CN1196932C - Preparation of reagent-free ampoul immuno sensor and use thereof - Google Patents
Preparation of reagent-free ampoul immuno sensor and use thereof Download PDFInfo
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- CN1196932C CN1196932C CN 03113053 CN03113053A CN1196932C CN 1196932 C CN1196932 C CN 1196932C CN 03113053 CN03113053 CN 03113053 CN 03113053 A CN03113053 A CN 03113053A CN 1196932 C CN1196932 C CN 1196932C
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Abstract
The present invention discloses a preparing method for a non-reagent ampere immune sensor and the application in the immune analysis. The preparing method has the preparing steps that an antigen to be measured is dissolved in buffer solution, the pretreatment is carried out to the surface of a carrier, and the solution is dropped and coated on the surface; the antigen is suspended above the liquid surface of a metal alkoxy compound, and the immune sensor can be obtained by sealing a constant temperature water bath of the system. The measuring condition of the antigen to be measured is optimized, and then the measuring standard curve of the antigen to be measured can be obtained by a series of treatment; the corresponding concentration can be found out on the measuring standard curve by the reduction of an electrochemical signal. In the method, because a middle body or other reagents do not need to be added in sample solution to be measured, the pollution of a middle body, etc. to an electrode can be avoided, an analysis system is simplified, and the analysis reaction can be completed by one step. Thus, the present invention has good precision, repeatability and stability.
Description
One, technical field
The present invention relates to a kind of preparation and application thereof of immunosensor, particularly relate to a kind of the do not have preparation of reagent ampere-type immunosensor and the application on immunoassay thereof.
Two, background technology
In recent decades, immuno analytical method combines characteristics such as the sensitivity, convenience of technology such as specific recognition reaction between antigen, antibody molecule and galvanochemistry, spectroscopy, acoustics, with its high selectivity and high sensitivity, become one of every field important analysis means such as clinical, biological chemistry, environmental analysis to analytes such as tumor markerses.Conventional immune analysis method mainly contains: radio immunoassay, enzyme-linked immunosorbent assay, fluoroimmunoassay, chemiluminescence immunoassay, flow-injection immunoassay etc.Said method mainly is to utilize mark radioactive isotope, enzyme molecule, fluorescent material etc. on antibody, when determined antigen and labelled antibody process specific reaction, after washing the plate separation, the radioactive isotope by measuring mark or the signal of fluorescent material, or measure the signal that marker enzyme acts on electroactive material that substrate produces, luminescent substance etc. and measure.Its operation steps mainly comprises first antibody to measuring antigen recognizing, and washing with mark two anti-reactions, is washed plate, adds substrate mensuration etc.
Immunosensor combines immunoassay and many characteristics such as spectrum or electrochemical techniques, can need not washing, separating step etc. in the operation without pre-service to the mensuration of analytic sample the time, has been subjected to extensive attention.Many kinds of sensors, particularly various electrochemical sensors have been brought into play important effect in every field.
Electrochemical immunosensor is divided into electric potential type immunosensor and ampere-type immunosensor usually.The electric potential type immunosensor is that antigen or antibody molecule are fixed on the electrode surface, is containing when this immunosensor in the solution of determined antigen and labelled antibody behind the incubation, and the amount of determined antigen can be measured by the variation of current potential on this immunosensor.The ampere-type immunosensor then is fixed on antigen or antibody molecule on the electrode surface, containing when this sensor in the solution of determined antigen and labelled antibody behind the incubation, be marked at the redox signal that enzyme on the antibody acts on the electroactive material that substrate produces by mensuration and measure determined antigen content.Horseradish peroxidase, alkaline phosphatase, laccase and glucose oxidase etc. utilize the corresponding substrate of their catalysis to produce electroactive material and prepare ampere immunity sensor through being often used as the marker enzyme of antibody.As: monoclonal antibody is fixed on sensor surface, utilizes the antibody of horseradish peroxidase mark,, measure the current signal of substrates such as oxygen in the horseradish peroxidase catalysis solution to be measured be marked on the antibody or hydrogen peroxide and measure by sandwich reaction; Antibody is fixed on sensor surface,, utilizes alkali phosphatase enzyme mark antibody the current signal of substrate such as naphthols, para-aminophenol phosphate in the solution to be measured etc. by the competitive immunoassay method.
As seen, conventional immune analysis method complex operation step, analysis time is longer, the sample consumption is big, cost of determination is higher, and existing ampere immunity sensor need add non-immunoreagents such as mediator or substrate in solution to be measured, even utilize the oxygen that dissolves in the solution to be measured as mediator, its measurement result also is subjected to the influence that environment temperature, pressure, solution composition etc. change.How to avoid the adding of mediator and substrate, simplify the mensuration system, thereby the step of simplifying the operation shortens analysis time, reduce cost of determination and become the problem that people pay close attention to.
The Direct Electrochemistry of immobilised enzymes has been widely used as the exploitation of biology sensor and no medium biology sensor, and this method has been avoided the pollution to electrode surface of in sample solution adding mediator and mediator.In order to overcome the shortcoming of existing immune analysis method, avoid the adding of mediator and substrate in immunoassay, simplify analytical procedure, shorten analysis time, reduce sample consumption, and then reduce cost of determination, realize online express-analysis clinically, we are applied to the Direct Electrochemistry character of immobilised enzymes in the immunoassay first, develop no reagent immune analysis method and have prepared immunosensor, are used for the detection of clinical tumor mark.
Three, summary of the invention
1, Fa Ming order: the present invention utilizes titanium colloidal sol vapour deposition technology of preparing (patent applied for, application number is 01138059.4), the gelatinization process of titanium colloidal sol is used for the embedding of antigen molecule at carrier surface, the structure new immune sensor, and utilize the horseradish peroxidase be marked on the antibody with the antigen generation immune response that is fixed on the electrode surface after the Direct Electrochemistry character that shows, developed a kind of new no reagent immuno analytical method.
2, technical scheme: specific implementation method of the present invention is as follows: the preparation method of no reagent ampere immunity sensor is characterized in that this preparation process is:
(1) determined antigen such as CA125 being dissolved in the pH value is that buffer solution can be selected phosphate buffered solution or Tris～HCl buffer solution or B～R buffer solution in 6.5～7.0 the buffer solution; Selected buffer solution is different because of antigen type, and standard is to make immunoreactive activity and electrochemical response maximum.
(2) to the surface of carrier such as glass carbon or graphite electrode+1.70V～+ 1.80V, preferred+1.75V, carry out potentiostatic deposition and handled 250 seconds-350 seconds, preferred 300 seconds, obtain the electrode surface of oxygen enrichment group; Pretreated requirement is to carrier surface, should obtain smooth, clean, hydrophilic surface;
(3) drip the solution of coating gained in (1) at carrier surface, and it is suspended from the top of metal alkoxide compound such as tetraisopropoxy titanium or tetramethoxy titanium liquid level, then that this system is airtight;
(4) above enclosed system is placed 15 ℃～35 ℃ water-bath, constant temperature 2～6 hours; Metal alkoxide compound steam in the enclosed system on the liquid level contacts with the solution of carrier surface, taking place slowly, hydrolysis reaction generates TiO 2 sol, with the antigen molecule embedding and be fixed in carrier surface, obtain novel immunologic function film and can be made into immunosensor behind the gelatine.
Utilize the above-mentioned no reagent ampere immunity sensor of producing to measure the method for determined antigen, it is characterized in that:
(1) under normal temperature or body temperature, through the incubation optimization of 40～70min;
(2) the compound concentration scope is at the determined antigen standard solution of 0～500U/ml and the reaction solution of fixed amount enzymic-labelled antibody, under the condition determination of when maximum current responds, measuring accordingly, immunosensor is respectively in these reaction solutions behind incubation 40～70min, take out, secondary water washing is washed, determine the reduction of enzyme Direct Electrochemistry signal on immunosensor in for the buffer solution of 6.5-7.0 such as phosphate buffered solution or Tris-HCl buffer solution or B-R buffer solution respectively in the pH value, obtain the bioassay standard curve of this determined antigen; See accompanying drawing 1.
(3) under the condition determination of when maximum current responds, measuring accordingly, with immunosensor incubation 40～70min in the reaction solution that contains determined antigen and fixed amount enzymic-labelled antibody, take out, after secondary water washing is washed, in the pH value is the reduction that determines enzyme Direct Electrochemistry signal on immunosensor in 6.5～7.0 buffer solution such as phosphate buffered solution or Tris-HCl buffer solution or the B-R buffer solution, finds respective concentration on the bioassay standard curve of this antigen.
The immunologic function film that influence is obtained and the principal element of immunosensor performance have four aspects:
(1) the pH value of buffer solution: only under certain acidity, antigen just has optimum activity.If the acidity of buffer solution departs from this numerical value, antigen molecule is dissolved in wherein will injures its activity, thereby influence the performance of immunologic function film and immunosensor.
(2) carrier surface character: whether carrier surface water wettability, to can generate evenly, effectively the immunologic function film is most important.If carrier surface shows hydrophobicity, contain the determined antigen drips of solution and be coated in can assemble automatically behind the carrier surface and form droplet, after the colloidal sol gelatine, the immunologic function film thickness of generation, clear-cut and inhomogeneous finally influences the performance of immunologic function film and immunosensor.
(3) temperature: when dripping the carrier scribble antigenic solution when being suspended from metal alkoxide compound such as tetraisopropoxy titanium or the tetramethoxy titanium liquid level, having two processes takes place simultaneously, the one, the metal alkoxide compound steam deposits at carrier surface, and another is the volatilization of moisture in the carrier surface solution.If temperature is too high, the vapour pressure of metal alkoxide compound is too big, and the speed of hydrolysis is too fast, and what form this moment is not colloidal sol just, but the solids precipitation of titania; If temperature is too low, the vapour pressure of metal alkoxide compound is too little, and the speed of moisture evaporation will be faster than the speed of vapor deposition, and carrier surface does not consequently have the steam generation hydrolysis reaction of enough water and metal alkoxide compound to generate the colloidal sol of wanting.
(4) time: time enough can make hydrolysis reaction abundant, also can guarantee the colloidal sol complete gelationization simultaneously.The too short time can make reaction insufficient, and gelatine is incomplete; The oversize time, just meaningless after the colloidal sol complete gelationization.
The optimization that utilizes no reagent immunosensor to measure the antigen condition comprises following three aspects:
(1) heated culture temperature: immune response generally takes place in human body or higher mammal body, and it is responsive to temperature.As normal temperature or human body temperature, immune response reaches maximal efficiency under the temperature that is fit to.Be lower than this temperature, antigen, antibody molecule activity are lower, react slower; Be higher than this temperature, antigen, the easy sex change of antibody molecule are until inactivation.
(2) the incubation time: the identification in the immune response between antigen, antibody, combination need certain hour to finish usually, be accuracy and the sensitivity that guarantees to measure, requirement provides the sufficiently long reaction time to guarantee that immune response is complete, and this time is generally 40-70min.
(3) amount of enzyme labelled antibody in the reaction solution: this mensuration is utilized the competitive immunoassay method, promptly compete, thereby enzyme labelled antibody and the antigen that is fixed on the immunosensor take place in the reaction solution reacting dose is reduced produce signal to fetch the amount of mensuration determined antigen between reducing by free determined antigen in the reaction solution and the antigen that is fixed on the immunosensor.The amount of enzyme labelled antibody should equal the amount of the fixing antigen of sensor surface in the reaction solution, at this moment, obtains maximum detection range and detects the sensitiveest.If the amount of enzyme labelled antibody is worth less than this, sensing range is dwindled; If the amount of enzyme labelled antibody is worth greater than this, can make measurement result less than normal.
3, beneficial effect
The Direct Electrochemistry response that this method utilizes the enzyme of specific immune response and labelled antibody to show on the TiO 2 sol gel mould has developed novel no reagent immune analysis method and has prepared immunosensor.Utilize TiO 2 sol gel vapour deposition process immobilized antigen molecule, and utilize the competitive immunoassay method, directly obtain the concentration of determined antigen by the reduction of direct assaying reaction Direct Electrochemistry response signal of the enzyme of labelled antibody to the immunosensor surface.The more existing immune analysis method of this method has the following advantages:
(1) need not in testing sample solution to add mediator or other reagent, avoided the pollution to electrode such as mediator, simplified analysis system;
(2) immune response one step finishes in the assay determination, reduces sample consumption, and need not washing, and steps such as separation have been simplified the operation steps of immunoassay, have shortened analysis time, are applicable to on-line quick detection clinically;
(3) detect or electroactive material directly is marked on the antibody by direct electrochemistry, can finish the direct mensuration of multiple different antigen molecules the enzyme of different labelled antibodies;
(4) this sensor sheet reveals good accuracy, and is repeated and stable, can develop into disposable immunoassays sensor and to marketing.
Four, description of drawings:
Accompanying drawing 1 is a CA125 bioassay standard curve.
Five, embodiment
1, the preparation of embodiment 1:CA125 immunosensor
Selecting the plate-like plane glass-carbon electrode of diameter 4mm to make carrier material, is the object that example is fixed as embedding with CA125:
(a) polishing electrode: earlier electrode surface is polished with abrasive paper for metallograph, use 1.0 respectively then, 0.3,0.05 the gamma oxidation aluminium paste of μ m polishes on chamois leather, then rinse well with secondary water, electrode surface is used 1: 1 nitric acid, acetone, the ultrasonic cleaning of secondary water successively, obtains fresh clean electrode surface.
(b) electrode shows pre-service: the electrode after will polishing is electrolysis after 300 seconds under+1.75V constant potential in 5.0 the phosphate buffered solution in the pH value, more respectively+0.3V～+ 1.3V and+0.3V～-the 1.3V potential range in scan round until obtaining the stable electrical flow valuve.Take out electrode, after the flushing of secondary water, nitrogen dries up.
(c) get 5 microlitre 500U/mL standard C A125 antigenic solutions, drip the electrode surface be coated in through (b) step process, then this electrode is suspended from the top of tetraisopropoxy titanium liquid level, with the airtight back of system in 25 ℃ of constant temperature 4 hours.
(d) in enclosed system, tetraisopropoxy titanium steam contacts with the antigenic solution of electrode surface, and taking place slowly, hydrolysis reaction generates TiO 2 sol.Colloidal sol in the process of gelling film forming with in the CA125 antigen embedding gel mould, thereby be fixed on electrode surface, make the CA125 immunosensor.
2. the mensuration of embodiment 2:CA125
(a) optimization of condition determination: change heated culture temperature respectively, the amount of HRP mark CA125 antibody in incubation time and the reaction solution, measure accordingly as optimum determining condition when selecting the maximum current response, experimental result shows, when at 33 ℃ of following incubation 40min, obtain maximum current response, when HRP mark CA125 antibody amount in the 50 microlitre reaction solutions greater than 83% the time, current-responsive reaches maximal value.
(b) mensuration of CA125: under the optimization experiment condition, utilize the competitive immunoassay method, measure series of standards CA125 antigen concentration, determine the typical curve that CA125 measures, utilize calibration curve method again, the concentration of CA125 antigen in the working sample.
Claims (8)
1, a kind of preparation method who does not have the reagent ampere immunity sensor is characterized in that preparation process is:
(1) determined antigen being dissolved in the pH value is in 6.5～7.0 the buffer solution;
(2) to carrier surface+1.70V～+ carry out potentiostatic deposition under the 1.80V condition to handle and obtained the electrode surface of oxygen enrichment group in 250 seconds～350 seconds;
(3) drip at carrier surface and be coated with above-mentioned solution, and it is suspended from the top of metal alkoxide compound liquid level, then that this system is airtight;
(4) above enclosed system is placed 15 ℃～35 ℃ water-bath, constant temperature 2～6 hours; Metal alkoxide compound steam in the enclosed system on the liquid level contacts with the solution of carrier surface, taking place slowly, hydrolysis reaction generates TiO 2 sol, with the antigen molecule embedding and be fixed in and carry this surface, obtain the immunologic function film and also can be made into immunosensor behind the gelatine.
2, the preparation method of no reagent ampere immunity sensor according to claim 1, wherein (1) described buffer solution is phosphate buffered solution or 2-amino-2-methylol-(1,3) propylene glycol-hydrochloric acid solution (Tris-HCl) buffer solution or acid mixture-sodium hydroxide buffer solution (B-R) buffer solution.
3, the preparation method of no reagent ampere immunity sensor according to claim 1, wherein (1) described determined antigen is CA125.
4, the preparation method of no reagent ampere immunity sensor according to claim 1, wherein (2) described voltage is+1.75V.
5, the preparation method of no reagent ampere immunity sensor according to claim 1, wherein (2) described processing time is 300 seconds.
6, the preparation method of no reagent ampere immunity sensor according to claim 1, wherein (2) described carrier is glass carbon or graphite electrode.
7, the preparation method of no reagent ampere immunity sensor according to claim 1, wherein (3) described metal alkoxide compound is tetraisopropoxy titanium or tetramethoxy titanium.
8, a kind of method of utilizing the described no reagent ampere immunity sensor of claim 1 to measure determined antigen is characterized in that:
(1) under normal temperature or body temperature, through the incubation optimization of 40～70min;
(2) the compound concentration scope is at the determined antigen standard solution of 0～500U/ml and the reaction solution of fixed amount enzymic-labelled antibody, measure accordingly during by the big current-responsive of selected amount as condition determination, utilize the competition immune response, immunosensor is respectively in these reaction solutions behind incubation 40～70min, take out, secondary water washing is washed, in the pH value is the reduction that determines enzyme Direct Electrochemistry signal on immunosensor in 6.5～7.0 phosphate buffered solution or Tris-HCl buffer solution or the B-R buffer solution respectively, obtains the bioassay standard curve of this determined antigen;
(3) under the condition determination of when maximum current responds, measuring accordingly, with immunosensor incubation 40～70min in the reaction solution that contains determined antigen and fixed amount enzymic-labelled antibody, take out, after secondary water washing is washed, in the pH value is the reduction that determines enzyme Direct Electrochemistry signal on immunosensor in 6.5～7.0 phosphate buffered solution or Tris-HCl buffer solution or the B-R buffer solution, finds respective concentration on the bioassay standard curve of this antigen.
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| CN100357727C (en) * | 2004-07-05 | 2007-12-26 | 南京大学 | Electrochemcial immunoassay for tumor marker and small size immunoassay chip |
| CN1312480C (en) * | 2004-09-24 | 2007-04-25 | 南京大学 | In-situ electrochemical immunological detecting method of tumor cell surface antigen |
| CN102818893B (en) * | 2012-08-28 | 2013-10-09 | 济南大学 | Preparation and application of Au-Pd core-shell material constructed lung cancer tumor marker immunosensor |
| CN104181292B (en) * | 2014-08-27 | 2015-12-02 | 湖南师范大学 | A kind of protein single molecules level ampere immunity analytical approach |
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