CN101271114B - Enzyme catalysis electricity-conducting immunity sensor based on micro-gap array electrode and its immunity detecting method - Google Patents
Enzyme catalysis electricity-conducting immunity sensor based on micro-gap array electrode and its immunity detecting method Download PDFInfo
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- CN101271114B CN101271114B CN2008100313212A CN200810031321A CN101271114B CN 101271114 B CN101271114 B CN 101271114B CN 2008100313212 A CN2008100313212 A CN 2008100313212A CN 200810031321 A CN200810031321 A CN 200810031321A CN 101271114 B CN101271114 B CN 101271114B
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Abstract
The invention provides an enzyme-catalyzed conductivity immune sensor based on a micro-gap array electrode and an immune detection method thereof, the immune sensor comprises a micro-gap array electrode, an electrode substrate which is carried out the silanization treatment and enzyme sediments with great conductivity; the invention utilizes the micro-gap electrode array immune sensor, fixes the antibody of the object to be detected on the electrode gap substrate and utilizes an enzyme-labeled antibody sandwich to carry out the detection of the protein to be detected. The method of the invention has the advantages of high sensitivity and simple operation, the device is portable and the price is cheap, which possibly provides the immune detection technology with rapidity, practicality, low cost, high sensitivity and high throughput for the early and on-site detection and application of the diseases.
Description
Technical field
The present invention relates to the preparation and the immunologic detection method thereof of microclearance electrod-array immune sensing device.
Background technology
Immunoassay is one of the most frequently used technology of biomedical Molecular Detection; Rapid screening and detection for pathogen (pathogenic bacteria, virus), disease marker, medicine and chemical-biological toxin etc. are extremely important, and are the main research tools in field such as clinical diagnosis, medical research, food and public safety, medicine toxicological analysis, environmental monitoring.The immuno analytical method of comparatively generally using at present mainly is ELISA, fluorescent immune method, gold mark sidestream immune chromatography etc.These detection method sensitivity are not high, are difficult to realize the diagnosis and the research of early stage disease, and these methods mostly need use exact instrument to carry out quantitative test, the demand of suitable field quick detection.
Summary of the invention
The objective of the invention is to overcome the deficiency that traditional immuno analytical method sensitivity is low, need the exact instrument detection by quantitative; A kind of enzyme catalysis conductance immune sensor and immunologic detection method thereof based on micro-gap array electrode proposed, have highly sensitive, fast, characteristics cheaply.
One of technical scheme of the present invention is that said enzyme catalysis conductance immune sensor based on micro-gap array electrode adopts the micro-gap array gold electrode to cook substrate, and adopts following steps to make:
1, the micro-gap array gold electrode of getting conventional lithographic printing method making is cooked substrate, and it is that interdigital is two
The utmost point, positive and negative electrode are two comb shape gold electrodes, composition micro-gap array gold electrode intersected with each other.
Comb shape tooth D
1Wide is 1 μ m-100 μ m, clearance D
2Be 1 μ m, 100 μ m;
2, the silanization of micro-gap array gold electrode is handled, and the steps include:
A. said micro-gap array gold electrode is cleaned three times each 1min with absolute ethyl alcohol;
B. this electrode is immersed in the NaOH solution of 1M, leave standstill 28min-32min;
C. clean this electrode three times with ultrapure water, each 1min; Dry up;
D. this electrode immersion is contained in the ethanolic solution of aminopropyl trimethoxysilane 5% (percent by volume), left standstill under the room temperature 23 hours-25 hours;
Can on the substrate between the micro-gap array gold electrode, form an amino silane self-assembled monolayer like this;
E. clean three times with ultrapure water, dry up, obtain the micro-gap array gold electrode of silanization;
3. the preparation process of immunosensor:
A. the micro-gap array gold electrode of said silanization being immersed massfraction is 5% glutaraldehyde water solution, leaves standstill under the room temperature 0.8 hour-1.2 hours, cleans three times with ultrapure water again;
B. on said electrode, drip the mouse monoclonal or the sheep polyclonal antibody solution of 30 μ L determinands, AC is 100 μ g/mL, and standing and reacting is 0.8 hour-1.2 hours under the room temperature; Make antibody be fixed on the substrate of micro-gap array gold electrode through the glutaraldehyde cross-linking agent like this; Said mouse monoclonal or sheep polyclonal antibody solution are said antibody is dissolved in 0.01M, pH 7.4 phosphate buffer solutions and gets;
C. clean said electrode three times with ultrapure water, dry up, obtain immunosensor; Be stored in 4 ℃ subsequent use.
Two of technical scheme of the present invention is that the analyte method that adopts above-mentioned enzyme catalysis conductance immune sensor based on micro-gap array electrode to detect the monoclonal antibody that alkali phosphatase enzyme mark is arranged adopts following steps:
1. with analyte sample or standard specimen solution (it is that said analyte sample or standard specimen are dissolved in 0.01M, pH 7.4 phosphate buffer solutions and get that this analyte sample or standard specimen solution dissolve), alkali phosphatase enzyme mark mouse monoclonal antibody solution (the different antigen determining of the antibody recognition site of fixing on this monoclonal antibody and the electrode; AC is 100 μ g/mL in the solution; This solution is said antibody is dissolved in 0.01M, pH 7.4 phosphate buffer solutions and gets;) each 20 μ L mixing back dropping is on immunosensor, standing and reacting is 0.4 hour-0.6 hour under the room temperature; Like this, analyte and alkali phosphatase enzyme mark monoclonal antibody are captured on the sensor surface through immune response respectively in the sample, and form the sandwich complex that a kind of monoclonal antibody-analyte-enzyme is marked second monoclonal antibody;
2. said sensor is placed deposition of silver solution, this deposition of silver solution is the 1mM ascorbic acid phosphoric acid esters, 2mM AgNO
30.1M glycocoll-NaOH buffer solution, pH9.0; Standing and reacting 8min-12min under the room temperature; Obtaining the electricity relevant with analyte concentration from this sensor then leads or resistance signal.
In the above-mentioned steps (2); Its substrate ascorbic acid phosphoric acid esters hydrolysis of alkaline phosphatase enzymatic produces the reductive agent ascorbic acid on the sensor surface; The latter becomes silver metal with silver ion reduction in the solution and is deposited on the micro-gap array gold electrode; Make the conducting of micro-gap array gold electrode positive and negative electrode part, thereby the electricity that increases the micro-gap array gold electrode is led (or reducing resistance), obtains the electricity relevant with analyte concentration and leads (or resistance) signal.
Three of technical scheme of the present invention is that the analyte method that adopts above-mentioned enzyme catalysis conductance immune sensor based on micro-gap array electrode to detect the monoclonal antibody of no alkaline phosphatase mark adopts following steps:
With analyte sample or standard specimen solution (this solution be said analyte sample or standard specimen are dissolved in 0.01M, pH 7.4 phosphate buffer solutions and get), (AC is 100 μ g/mL to determinand mouse monoclonal antibody solution; This solution is that said monoclonal antibody is dissolved in 0.01M, pH 7.4 phosphate buffer solutions and gets) each 20 μ L mixes the back and drips on the immunosensor that is fixed with determinand sheep polyclonal antibody, and standing and reacting is 0.5 hour under the room temperature; Analyte and monoclonal antibody are captured on the sensor surface through immune response respectively in the sample like this, and form the sandwich complex of a kind of resisting-analyte-monoclonal antibody more;
2. drip the sheep anti-mouse igg antibody of 20 μ L alkali phosphatase enzyme marks at said sensor surface, concentration is 10 μ g/mL, is dissolved in phosphate buffer solution, wherein Na
3PO
4Be 0.01M, pH 7.4, and standing and reacting is 0.4 hour-0.6 hour under room temperature; ELIAS secondary antibody through with sandwich complex in monoclonal antibody react and be captured on the sensor surface;
3. said sensor is placed deposition of silver solution, this deposition of silver solution is the 1mM ascorbic acid phosphoric acid esters, 2mM AgNO
30.1M glycocoll-NaOH buffer solution, pH9.0, standing and reacting 8min-12min under the room temperature; Obtaining the electricity relevant with analyte concentration from said sensor again leads or resistance signal.
In the above-mentioned steps (3); Its substrate ascorbic acid phosphoric acid esters hydrolysis of alkaline phosphatase enzymatic produces the reductive agent ascorbic acid on the sensor surface; The latter becomes silver metal with silver ion reduction in the solution and is deposited on the micro-gap array gold electrode; Make the conducting of micro-gap array gold electrode positive and negative electrode part, thereby the electricity that increases the micro-gap array gold electrode is led (or reducing resistance), obtains the electricity relevant with analyte concentration and leads (or resistance) signal.
The enzyme catalysis conductance immune sensing detection technology that the present invention sets up based on micro-gap array electrode; Highly sensitive; Can realize the detection of fg/mL protein molecular, and need not exact instrument and carry out detection by quantitative that being expected to detect to use for disease is early stage and on-the-spot provides effective technical basis.
Known that by above the present invention is a kind of enzyme catalysis conductance immune sensor and immunologic detection method thereof based on micro-gap array electrode, its advantage has:
1. micro-gap array electrode preparation is simple, can be in enormous quantities, low-cost production;
2. it is simple to detect step, and detection time, detecting required instrument can be with universal electric meter or the resistance measuring equipment of developing voluntarily, portable and cheap in 1h;
3. this immunosensor is highly sensitive, can realize the detection of fg/mL protein molecular, and background interference is very little.
Description of drawings
Fig. 1 is micro-gap array electrode pattern (black region is golden film, and white portion is exposed substrate).The mask pattern is (black region is the clear area, and white portion is the opacity) in contrast.It is the interdigital bipolar electrode, and positive and negative electrode is two comb shape gold electrodes, intersects through the micron order clearance for insulation each other and forms the array gold electrode.5 to 20 of the comb shape numbers of teeth of positive and negative electrode, wide is 1 μ m-100 μ.Electrode gap is 1 μ m-100 μ m.
Fig. 2 is based on the immunosensor photo in kind (black region is golden film, and white portion is exposed substrate, the fixing analyte antibody in arrow points zone wherein, the gold electrode two ends are wired to pick-up unit) of micro-gap array electrode.
Fig. 3 is based on the immunosensor of micro-gap array electrode to variable concentrations hIgG corresponding response curve (current potential-current curve, its slope representative response electricity is led); Visible by this figure, the electricity of sensor is led response and is increased with analyte hIgG concentration, detects lower limit and can reach 1fg/mL.
Embodiment
Embodiment 1: the immunosensor based on micro-gap array electrode detects immunoglobulin G while (hIgG).
1. the preparation of immunosensor:
Get the micro-gap array gold electrode of conventional lithographic printing method making and cook substrate, it is the interdigital bipolar electrode, and positive and negative electrode is two comb shape gold electrodes, composition micro-gap array gold electrode intersected with each other.The comb shape facewidth is 1 μ m-100 μ m, and the gap is 1 μ m--100 μ m;
After the micro-gap array gold electrode cleaned three times (each 1min) with absolute ethyl alcohol (100%), electrode is immersed 30min in the NaOH solution of 1M; Use the ultrapure water cleaning electrode again three times (each 1min), dry up.Then, the electrode immersion is contained in the ethanolic solution of aminopropyl trimethoxysilane 5% (percent by volume), left standstill under the room temperature 24 hours.Dry up after cleaning three times with ultrapure water, obtain the micro-gap array gold electrode of silanization.
The micro-gap array gold electrode of silanization is immersed glutaraldehyde water solution (massfraction 5%), left standstill under the room temperature 1 hour; After ultrapure water cleaning three times, (AC is 100 μ g/mL to goat anti-human igg's polyclonal antibody solution of dropping 30 μ L hIgG, is dissolved in phosphate buffer solution, wherein Na on electrode
3PO
4Be 0.01M, pH 7.4), standing and reacting is 1 hour under the room temperature.Dry up subsequent use after cleaning three times with ultrapure water.
2.hIgG detection:
Standard specimen solution, each 20 μ L of mouse-anti hIgG monoclonal anti liquid solution of hIgG are mixed the back dropping on the immunosensor of as above preparation, and standing and reacting is 0.5 hour under the room temperature; (enzyme labelled antibody concentration is 10 μ g/mL, phosphate buffer solution, Na to drip the sheep anti-mouse igg polyclonal antibody solution of 20 μ L alkali phosphatase enzyme marks at sensor surface
3PO
4Be 0.01M, pH 7.4), standing and reacting is 0.5 hour under room temperature.Sensor is placed deposition of silver solution (1mM ascorbic acid phosphoric acid esters, 2mM AgNO
30.1M glycocoll-NaOH buffer solution, pH9.0) in, standing and reacting 10min under the room temperature.Then; With the immunosensor both positive and negative polarity respectively with the working electrode of electrochemical workstation (CHI 760C) with electrode (contrast electrode is with compound to electrode) is connected; Adopt linear sweep voltammetry to detect in 0~50mV potential range; Write down the response curve (as shown in Figure 3) of sensor current, and calculate its slope (being electric conductivity value) with potential change.According to the electric conductivity value of variable concentrations hIgG standard specimen, obtain working curve.
We use the immunosensor developed as above method 10 hIgG blood serum samples are measured, electricity is per sample led response and working curve, tries to achieve corresponding hIgG concentration.With the contrast of ELISA method, the relative error of the result of the immunosensor of being developed and the measured value of ELISA is all in 5.6%.
Embodiment 2: the immunosensor based on micro-gap array electrode detects PSA (PSA).
1. the preparation of immunosensor:
After the micro-gap array gold electrode cleaned three times (each 1min) with absolute ethyl alcohol (100%), electrode is immersed 30min in the NaOH solution of 1M; Use the ultrapure water cleaning electrode again three times (each 1min), dry up; Then, the electrode immersion is contained in the ethanolic solution of aminopropyl trimethoxysilane 5% (percent by volume), left standstill under the room temperature 24 hours.Dry up after cleaning three times with ultrapure water, obtain the micro-gap array gold electrode of silanization.
The micro-gap array gold electrode of silanization is immersed glutaraldehyde water solution (massfraction 5%), left standstill under the room temperature 1 hour; After ultrapure water cleaning three times, (concentration is 100 μ g/mL, is dissolved in phosphate buffer solution, wherein Na on electrode, to drip 30 μ L mouse-anti people PSA monoclonal anti liquid solutions
3PO
4Be 0.01M, pH 7.4), standing and reacting is 1 hour under the room temperature; After ultrapure water cleaning three times, dry up, subsequent use.
2.PSA detection:
(fixing monoclonal antibody is discerned different antigen determining site on this monoclonal antibody and the electrode, and AC is 100 μ g/mL, is dissolved in phosphate buffer solution, wherein Na with analyte PSA standard specimen solution, alkali phosphatase enzyme mark mouse-anti people PSA monoclonal anti liquid solution
3PO
4Be 0.01M, pH 7.4) each 20 μ L mixing back dropping is on immunosensor, and standing and reacting is 0.5 hour under the room temperature; Sensor is placed deposition of silver solution (1mM ascorbic acid phosphoric acid esters, 2mM AgNO
30.1M glycocoll-NaOH buffer solution, pH9.0) in, standing and reacting 10min under the room temperature.Electricity according to the method determination sensor of case study on implementation 1 is led, and obtain working curve, and the electricity of sensor is led in 1fg~10ng/mL scope linear with PSA concentration.
The immunosensor that the present invention's use is developed as above method is measured 8 normal persons and 10 prostate cancer patients' blood serum sample, and electricity is per sample led response and working curve, tries to achieve corresponding PSA concentration.With the contrast of ELISA method, the relative error of the result of the immunosensor of being developed and the measured value of ELISA is all in 7.9%.
Traditional lithographic printing method is adopted in the micro-gap array electrode preparation: on the substrate (quartz, conventional ceramic or glass material) of the 4 * 6mm that cleans is gone up with photoresist spinner, be coated with last layer photoresist (positive glue), rotating speed 3000r/min, 80 ℃ of baking 15min.In litho machine, mask is aimed at substrate, uv-exposure is transferred to the figure on the mask (like Fig. 1) on the substrate.Develop and remove the photoresist of exposed portion, after nitrogen stream dries up, adopt vacuum sputtering on substrate, to spray thick Ti layer of 30nm and the thick Au layer of 120nm successively.After removing on-chip photoresists with the organic solvent chloroform then, can obtain micro-gap array gold electrode as shown in Figure 1.Width between wherein single gold ribbon width and adjacent two gold ribbons can be adjusted between 1~100 μ m as required.
Claims (1)
1. the enzyme catalysis conductance immune sensor based on micro-gap array electrode is characterized in that, adopts the micro-gap array gold electrode to cook substrate, and adopts following steps to make:
(1) the micro-gap array gold electrode of getting conventional lithographic printing method making is cooked substrate; It is the interdigital bipolar electrode, and positive and negative electrode is two comb shape gold electrodes, composition micro-gap array gold electrode intersected with each other; The comb shape facewidth is 1 μ m-100 μ m, and the gap is 1 μ m-100 μ m;
(2) silanization of micro-gap array gold electrode is handled, and the steps include:
A. said micro-gap array gold electrode is cleaned three times each 1min with absolute ethyl alcohol;
B. this electrode is immersed in the NaOH solution of 1M, leave standstill 28min-32min;
C. clean this electrode three times with ultrapure water, each 1min; Dry up;
D. this electrode immersion is contained in the ethanolic solution of aminopropyl trimethoxysilane 5% (percent by volume), left standstill under the room temperature 23 hours-25 hours;
Can on the substrate between the micro-gap array gold electrode, form an amino silane self-assembled monolayer like this;
E. clean three times with ultrapure water, dry up, obtain the micro-gap array gold electrode of silanization;
(3) preparation process of immunosensor:
A. the micro-gap array gold electrode of said silanization being immersed massfraction is 5% glutaraldehyde water solution, leaves standstill under the room temperature 0.8 hour-1.2 hours, cleans three times with ultrapure water again;
B. on said electrode, drip the mouse monoclonal or the sheep polyclonal antibody solution of 30 μ L determinands, standing and reacting is 0.8 hour-1.2 hours under the room temperature; Make antibody be fixed on the substrate of micro-gap array gold electrode through the glutaraldehyde cross-linking agent like this; AC in said mouse monoclonal or the sheep polyclonal antibody solution is 100 μ g/mL, and this antibody-solutions is said antibody is dissolved in 0.01M, pH 7.4 phosphate buffer solutions and gets;
C. clean said electrode three times with ultrapure water, dry up, obtain immunosensor; Be stored in 4 ℃ subsequent use.
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CN101858918B (en) * | 2010-06-09 | 2013-03-20 | 长沙市食品质量安全监督检测中心 | Method for detecting ractopamine in animal-derived food by microgap array electrode-based electrochemical immunosensor |
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CN108152485B (en) * | 2017-12-12 | 2019-11-15 | 江南大学 | A kind of immunoassay method reacting triggering CdS photoelectric current based on alkaline phosphate ester enzymatic |
CN111122683A (en) * | 2019-12-12 | 2020-05-08 | 同济大学 | Electrochemical method for detecting cephalosporin by three-dimensional interdigital printed electrode |
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