal.The concentration of employed electronic transmission medium and substrate remains on usually than required higher of enzymatic reaction.With this understanding, the steady current that circulation produced of electrode surface TMB is the amount corresponding to HPR.Because the solid phase that sandwich of layers depended on also can be used to measure electric current, as passing through electrode joint detection instrument, just can produce steady current within several seconds at the adding substrate solution.So the electric current that produces is directly proportional with the amount of the HRP compound that is cured in electrode surface by analyte.Substrate solution can add detection zone (Fig. 1) by hand when detecting, also substrate solution can be sealed in reservoir, in alumna pouch, is pre-installed on the sensor.In a kind of mode in back, when detecting, can tear and pack to allow solution flow out with the method for extruding or with mechanical means.
When using disposable immunosensor to carry out the enzyme immune detection, the sample that will contain target analytes places sample region.Second bond as enzyme-antibody complex, is released when contacting with sample.The sample and second bond subsequently infiltrate be installed on the sensor diaphragm the capillary film be cured on the sensor antibodies together.Cross device if Yi Lu is installed, for example as blood separation, this device then should be installed on the composite pad, and blood plasma is separated from blood.The liquid sample of any surplus of oozing out from the capillary film all can be absorbed pad and absorb.In the sandwich immunologic detection method, analyte is sandwiched between the antibody and antibody complex that is solidificated in sensor surface.The quantity of the analyte that separates from liquid sample is directly proportional with the analyte quantity that is fixed in the sensor electrode surface through antigen-antibody interaction, and can be detected by antibody-multienzyme complex that thing by analysis is cured in sensor surface.
The also available a kind of substrate sensor of immune detection is carried out, and will adsorb antibody (first antibody) and be solidificated on the working electrode.In this detection method, earlier a kind of sample that contains target analytes is mixed mutually with antibody-multienzyme complex, again potpourri is added sensor and carry out hatching of short time, incubation time should be enough to make analyte in the sample and antibody-multienzyme complex to combine and be adsorbed antibody absorption, and incubation time is about 5 to 30 minutes.Hatch finish after, the analyte and the compound that do not adsorb with flush away with the buffer solution cleaning sensor.Add the amount that the detection solution that contains zymolyte and electronic transmission medium is measured the analyte that is adsorbed then.Can measure with the readable instrument of the electric current of routine by steady current on the electrode of enzymatic electrochemical reaction generation.The size of electric current is directly proportional with analyte quantity in the liquid sample.
In another embodiment of the invention, use said method, hatch finish after, need not to use the buffer solution for cleaning sensor, but directly poor by the magnitude of current that galvanometer is measured between the working electrode and second working electrode as contrast electrode with second working electrode, this magnitude of current difference is directly proportional with the amount of analyte in the sample.
In yet another embodiment of the present invention, can use complete detecting instrument to quantize immune detection, for example use biology of the present invention (immunity) sensor.This immunosensor comprises the disposable sensor diaphragm that contains a detection zone at least, and its working electrode surface is solidified with first bond.This immunosensor also can comprise other assembly, as hand-held monitor, the standard control of analyte, buffer solution or the like.
The present invention is particularly useful for any analyte that produces antigen-antibody response applicable to any analyte that produces biological respinse almost.Here " analyte " speech refers to any sample or any sample that carries out making nucleic acid molecular hybridization that causes immunogenic response widely.Representational analyte includes but not limited to: anesthetic, hormone, protein, bacterium, virus, cancer marker or the like.Utilize biology sensor of the present invention and method thereof to include but not limited to by detected analyte: the prostate class Detection of antigen during prostate cancer detects; With alpha-fetoprotein (AFP), human chorionic gonadotrophin (HCG), free estriol (estriol) is the examination of science of heredity in utero of sign; With the suchlike detections such as acute myocardial infarction of myocardium calcium protein I as sign; And be that the molecular genetics of sign detects with DNA.Analyte can be because of the difference of various liquid samples difference to some extent, and they include but not limited to: various body fluid, for example analyte in serum, urine, saliva etc. and the various biofluids etc.
Here why with the instantiation of antigen-antibody response as biology sensor of the present invention, be because they are very representative and be convenient to understand, but be not only limited to protein-based but biology sensor of the present invention and quantified detection method thereof are used with reagent analyte generation specific reaction, can also comprise other natural synthetic or synthetic high score subclass, as the synthetic acceptor, the synthetic class of carbohydrates/protein, nucleic acid and nonprotein class can generate the material of target compound by compound or cross reaction until those.
An outstanding advantage of the present invention is that the layman just can carry out such quantitative testing, and only need finish adding sample solution or the such simple steps of detection agent.In the detection for a long time sample hatch and detachment process, whole testing process just can finish to dozens of minutes in a few minutes, really realized single stage method and need not the modern medicine test requirements of sample separation.
Below, we will be described in more detail the present invention by following examples, make advantage of the present invention and effect apparent.
Embodiment 1
Preliminary work: print sensor
Substrate sensor shown in Fig. 1 can be bought from market, also can conductive material be printed/be coated on a corresponding substrate by serigraphy, for example makes by oneself on the plastics.The method that used sensor is to use polyester film and masking liquid material maker to be recommended in the test is printed.The masking liquid material comprise silver conductor and carbon conductor (Polymer Thick film Compositions, 5000 and 7102, DuPont, Research Triangle Park, NC), Mylar is adopted in substrate
(mylar) plastics.Working electrode and third electrode are all printed by the carbonaceous masking liquid and are formed, and contrast electrode is printed by silver masking liquid and formed.Want to make working electrode and inoperative electrode to obtain better electric conductivity, can be chosen in the following silver coating of last layer that is coated with earlier of carbonaceous coatings.Also can on lead, be coated with the last layer dielectric film to form insulation course.The sensor made from the method need not further process aspect the curing of absorption antibody.
Embodiment 2
Antibody on the sensor of printing by silk screen print method solidifies
This example describe to use fatty alcohol solution that antibody is solidificated in method on the sensor that serigraphy prints.Fatty alcohol solution helps the wetting electrode surface, reduces static, quickens the decomposition of protein in the solution, makes it easier of electrode surface.
By using a kind of buffering antibody-solutions (as PBS) that contains fatty alcohol antibody can be directly fixed in the carbonaceous sensor surface.Preferred fatty alcohol is an isopropyl alcohol.Antibody to be solidificated on the sensor of serigraphy printing, need drip the phosphate solution that contains isopropyl alcohol that last layer is mixed into the absorption antibody-solutions of 3 μ l in the working electrode district.Volatilize in air under this fatty alcohol solution room temperature, deposition one deck is attached to the antibody of electrode surface.
The amount that adds the antibody of sensor needs to determine according to each absorption antibody.In general, in sensor, add the antibody of q.s, can obtain best sensor response so that form the individual layer antibody layer at sensor surface.The antibody that is solidificated in sensor surface by this method can keep the biologically active of self usually in a short time.
For further stablizing the antibody that is cured on the sensor, use in order to long-term, the sensor surface that has antibody to adhere to can be immersed in and contain 25% StabilCoat
Insulation ten minutes under the room temperature in the solution of (SurModics, Inc., EdenPrairie MN) and 0.01% Tween20.Solution evaporation after ten minutes, sensor becomes dry fully, puts it into then in the airtight container of drying agent.
The content of alcohol also affects the sensitivity of sensor response in the curing solution. and we have done many tests and have contrasted isopropanol content in the antibody curing solution to influence that it produced.Method in the use described in the joint, the monoclonal anti-alpha-fetoprotein antibody in the PBS impact damper that contains various different isopropyl alcohols (v/v%) has been cured on the sensor.This sensor that is cured put under the mixed solution room temperature that contains 200ng/ml alpha-fetoprotein (AFP) and AFP-HRP compound hatched 15 minutes, then with the PBS solution flushing of pH7.4/0.5%Tween 20.The instant electric current that AFP-HRP produced that solidifies on each sensor is measured by the method described in the use embodiment 3.Following table 1 explanation sensor is corresponding to the signal value that content obtained of isopropyl alcohol in the antibody curing solution.The result shows that 25% content is blanket in the PBS solution, so it is to solidify the optimal liquid proportional of using of great majority absorption antibody at present.
Table 1
| The number percent of isopropyl alcohol (%) | Corresponding real-time signal value signal number percent (%) |
| 0 5 10 20 25 30 | 47.7 51.5 58.4 70.1 100 64.5 |
Experimental results show that, methyl alcohol, ethanol and ethane acetate all are that antibody is solidificated in ideal solvent on the electrode, but the result of solidifying test shows, pass in time, the decline rate of the stability of the sensor of usefulness ethanolic solution curing antibody is more faster than the decline rate of the stability of the sensor of using the aqueous isopropanol curing antibody.
Curing antibody also can use except that dripping the technology cover the coating and realize, for example, the curing of antibody also can be used and antibody-solutions is sprayed at electrode surface allows the method for this solution evaporation then.Using the method for spraying to be deposited on the uniformity coefficient of antibody on the sensor can be by controlling the selection of solvent and spray condition.And this spray-painting technology also is applicable to the batch process of antibody cure sensor.
Embodiment 3
The Electrochemical Detection of HRP compound
By the compound of second antibody and a kind of substrate target analytes and curing antibody mode sticking and together can be formed a kind of sandwich structure, this structure is achieved by the compound quantitative determination of target analytes that makes of enzyme.
The activity of enzyme is that the electrochemical reduction character by electronic transmission medium is determined.The electronic transmission medium that may use among the present invention comprises dimethylarminomethyl ferrocene, ascorbic acid, benzoquinones, 3,3 ', 5, and 5 '-tetramethyl benzidine (TMB).The preferred medium of carrying out the active ammeter detection of HRP is TMB.ELISA reported once that TMB was adapted at using in Spectrophotometric, and be used as a kind of electrochemical mediators be used for HRP be enzyme immune detection (G.Volpeet al, supra).Find that in the test of sandwich check system TMB is the good substrates of low content HRP being carried out Electrochemical Detection.
By the TMB that contains 40 μ M, and the 0.1M of 5%-10% dimethyl sulfoxide, a kind of preferred common substrate that PH6.0 sodium acetate solution and 0.01% hydrogen peroxide mix is in order to the HRP enzymatic reaction.Perhaps, also can buy the substrate solution finished product that contains TMB, buffer solution and hydrogen peroxide.This class final mean annual increment solution comprises K-Blue Substrate
RReady-to-use (TMB) and 1-Step
TMTurboTMB (Pierce, Rockford, IL).
(West Lafayette IN) measures for Bioanalytical Systems, Inc by Petite Ampere analyzer to be fixed in the enzymatic activity of HRP on the sensor.To with sensor that monitor links to each other in add the HRP substrate solution after, apply again-electromotive force of 50mV.Add substrate solution after 5 seconds, can measure the electric current of the HRP generation of being fixed on the sensor.
The selection of film
By of the comparison of several dissimilar films, can determine the characteristic of the capillary film of different materials to the damping fluid rate of capillary flow.Here (microporous membrane that Massachusetts) provides compares with the nylon net filter device with different mesh sizes for Millipore Corporation, Bedford with manufacturer of a few family.It is long that film is cut into 4.5cm, the strip that 4mm is wide.Each diaphragm is fixed on the one end on the plastic-substrates by a transparent faciola.(phosphate-buffered salt PBS/0.5% casein pH7.4) is added in the stiff end of diaphragm with buffer solution.Note flow of liquid and spend the required time of 4cm diaphragm to be detected.As shown in table 2, the flow velocity of microporous membrane is usually less than the flow velocity of reticulate texture film.And in the mesh size scope of being surveyed, the flow velocity of buffer solution directly is directly proportional with the size of aperture openings.Although the actual speed of test depends on the material of used film, the result of these tests shows that any tested film can be used to provide fast detecting.In addition, all tested films all show stable flowability.
Table 2
| The type of film | Pore size | Damping fluid flows through the time of 4cm film |
| Durapore, SV type Durapore, SV type Satorius, cellulose nitrate Satorius, acetate fiber Whatman, cellulose nitrate Whatman, cellulose nitrate nylon net filter device pore, the nylon wire pore, the nylon wire pore, | 4 μ m, 1 μ m, 8 μ m, 8 μ m, 5 μ m, 3 μ m aperture openings sizes, 11 μ m, 50 μ m, 60 μ m | 3’26” 3’45” 2’32” 2’59” 3’47” 3’42” 1’33’ 1’05” 49” |
| Pore, | 80μm | 40” |
The contrast of tested film sensors reactivity worth is by being used for each film to compare with a kind of sensor device.Anti-alpha-fetoprotein (AFP) antibody according to front example 2 described steps be fixed on sensor.As the second antibody that sandwich detects, anti-AFP-HRP compound is deposited and is dry on a composite pad.The preparation of composite pad and various tested sensor diaphragm is carried out according to back example 7 described steps.Used film all is the film of handling without retarding agent in this example.
Be dissolved in the caseic 300ng/mlAFP solution of PBS/0.5% to what each sensor diaphragm added 100 μ l pH7.4.This sample flow is crossed the tested person film that is assembled on the sensor diaphragm, and reacts with antibody that is fixed on sensor surface and HRP antibody complex.Use TMB/H
2O
2Substrate solution is measured electrochemical signals.And contrast the background response that non-specific signal causes and the reaction of analyte by the method that adds damping fluid to the sensor diaphragm that does not have analyte.Table 3 shows the result of this test.
Table 3
| Film type | Pore size | Signal intensity μ A | Background μ A |
| Durapore, SV type Satorius, cellulose nitrate Satorius, acetate fiber Whatman, cellulose nitrate Whatman, the cellulose nitrate pore, nylon wire | 4μm 8μm 8μm 5μm 3μm 50μm | 4.01 2.00 1.83 4.36 0.97 3.03 | 2.51 1.12 0.99 2.44 0.55 0.47 |
Test findings shows that background signal that the multi-cellular structure film produced is generally greater than the reticulate texture film.On the other hand,, but still can produce stronger signal, demonstrate antibody-antigen compound condition more efficiently although the flow velocity of test solution in the reticulate texture film is higher than the film (seeing Table 2) of multi-cellular structure far away.These data presentation go out, and compare with microporous membrane, and the reticulate texture film has minimum background signal and the highest signal to noise ratio (S/N ratio), prove that cancellated film is more suitable at present in this biology sensor and method thereof.
Though reticulate texture film such as nylon wire are more suitable in the material that is used as this biology sensor, but should see that also other has the material of similar structures, even if the material of multi-cellular structure, optimizing in process later on still can be in order to obtain similar effects to subdue its nonspecific property signal response.
The selective membrane retarding agent is to reduce non-specific signal
Can further improve the reactivity worth of biology sensor by the diaphragm in the retardance sensor chip.The retardance of film is based on following purpose: the non-specific binding that 1) reduces antibody complex or analyte and film surface.2) suction again and the storage characteristics of raising off-the-shelf hardware.Usually can be by adding protein (as casein, or bovine serum albumin), surfactant (as Tween 20, or TritonX-100), or high molecular polymer (as polyvinyl alcohol (PVA)) blocks to lower non-specific the adhering on the film.
Preferred retarding agent is a kind of polymkeric substance with hydrophobic nature middle-end and hydrophilic end among the present invention, and its structure is PEG-PPG-PEG, and wherein PEG is a polyglycol, and PPG is a polypropylene glycol.This base polymer has Pluronic
TMAnd Poloxamer
TMDeng.The middle-end of hydrophobic nature is adsorbable on the surface of hydrophobic nature, be not adsorbed in its surperficial hydrophilic end then can resemble as the marine alga unmanaged flexibility.The covering of hydrophobic nature middle-end and hydrophilic end float can be jointly retardation film surface effectively, and form the top layer that does not absorb protein.
For the check retarding agent to the effect that non-specific signal produced, with condition like the biology sensor detection type under, carried out some the experiment, to determine to residue in the quantity of the HRP antibody complex on the blocked film.Employed retardance reagent comprises (a) a kind of PEG-PPG-PEG polymkeric substance (average M
a8,400), (b) Triton X-100, (c) PVA (M
w13,000-23,000), (d) Tween20.Above reagent is all by Aldrich Chemical Company, Inc, and Milwaukee, WI provides.First film with drying (nylon wire, 30 μ m mesh Millipore) are infiltrated in the solution that contains 1% retarding agent, and overnight depositing under 4C ° temperature makes its evenly diffusion naturally under the friction condition then.Next again film is at room temperature dried, be cut into the long diaphragm of the wide 2cm of 0.5cm then, and be assembled on the based sensor that solidifies by the absorption antibody of myocardium calcium protein I, other device also has an absorption pad and a compound pad that contains the pre-dried anti-myocardium calcium protein I HRP compound of 30ng/pad.The PBS pH7.4/0.5% casein buffer solution that adds 150 μ l is in acquisition zone and it is flowed.Add 3 after 5 minutes again and contain TMB/H
2O
2Substrate solution in detection zone.The current signal of each sensor diaphragm is recorded by the electrochemical method described in the example 3.
Experimental result shows that for the film of not retardance, because the adhewsive action of HRP compound on the film, all retardance reagent all possesses the effect of certain non-specific signal of reduction.The effect of retardance is followed successively by: PEG-PPG-PEG＞＞PVA＞Triton X-100＞Tween 20.Find also that simultaneously the consumption of retardance reagent equally has influence on the quantity of non-specific signal.Usually, the increase of the consumption of retardance reagent can cause weakening of non-specific signal.
Embodiment 6
Antibody--HRP compound
Many antibody--HRP compound market is on sale.Alternatively, also can use Pierce ' sEZ-Link
TMPlus activation peroxidase kit (Pierce Chemical, Rockford IL) obtains through following steps:
1. IgG that will about 1mg is with the phosphate buffered saline(PBS) dissolving of 0.5-1.0ml.
2. the EZ-Link Plus of reorganization 1mg freeze-drying
TMThe activation peroxidase is in 100 μ l water and add in the IgG solution.
3. add 10 μ l cyano group sodium borohydride solution immediately, it consists of 5M NaCNBH3 than 1M NaOH.
4. at room temperature this solution was hatched 1 hour.
5. add 20 μ l and contain 3M amido ethanol, pH9.0 separates buffering, and at room temperature reacts 15 minutes.
6. the compound in the dialysis PBS solution.Use DispoDialyzer analyses the molecular weight 100,000 (MWCO) in composing, so that free HRP is removed from complex solution.
7. with Pierce ' s SuperFreeze
TMThe agent of peroxidase stable composite joins complex solution, and with this solution long term storage in refrigerator.
Embodiment 7
The compound release conditions
The manufacturing materials of composite pad was diversified during biology sensor of the present invention detected, for example by the bonding borosilicate fiber of polyacrylic acid ethene, or the polyester matrix material.The material of making composite pad should possess character such as compound, the stable flowability of low protein, stabilized complex release.(KK0141 Gelman Sciences, Ann Arbor MI) are a kind of more satisfactory compound releasable material to Loprosorb.
In the detection of following introduction, antibody complex is dry on composite pad, and available spray bottle is sprayed on complex solution on the composite pad, and also available micro pipette drops in composite solution on the compound releasable material by hand.Antibody in this example--HRP complex solution is diluted by 20% sucrose damping fluid.Composite pad should keep dry before the capillary film in contact sample collection district.After liquid sample added pickup area, compound was by rehydration and infiltrate film.
Survey the disposable sensor device of prostate class antigen.
Substrate sensor shown in Fig. 2 can be carried out the detection of analytes in the various liquid separately, and the sample that will contain target analytes directly adds the detection zone that curing antibody is arranged.In this example, such substrate sensor is solidified the monoclonal anti-PSA antibody in this sensor diaphragm by using above example 2 described methods, detects to carry out prostate class antigen (PSA).PSA in the casein solution of PBS PH7.4/0.5% and serum sample and second monoclonal anti-PSA antibody-HRP compound are hatched simultaneously, and the result forms compound absorption on the substrate sensor surface.Through after the hatching of room temperature following ten minutes, with PBS 0.05%Tween 20, the solution of PM7.4 is cleaned, and then splashes into the substrate solution that contains hydrogen peroxide and TMB with sensor diaphragm.(West Lapayette IN) connects for Petite ampere, Booaralytical Systems Inc., and the electric current of generation five seconds after adding substrate solution records with sensor and readable instrument with electrode.As shown in Figure 4, the size of induction by current is directly proportional with PSA content in the test sample book.The analyte measured of method include but not limited to human chorionic gonadotrophin (HCG), myocardium calcium protein (CRP) and alpha-fetoprotein (AFP) thus.
Embodiment 9
Use the sandwich of disposable sensor to detect
Use the disposable film sensors among the present invention also can carry out the sandwich immune detection to alpha-fetoprotein (AFP).
In this example, according to Boorsman, D.M.et.al (J.Histochem.Cytochem.23:200-207,1976) method is that anti--AFP-HRP compound of 15ug/ml is deposited on that (Gelman Sciences produces (Loprosorb, KK0141)) on the low protein combination composite pad through the 3ul of purified treatment proportion.This composite pad at room temperature dried in the air 20 minutes, was installed on the sensor diaphragm in the indoor storage one night side of controlled humidity then, and the monoclonal anti-AFP with 30ng/sensor is fixed on the sensor then, and sensor device just installs like this.
When using disposable biological sensor to carry out the enzyme immunoassay, add the 200ulPH value in the specimen sample district and be 7.4 the caseic AFP sample solution of PBS/0.5%.The capillary film that sample flow is crossed on the sensor diaphragm reacts with the antibody and the abzyme compound that are solidificated in sensor surface.After a few minutes, adding contains TMB/H in the sensor district
2O
2The 100ul/sensor substrate solution.Behind the five seconds, (Petite Ampere BioanalyticalSystems, Lafayette IN) measures electric current to use the readable instrument that links to each other with sensor diaphragm.As shown in Figure 5, the signal in the detection zone is assembled constantly reinforcement with analyte in the liquid sample.
Principle is similar, and biology sensor of the present invention also can be used for detecting cardiac marker myocardium calcium protein (Tn1), is used for the detection of heart injury illness.This with the biology sensor of described method assembling by a substrate sensor, a composite pad, a nylon reticular membrane (30um Millipore) combine and assemble forms.Each sensor has all been fixed the monoclonal antibody of about 45ng at its working electrode surface; Composite pad contains the second antibody of the anti-and TnI that HRP is mutually compound of 30ng.Used film blocks in advance through 2% PEG-PPG-PEG (nm8400) solution in this example, add 150ul in sample region in the detection and contain the TnI sample liquid, after five minutes, add K-Blue substrate finished product TMB (Neogen Corporation at detection zone, Lexington, KY).Behind the five seconds, (Petite Ampere Bioanalytical Systems, Lafayette IN) measures electric current to use the readable instrument that links to each other with sensor diaphragm.The testing result explanation in clinical scope, can be carried out quantitative determination with this biology sensor to the amount of TnI in the liquid sample as shown in Figure 5.
The present embodiment explanation is the preparation and the detection of the DNA sensor of specific reaction thing and detecting reactant with DNA.
Known dilution Escherichia coli are added in polymerase chain reaction,PCR (PCR) potpourri of standard, according to the operating guidance preparation (Amplitaq Kit, Perkin Elmer Cetus, Emeryville, Calif.).Two oligonucleotide primers that are used to react (each 5 μ l) are 18mers, itself and the terminal homology of each 1kb fragment of the DNA that represents tetracycline resistance gene in bacterium.This is reflected at following condition and carries out: 94 ℃ 1 minute, 54 ℃ 1 minute and 74 ℃ 1 minute, repeat above-mentioned three step 30 circulations.
End eventually in this PCR reaction adds two new primers in potpourri, each 5 μ l then carries out 10 round-robin PCR.Regional homology on this new primer and the tetracycline resistance gene that is arranged in the zone that is combined by first group of primer.Covalently bound with one 5 ' end of these second primers is a biotin-HRP compound label, and hold covalently bound with 5 ' of another primer is one " general binding site ", it is made up of 10 base sequences, and this sequence representative is corresponding to the specific site of a double-stranded DNA in conjunction with albumen (dsDNA BP).
Behind second time PCR, get 10 μ l reaction mixture liquid 0.7% Ago-Gel, 120V electrophoresis 30 minutes, ethidium bromide staining is observed under ultraviolet light.
Then 5 μ l reaction mixtures are mixed with equal-volume methyl alcohol, place on the composite pad of a membrane electrode, in 37 ℃ of moistening casees, hatched 30 minutes, measure the strength of current of specific bond, by reader read current intensity.Described membrane electrode wraps in 37 ℃ of moistening incubators by 1 hour with the 100 μ g/ml dsDNA BP solution of 10 μ l earlier, then of short duration rinsing in the phosphate buffer of PH7.4, and the 0.25%BLOTTO in 6 * SSC (Maniatis, 1990) was 37 ℃ of sealings 15 minutes.
The gel of ethidium bromide staining is presented at a single band in the correct molecular weight ranges, show with technology of the present invention successfully to have amplified target DNA from few to single bacterium.Use radiolabeled dsDNA BP to show that this albumen and PVDF and glass have good binding.Contain corresponding to the DNA of the amplification of dsDNA BP binding site successfully with the immobilization protein combination, still underway for the research of this optimum reaction condition.
As mentioned above, with the technology of the present invention from single bacterium successfully the amplification to target DNA in the gel of ethidium bromide staining, proved, proved that by the galvanometer reading target DNA this amplification, that modify can be attached to the surface of sensor by dsDNA BP.Therefore, for two steps required for the present invention---the specific amplified of target DNA and modify after with the target DNA of amplification by a double-stranded DNA-in conjunction with protein combination to sensor surface, all possess.
More than to detailed description of the present invention and embodiment explanation, do not limit the present invention, those skilled in the art can make various modifications and variations according to the present invention, only otherwise exceed essence spirit of the present invention, all should belong to scope of the present invention.
Claims (39)
1, a kind of biology sensor that is used for fluid sample quantitative detecting analysis thing is characterized in that, described sensor comprises at least
A substrate sensor, on a working electrode on the plastic-substrates of being coated on, a contrast electrode and at least one third electrode are arranged, be fixed with first bond that can combine on the described working electrode with analyte, each electrode of described substrate sensor links to each other with readable instrument, carries out electrochemical measurement;
A capillary film is arranged at described substrate sensor top, is used for detecting the horizontal direction transmission of sample;
A composite pad, include in the described composite pad can with second bond of analyte specific bond.
2, biology sensor according to claim 1 is characterized in that being furnished with on described substrate sensor:
One sample region (14 '), its top is provided with described composite pad (20), is used to gather the liquid sample;
One detection zone (16 '), the liquid sample of collection comes in contact reaction at this and described all electrodes;
Described capillary film couples together described sample region and detection zone.
3, biology sensor according to claim 2 is characterized in that it also comprises a waste liquid absorption pad (18), is used to receive the liquid sample of detection.
4, biology sensor according to claim 2 is characterized in that, on the described working electrode the first fixing bond be can with nucleic acid, the protein of analyte specific bond.
5, biology sensor according to claim 4 is characterized in that first fixing on the described working electrode bond is for having immunocompetent antibody or antibody fragment.
6, biology sensor according to claim 5 is characterized in that first fixing on the described working electrode bond is a monoclonal antibody.
7, biology sensor according to claim 4 is characterized in that first fixing on the described working electrode bond is DNA.
8, biology sensor according to claim 1 is characterized in that, described third electrode is a correcting electrode, is used for the error of correct detection system.
9, biology sensor according to claim 1 is characterized in that, described third electrode is one second working electrode, is used for the blank of described working electrode.
10, biology sensor according to claim 1 is characterized in that described second bond is deposited in advance or solidifies in described composite pad.
11, biology sensor according to claim 10, it is characterized in that described second bond for through the label mark can with nucleic acid, the protein of analyte specific bond.
12, biology sensor according to claim 11 is characterized in that described label is for producing the enzyme of Electrochemical Detection signal with substrate and electronic transmission medium.
13, biology sensor according to claim 12 is characterized in that described enzyme is horseradish peroxidase or alkaline phosphatase.
14, biology sensor according to claim 11 is characterized in that described protein is antibody or antibody fragment.
15, biology sensor according to claim 11 is characterized in that described nucleic acid is DNA.
16, biology sensor according to claim 12 is characterized in that described substrate is the substrate solution that contains hydrogen peroxide; Described electronic transmission medium is a kind of derivant, benzoquinones, the ascorbic acid or 3 of the pig iron or the pig iron, 3 ', 5, and 5 '-tetramethyl benzidine.
17,, it is characterized in that described capillary film is a biofilm according to right 1 described biology sensor.
18, biology sensor according to claim 1 is characterized in that a filtering device is set on described composite pad, is used for the filter liquide sample.
19, biology sensor according to claim 2, it is characterized in that, it also comprises a sensor cover (12), on it corresponding to described sample collection district also detection zone one liquid sample be set respectively gather a hole (14) and a detection hole (16), be used for application of sample and observing response process; And an absorption cell, this groove links to each other with the capillary film is local at the end of liquid flow path, is used for the excess liquid in absorption detecting district.
20, biology sensor according to claim 1 is characterized in that, described working electrode and/or auxiliary electrode comprise a carbon conductor of printing by serigraphy, and third electrode comprises a silver or the silver/silver chloride conductor printed by serigraphy.
21, biology sensor according to claim 1 wherein also comprises:
A) can be solidificated in described working electrode surface by fatty alcohol solution with first bond of analyte generation idiosyncrasy through buffered;
B) can be increased the stabilizing agent of stability so that first bond that is cured is further stable at the working electrode surface bag that has solidified first bond by one deck.
22, biology sensor according to claim 21, wherein said fatty alcohol are isopropyl alcohol; Described stabilizing agent contains sugar.
23, a kind of method that analyte in the liquid sample is quantized to detect may further comprise the steps:
A) with sample with contain can contact to form a kind of test mixture through the label mark with the solution of second bond of analyte specific bond;
B) at described biology sensor above-mentioned test mixture is hatched,, make analyte be clipped in described second bond and solidify between first bond of working sensor electrode surface through the time of one section abundance;
C) described biosensor surface add contain substrate and electronic transmission medium detection solution, to promote the electric transmission reaction.
D) by measuring the induction by current that electronic transmission medium produced, come the analyte content of further working sample by the analyte catalysis that has label.
24, method according to claim 23, wherein said through being deposited in advance with second bond of analyte specific bond or solidify in described composite pad of label mark, step a) is carried out on the composite pad of described biology sensor.
25, method according to claim 23, wherein said step d) is measured by readable galvanometer device.
26, method according to claim 23, wherein after step b) with second working electrode of described sensor as contrast electrode, poor by the magnitude of current of measuring between the working electrode and second working electrode, and then the analyte content in the working sample.
27, method according to claim 23 wherein also comprises with damping fluid after step b) and washes sensor surface.
28, method according to claim 23, wherein said first bond is nucleic acid, protein.
29, method according to claim 28, wherein said nucleic acid are DNA.
30, method according to claim 28, wherein said protein are antibody or antibody fragment.
31, method according to claim 30, wherein said antibody are monoclonal antibody.
32, method according to claim 23, wherein said second bond is nucleic acid, protein.
33, method according to claim 32, wherein said protein are antibody or antibody fragment.
34, method according to claim 32, wherein said nucleic acid are DNA.
35, method according to claim 23, wherein said liquid sample are body fluid.
36, method according to claim 35, wherein said body fluid are blood, serum, blood plasma, urine or prostatic fluid.
37, method according to claim 23, wherein said analyte are alpha-fetoprotein, prostate class antigen, cardiac troponin, carbon activated protein, human chorionic gonadotrophin or nucleic acid.
38, method according to claim 23, wherein the electronic transmission medium described in the step c) is a kind of derivant, benzoquinones, the ascorbic acid or 3 of the pig iron or the pig iron, 3 ', 5,5 '-tetramethyl benzidine.
39, method according to claim 23, wherein the substrate described in the step c) is the substrate solution that contains hydrogen peroxide.
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| EP1739407B1 (en) * | 2004-05-06 | 2018-05-30 | Panasonic Intellectual Property Management Co., Ltd. | Sensor, measuring equipment and measuring method |
| CN101126734B (en) * | 2006-08-15 | 2011-06-08 | 中国科学院化学研究所 | Biosensor based on aptamer modified conducting polymer and its preparation method and uses |
| CN103602755A (en) * | 2013-08-21 | 2014-02-26 | 中山大学达安基因股份有限公司 | Kit for detecting 20 subtypes of human papillomaviruses by electrochemical gene sensor method |
| CN106999929A (en) * | 2014-09-11 | 2017-08-01 | 克忧公司 | For detecting the system and method with analyte quantification |
| MX2019003929A (en) * | 2016-10-07 | 2019-06-10 | Boehringer Ingelheim Vetmedica Gmbh | Analysis system and method for testing a sample. |
| EP3862750B1 (en) * | 2018-09-29 | 2025-02-26 | Boe Technology Group Co., Ltd. | Electrochemical sensor and detection device for body fluid detection |
| CN113272647A (en) * | 2018-10-25 | 2021-08-17 | 以色列国农业和农村发展部农业研究组织(Aro)(佛卡尼中心) | Device and method for the point-of-care analysis of liquid samples |
| CN114113247B (en) * | 2020-08-31 | 2024-05-14 | 浙江纳智汇生物科技有限公司 | Electrochemical detection method, detection chip, detection kit and detection system based on enzyme inhibition method |
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