CN101271114A - 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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- CN101271114A CN101271114A CNA2008100313212A CN200810031321A CN101271114A CN 101271114 A CN101271114 A CN 101271114A CN A2008100313212 A CNA2008100313212 A CN A2008100313212A CN 200810031321 A CN200810031321 A CN 200810031321A CN 101271114 A CN101271114 A CN 101271114A
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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 mainly is euzymelinked immunosorbent assay (ELISA), fluorescent immune method, gold mark sidestream immune chromatography etc. at present.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 to use exact instrument to carry out quantitative test, are not suitable for the demand of 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 described 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 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.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. described 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 described silanization is immersed massfraction and be 5% glutaraldehyde water solution, left standstill under the room temperature 0.8 hour-1.2 hours, clean three times with ultrapure water again;
B. drip the mouse monoclonal or the sheep polyclonal antibody solution of 30 μ L determinands on described electrode, antibody concentration is 100 μ g/mL, leaves standstill reaction 0.8 hour-1.2 hours under the room temperature; Make antibody be fixed on the substrate of micro-gap array gold electrode by the glutaraldehyde cross-linking agent like this; Described mouse monoclonal or sheep polyclonal antibody solution are described antibody is dissolved in 0.01M, pH7.4 phosphate buffer solution and gets;
C. clean described electrode three times with ultrapure water, dry up, obtain immunosensor; Be stored in 4 ℃ standby.
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 (this analyte sample or standard specimen solution are molten be described analyte sample or standard specimen are dissolved in 0.01M, pH7.4 phosphate buffer solution and get), alkali phosphatase enzyme mark mouse monoclonal antibody solution (fixing antibody recognition synantigen decision bit point not on this monoclonal antibody and the electrode, antibody concentration is 100 μ g/mL in the solution, this solution is described antibody is dissolved in 0.01M, pH7.4 phosphate buffer solution and gets,) respectively drip on immunosensor after 20 μ L mixing, leave standstill reaction 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 by 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. described 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; Leave standstill reaction 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 described analyte sample or standard specimen are dissolved in 0.01M, pH7.4 phosphate buffer solution and get), (antibody concentration is 100 μ g/mL to determinand mouse monoclonal antibody solution, this solution is described monoclonal antibody is dissolved in 0.01M, pH7.4 phosphate buffer solution and gets) each 20 μ L mixes the back and drips on the immunosensor that is fixed with determinand sheep polyclonal antibody, leaves standstill reaction 0.5 hour under the room temperature; Analyte and monoclonal antibody are captured on the sensor surface by 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 described sensor surface, concentration is 10 μ g/mL, is dissolved in phosphate buffer solution, wherein Na
3PO
4Be 0.01M, pH7.4 leaves standstill reaction 0.4 hour-0.6 hour under room temperature; ELIAS secondary antibody by with sandwich complex in monoclonal antibody react and be captured on the sensor surface;
3. described 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, leave standstill under the room temperature reaction 8min-12min; Obtaining the electricity relevant with analyte concentration from described 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.
As known from the above, the present invention is a kind of enzyme catalysis conductance immune sensor and immunologic detection method thereof based on micro-gap array electrode, and 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 by 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); By this figure as seen, 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 1 M; 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, (antibody concentration 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, pH7.4), leave standstill reaction 1 hour under the room temperature.Dry up standby 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, leave standstill reaction 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, pH7.4), under room temperature, leave standstill reaction 0.5 hour.Sensor is placed deposition of silver solution (1mM ascorbic acid phosphoric acid esters, 2mM AgNO
30.1M glycocoll-NaOH buffer solution, pH9.0) in, leave standstill under the room temperature reaction 10min.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 prostate specific antigen (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 1 M; 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 to drip 30 μ L mouse-anti people PSA monoclonal anti liquid solutions on electrode
3PO
4Be 0.01M, pH7.4), leave standstill reaction 1 hour under the room temperature; After ultrapure water cleaning three times, dry up, standby.
2.PSA detection:
(fixing monoclonal antibody is discerned not synantigen decision bit point on this monoclonal antibody and the electrode with analyte PSA standard specimen solution, alkali phosphatase enzyme mark mouse-anti people PSA monoclonal anti liquid solution, antibody concentration is 100 μ g/mL, be dissolved in phosphate buffer solution, wherein Na
3PO
4Be 0.01M, pH7.4) each 20 μ L mixes the back dropping on immunosensor, leaves standstill reaction 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, leave standstill under the room temperature reaction 10min.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: be coated with last layer photoresist (positive glue) on the substrate (quartz, conventional ceramic or glass material) of the 4 * 6mm that cleans is gone up with photoresist spinner, 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 (as 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 (3)
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. described 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 described silanization is immersed massfraction and be 5% glutaraldehyde water solution, left standstill under the room temperature 0.8 hour-1.2 hours, clean three times with ultrapure water again;
B. on described electrode, drip the mouse monoclonal or the sheep polyclonal antibody solution of 30 μ L determinands, leave standstill reaction 0.8 hour-1.2 hours under the room temperature; Make antibody be fixed on the substrate of micro-gap array gold electrode by the glutaraldehyde cross-linking agent like this; Antibody concentration in described mouse monoclonal or the sheep polyclonal antibody solution is 100 μ g/mL, and this antibody-solutions is described antibody is dissolved in 0.01M, pH7.4 phosphate buffer solution and gets;
C. clean described electrode three times with ultrapure water, dry up, obtain immunosensor; Be stored in 4 ℃ standby.
2, a kind of analyte method that adopts the described enzyme catalysis conductance immune sensor based on micro-gap array electrode of claim 1 to detect the monoclonal antibody that alkali phosphatase enzyme mark is arranged is characterized in that it adopts following steps:
(1) analyte sample or standard specimen solution, each 20 μ L of alkali phosphatase enzyme mark mouse monoclonal antibody solution are mixed the back and drip on immunosensor, leave standstill reaction 0.4 hour-0.6 hour under the room temperature; Described analyte sample or standard specimen solution are this analyte sample or standard specimen are dissolved in 0.01M, pH7.4 phosphate buffer solution and get; Described alkali phosphatase enzyme mark mouse monoclonal antibody solution is described antibody is dissolved in 0.01M, pH7.4 phosphate buffer solution and gets, and antibody concentration is 100 μ g/mL, fixing antibody recognition synantigen decision bit point not on this monoclonal antibody and the electrode;
(2) described 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; Leave standstill reaction 8min-12min under the room temperature; Obtaining the electricity relevant with analyte concentration from this sensor then leads or resistance signal.
3, a kind of analyte method that adopts the described enzyme catalysis conductance immune sensor based on micro-gap array electrode of claim 1 to detect the monoclonal antibody of no alkaline phosphatase mark is characterized in that it adopts following steps:
(1) analyte sample or standard specimen solution, each 20 μ L of monoclonal antibody solution are mixed the back and drip on the immunosensor that is fixed with the determinand polyclonal antibody, leave standstill reaction 0.5 hour under the room temperature;
(2) drip the second antibody solution of 20 μ L alkali phosphatase enzyme marks at described sensor surface, under room temperature, leave standstill reaction 0.4 hour-0.6 hour; The sheep anti-mouse igg antibody solution that described second antibody solution is alkali phosphatase enzyme mark, antibody concentration are 10 μ g/mL, are described antibody is dissolved in 0.01M, pH 7.4 phosphate buffer solutions and gets;
(3) described 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, leave standstill under the room temperature reaction 8min-12min; Obtaining the electricity relevant with analyte concentration from described sensor again leads or resistance signal.
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