CN105699464B - A kind of preparation method and application of the immunosensor based on cuprous oxide doping Pd nano particle mark - Google Patents
A kind of preparation method and application of the immunosensor based on cuprous oxide doping Pd nano particle mark Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method and application of the immunosensor based on cuprous oxide doping Pd nano particle mark.Belong to new function material and bio-sensing detection technique field.It is of the invention specific be utilized in prepare cuprous nano particle on the basis of, in-situ reducing palladium bichloride forms the compound of cuprous oxide doping Pd nano particle, and optimum doping amount is have studied, the highly sensitive detection to prostate antigen is realized, the early diagnosis to prostate has great importance.
Description
Technical field
The present invention relates to a kind of preparation method and application of the immunosensor of cuprous oxide doping Pd nano particle mark.
Specific to be utilized in in-situ reducing palladium bichloride in cuprous oxide mother liquor, the nano-complex of synthesis cuprous oxide doping palladium, prepares
A kind of electrochemical immunosensor for detecting prostate antigen, belongs to new function material and bio-sensing detection technique field.
Background technology
A variety of methods such as ELISA, RIA, fluorescent polarization immunoassay, immunoluminescence technology, automation immunoassay at present
Applied to detection PSA.Wherein, electro-chemistry immunity detection is quick and easy to operate etc. excellent with high sensitivity, high selectivity, analysis
Point, it is combined using antigen with the immunological method specifically bound between antibody with electrochemical techniques detection.In electrochemistry
In detection, label plays key effect, therefore various labels come into being.Find that a kind of step is simple, condition
Gently, it is particularly important that product is easy to get, electrochemical response signal strong label, the present invention have synthesized a kind of cuprous oxide doping palladium
Nano-particle compound, is prepared for a kind of electrochemical immunosensor of cuprous oxide doping Pd nano particle labelled antibody, real
The detection to prostate antigen is showed.
The present invention carries out hydrogen peroxide using cuprous oxide and Pd nano particle the performance of electrochemical catalysis, and oxidation is sub-
Copper is combined with Pd nano particle, obtains the nano-complex of cuprous oxide doping palladium, and is prepared for cuprous oxide doping not
The palladium nano-complex of same amount, and optimum doping amount is found, synthesis step is to prepare the basis of cuprous nano particle
On, step reduction palladium bichloride in situ, has obtained the nano-complex of cuprous oxide doping palladium, this method step is simple, condition temperature
With product is easy to get.It this method solve and cuprous oxide is used alone and palladium nano particle catalysis hydrogen peroxide electric signal is small asks
Topic.Using the nano combined substance markers prostate antibody of cuprous oxide doping palladium, the association of cuprous oxide and Pd nano particle is utilized
Same-action, enhances the electrochemical catalysis performance of catalyzing hydrogen peroxide reduction.Generate during row gland and progressively put before detection
Big electrochemical signals, effectively enhance the sensitivity of electrochemical immunosensor.This method cost is low, high sensitivity, spy
The advantages that opposite sex is good, detection speed is fast, and preparation process is simple and easy to get, overcomes current prostate antigen detection method not
Foot.
The content of the invention
An object of the present invention is the immunosensor based on cuprous oxide doping Pd nano particle mark, constructs one
Kind without enzyme, quick and overdelicate interlayer type electrochemical immunosensor.
The second object of the present invention is that the interlayer type electrochemical immunosensor is applied to the detection of prostate antigen.
Technical scheme is as follows：
1. the preparation method of the immunosensor of cuprous oxide doping Pd nano particle mark：
（1）Polished with the glass-carbon electrode of 1.0,0.3,0.05 μm of a diameter of 4 mm of alumina powder foot couple, existed respectively successively
It is cleaned by ultrasonic in ultra-pure water and absolute ethyl alcohol, nitrogen drying；
（2）5 ~ 15 μ L, solution of gold nanoparticles are dripped into electrode surface, dried at room temperature；
（3）When electrode is closely dry, 6 μ L, the prostate-specific capture antibody that concentration is 1 ~ 3 μ g/mL are added dropwise to and repaiied
Electrode surface is adornd, when hatching 1 is small in 4 DEG C of refrigerators, is cleaned up with ultra-pure water；
（4）Be added dropwise 6 μ L, mass fraction be 1.0 ~ 3.0% bovine serum albumin solution in electrode surface, close non-spy
Specific activities site, dries in 4 DEG C of refrigerators, is cleaned up with ultra-pure water；
（5）6 μ L, 10 are added dropwise-5A series of prostate-specific antigen mark of various concentrations of the ng/mL of ng/mL ~ 100
Quasi- solution, dries in 4 DEG C of refrigerators, is cleaned up with ultra-pure water；
（6）6 μ L are added dropwise, the detection antibody-solutions that the cuprous oxide doping Pd nano particle of 1 ~ 3 mg/mL marks, put
Dried in 4 DEG C of refrigerators, that is, a kind of immunosensor based on cuprous oxide doping Pd nano particle mark is made.
2. the preparation method of the detection antibody-solutions of cuprous oxide doping Pd nano particle mark
（1）Cuprous oxide adulterates the preparation of Pd nano particle
In beaker, 0.100 ~ 0.300 g copper acetates are dissolved in 20 ~ 60 mL ultra-pure waters, magnetic agitation, obtains
The solution of clarification；The N of 4.0 ~ 12.0 mL, 0.1 mol/L2H4·H2O solution is added rapidly to above-mentioned mixed under being vigorously stirred
Compound, color change to glassy yellow from blueness immediately；Gained mixture continues stirring 30 minutes, centrifugation, respectively with ultra-pure water and nothing
Water-ethanol is washed several times, and in 60 DEG C of baking ovens it is dry 4 it is small when；It is molten to add 0.5 ~ 4.0 mL, the palladium bichloride of 0.05 mol/L
Liquid, magnetic agitation 30 minutes, centrifuge washing；
（2）The preparation of cuprous oxide doping Pd nano particle mark detection antibody-solutions
It is 1 ~ 3 mg/mL cuprous oxide/Pd nano particle solution and 1 mL, concentration 0.5 by 1 mL of preparation, concentration
The detection antibody mixing of ~ 1.5 μ g/mL, when concussion 12 is small, is added dropwise the ox that 0.5 ~ 1.5 mL mass fractions are 1.0 ~ 3.0%
Serum albumin solution, continue concussion 12 it is small when, centrifugation, with pH be 7.4 phosphate buffer solution wash, obtain cuprous oxide
The detection antibody incubation content of Pd nano particle mark is adulterated, is scattered in the phosphate buffer solution that 1 mL, pH is 7.4,
The detection antibody-solutions of cuprous oxide doping Pd nano particle mark are made, are stored for future use in 4 DEG C of refrigerators.
3. the detecting step of prostate antigen
（1）Tested using electrochemical workstation with three-electrode system, platinum electrode is auxiliary electrode, and saturation calomel is electric
Extremely reference electrode, prepared modified electrode are working electrode, are carried out in the phosphate buffer solution that 10 mL, pH are 7.4
Test；
（2）Selection chronoamperometry is detected prostate antigen, and input voltage is arranged to -0.4 V, sampling interval
It is arranged to 0.1 second, run time is arranged to 200 seconds；
（3）After background current tends towards stability, 10 μ L concentration were injected for 5 into phosphate buffer solution every 50 seconds
The hydrogenperoxide steam generator of mol/L, then record current change with time, drawing curve；
（4）Testing sample solution is detected instead of prostate antigen standard solution.
The useful achievement of the present invention
（1）Cuprous oxide doping Pd nano particle synthesis step is established on the basis of cuprous nano particle is prepared,
Step reduction palladium bichloride in situ, has obtained the nano-complex of cuprous oxide doping palladium, and probes into optimum doping amount, this method step
Rapid simple, mild condition, product are easy to get.
（2）The present invention carries out hydrogen peroxide using cuprous oxide and Pd nano particle the performance of electrochemical catalysis, by oxygen
Change it is cuprous be combined with Pd nano particle, obtain cuprous oxide doping palladium nano-complex, this method solve exclusive use
The problem of cuprous oxide and small palladium nano particle catalysis hydrogen peroxide electric signal.Answered using the nano-particle of cuprous oxide doping palladium
Compound marks prostate antibody, using the synergistic effect of cuprous oxide and Pd nano particle, enhances catalyzing hydrogen peroxide reduction
Electrochemical catalysis performance.
（3）The interlayer type electrochemical immunosensor of preparation is used for the detection of prostate antigen by the present invention, and test limit is low,
The range of linearity is wide, it is possible to achieve simple, quick, sensitive and specific detection.The present invention detects the range of linearity to prostate antigen
For 10-5~ 100 ng/mL, test limit reach 2 fg/mL.
Embodiment
A kind of 1 cuprous oxide of embodiment adulterates preparation method of the Pd nano particle as the immunosensor of label
（1）Polished with the glass-carbon electrode of 1.0,0.3,0.05 μm of a diameter of 4 mm of alumina powder foot couple, existed respectively successively
It is cleaned by ultrasonic in ultra-pure water and absolute ethyl alcohol, nitrogen drying；
（2）5 μ L, solution of gold nanoparticles are dripped into electrode surface, dried at room temperature；
（3）When electrode is closely dry, 6 μ L, the prostate-specific antibody that concentration is 1 μ g/mL are added dropwise to modified electrode table
Face, when hatching 1 is small in 4 DEG C of refrigerators, is cleaned up with ultra-pure water；
（4）6 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 1.0% is lived in electrode surface, closing non-specificity
Property site, dries in 4 DEG C of refrigerators, is cleaned up with ultra-pure water；
（5）6 μ L, 10 are added dropwise-5A series of prostate-specific antigen mark of various concentrations of the ng/mL of ng/mL ~ 100
Quasi- solution, dries in 4 DEG C of refrigerators, is cleaned up with ultra-pure water；
（6）6 μ L are added dropwise, the detection antibody-solutions that the cuprous oxide doping Pd nano particle of 1 mg/mL marks, are placed in 4
Dry, store for future use in DEG C refrigerator.
A kind of 2 cuprous oxide of embodiment adulterates preparation method of the Pd nano particle as the immunosensor of label
（1）Polished with the glass-carbon electrode of 1.0,0.3,0.05 μm of a diameter of 4 mm of alumina powder foot couple, existed respectively successively
It is cleaned by ultrasonic in ultra-pure water and absolute ethyl alcohol, nitrogen drying；
（2）10 μ L, solution of gold nanoparticles are dripped into electrode surface, dried at room temperature；
（3）When electrode is closely dry, 6 μ L, the prostate-specific antibody that concentration is 2 μ g/mL are added dropwise to modified electrode table
Face, when hatching 1 is small in 4 DEG C of refrigerators, is cleaned up with ultra-pure water；
（4）6 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 2.0% is lived in electrode surface, closing non-specificity
Property site, dries in 4 DEG C of refrigerators, is cleaned up with ultra-pure water；
（5）6 μ L, 10 are added dropwise-5A series of prostate-specific antigen mark of various concentrations of the ng/mL of ng/mL ~ 100
Quasi- solution, dries in 4 DEG C of refrigerators, is cleaned up with ultra-pure water；
（6）6 μ L are added dropwise, the detection antibody-solutions that the cuprous oxide doping Pd nano particle of 2 mg/mL marks, are placed in 4
Dry, store for future use in DEG C refrigerator.
A kind of 3 cuprous oxide of embodiment adulterates preparation method of the Pd nano particle as the immunosensor of label
（1）Polished successively with the glass-carbon electrode of 1.0,0.3,0.05 μm of a diameter of 4 mm of alumina powder foot couple, respectively super
It is cleaned by ultrasonic in pure water and absolute ethyl alcohol, nitrogen drying；
（2）15 μ L, solution of gold nanoparticles are dripped into electrode surface, dried at room temperature；
（3）When electrode is closely dry, 6 μ L, the prostate-specific antibody that concentration is 3 μ g/mL are added dropwise to modified electrode table
Face, when hatching 1 is small in 4 DEG C of refrigerators, is cleaned up with ultra-pure water；
（4）6 μ L are added dropwise, the bovine serum albumin solution that mass fraction is 3.0% is lived in electrode surface, closing non-specificity
Property site, dries in 4 DEG C of refrigerators, is cleaned up with ultra-pure water；
（5）6 μ L, 10 are added dropwise-5A series of prostate-specific antigen mark of various concentrations of the ng/mL of ng/mL ~ 100
Quasi- solution, dries in 4 DEG C of refrigerators, is cleaned up with ultra-pure water；
（6）6 μ L are added dropwise, the detection antibody-solutions that the cuprous oxide doping Pd nano particle of 3 mg/mL marks, are placed in 4
Dry, store for future use in DEG C refrigerator.
The preparation method of the detection antibody-solutions of 4 cuprous oxide of embodiment doping Pd nano particle mark
（1）Cuprous oxide adulterates the preparation of Pd nano particle
In beaker, 0.100 ~ 0.300 g copper acetates are dissolved in 20 ~ 60 mL ultra-pure waters, magnetic agitation, obtains
The solution of clarification；The N of 4.0 ~ 12.0 mL, 0.1 mol/L2H4·H2O solution is added rapidly to above-mentioned mixed under being vigorously stirred
Compound, color change to glassy yellow from blueness immediately；Gained mixture continues stirring 30 minutes, centrifugation, respectively with ultra-pure water and nothing
Water-ethanol is washed several times, and in 60 DEG C of baking ovens it is dry 4 it is small when；It is molten to add 0.5 ~ 4.0 mL, the palladium bichloride of 0.05 mol/L
Liquid, magnetic agitation 30 minutes, centrifuge washing；
（2）The preparation of cuprous oxide doping Pd nano particle mark detection antibody-solutions
It is 1 ~ 3 mg/mL cuprous oxide/Pd nano particle solution and 1 mL, concentration 0.5 by 1 mL of preparation, concentration
The detection antibody mixing of ~ 1.5 μ g/mL, when concussion 12 is small, is added dropwise the ox that 0.5 ~ 1.5 mL mass fractions are 1.0 ~ 3.0%
Serum albumin solution, continue concussion 12 it is small when, centrifugation, with pH be 7.4 phosphate buffer solution wash, obtain cuprous oxide
The detection antibody incubation content of Pd nano particle mark is adulterated, is scattered in the phosphate buffer solution that 1 mL, pH is 7.4,
The detection antibody-solutions of cuprous oxide doping Pd nano particle mark are made, are stored for future use in 4 DEG C of refrigerators.
By 1 mL concentration be 1 mg/mL cuprous oxide/Pd nano particle solution and 1 mL concentration is that 0.5 μ g/mL detections are anti-
Body mix, concussion 12 it is small when, be added dropwise 0.5 mL, mass fraction be 1.0% bovine serum albumin solution, continue concussion 12 it is small when,
Centrifugation, is washed with the phosphate buffer solution that pH is 7.4, and the detection antibody for obtaining cuprous oxide doping Pd nano particle mark is incubated
Compound, is scattered in the phosphate buffer solution that 1 mL, pH is 7.4, and cuprous oxide doping Pd nano particle mark is made
Detection antibody-solutions, stored for future use in 4 DEG C of refrigerators.
The preparation method of the detection antibody-solutions of 5 cuprous oxide of embodiment doping Pd nano particle mark
（1）Cuprous oxide adulterates the preparation of Pd nano particle
In beaker, 0.100 ~ 0.300 g copper acetates are dissolved in 20 ~ 60 mL ultra-pure waters, magnetic agitation, obtains
The solution of clarification；The N of 4.0 ~ 12.0 mL, 0.1 mol/L2H4·H2O solution is added rapidly to above-mentioned mixed under being vigorously stirred
Compound, color change to glassy yellow from blueness immediately；Gained mixture continues stirring 30 minutes, centrifugation, respectively with ultra-pure water and nothing
Water-ethanol is washed several times, and in 60 DEG C of baking ovens it is dry 4 it is small when；It is molten to add 0.5 ~ 4.0 mL, the palladium bichloride of 0.05 mol/L
Liquid, magnetic agitation 30 minutes, centrifuge washing；
（2）The preparation of cuprous oxide doping Pd nano particle mark detection antibody-solutions
It is 1.0 μ g/mL detections by solution that 1 mL concentration is 2 mg/mL cuprous oxide/Pd nano particle and 1 mL concentration
Antibody mixes, and when concussion 12 is small, 1.0 mL is added dropwise, the bovine serum albumin solution that mass fraction is 2.0%, it is small to continue concussion 12
When, centrifugation, is washed with the phosphate buffer solution that pH is 7.4, and the detection for obtaining cuprous oxide doping Pd nano particle mark resists
Body incubation content, is scattered in the phosphate buffer solution that 1 mL, pH is 7.4, and cuprous oxide doping Pd nano particle is made
The detection antibody-solutions of mark, store for future use in 4 DEG C of refrigerators.
The preparation method of the detection antibody-solutions of 6 cuprous oxide of embodiment doping Pd nano particle mark
（1）Cuprous oxide adulterates the preparation of Pd nano particle
In beaker, 0.100 ~ 0.300 g copper acetates are dissolved in 20 ~ 60 mL ultra-pure waters, magnetic agitation, obtains
The solution of clarification；The N of 4.0 ~ 12.0 mL, 0.1 mol/L2H4·H2O solution is added rapidly to above-mentioned mixed under being vigorously stirred
Compound, color change to glassy yellow from blueness immediately；Gained mixture continues stirring 30 minutes, centrifugation, respectively with ultra-pure water and nothing
Water-ethanol is washed several times, and in 60 DEG C of baking ovens it is dry 4 it is small when；It is molten to add 0.5 ~ 4.0 mL, the palladium bichloride of 0.05 mol/L
Liquid, magnetic agitation 30 minutes, centrifuge washing；
（2）The preparation of cuprous oxide doping Pd nano particle mark detection antibody-solutions
The 1 mL concentration for adding palladium bichloride preparation is dense for the solution of 3 mg/mL cuprous oxide/Pd nano particle and 1 mL
Spend and detect antibody mixing for 1.5 μ g/mL, when concussion 12 is small, 1.5 mL are added dropwise, the bovine serum albumin(BSA) that mass fraction is 3.0%
Solution, continue concussion 12 it is small when, centrifugation, with pH be 7.4 phosphate buffer solution wash, obtain cuprous oxide adulterate palladium nanometer
The detection antibody incubation content of particle label, is scattered in the phosphate buffer solution that 1 mL, pH is 7.4, it is sub- that oxidation is made
The detection antibody-solutions of Copper-cladding Aluminum Bar Pd nano particle mark, store for future use in 4 DEG C of refrigerators.
The detecting step of 7 prostate antigen of embodiment
（1）Tested using electrochemical workstation with three-electrode system, platinum electrode is auxiliary electrode, and saturation calomel is electric
Extremely reference electrode, prepared modified electrode are working electrode, are carried out in the phosphate buffer solution that 10 mL, pH are 7.4
Test；
（2）Selection chronoamperometry is detected prostate antigen, and input voltage is arranged to -0.4 V, sampling interval
It is arranged to 0.1 second, run time is arranged to 200 seconds；
（3）After background current tends towards stability, it is 5 mol/ to inject 10 μ L concentration into phosphate buffer solution every 50 seconds
The hydrogenperoxide steam generator of L, then record current change with time, drawing curve；
（4）Testing sample solution is detected instead of prostate antigen standard solution.
（5）The electrochemical immunosensor is 10 to the prostate antigen detection range of linearity-5~ 100 ng/mL, test limit
2 fg/mL。
Claims (3)
- A kind of 1. preparation method of the immunosensor based on cuprous oxide doping Pd nano particle mark, it is characterised in that bag Include following steps：(1) polished successively with the glass-carbon electrode of 1.0,0.3,0.05 μm of a diameter of 4mm of alumina powder foot couple, respectively in ultra-pure water It is cleaned by ultrasonic with absolute ethyl alcohol, nitrogen drying；(2) 5 ~ 15 μ L, solution of gold nanoparticles are dripped into electrode surface, dried at room temperature；(3) when electrode is closely dry, 6 μ L, the prostate-specific that concentration is 1 ~ 3 μ g/mL is captured into antibody and are added dropwise to modification electricity Pole surface, when hatching 1 is small in 4 °C of refrigerators, is cleaned up with ultra-pure water；(4) it is non-specific in electrode surface, closing for 1.0 ~ 3.0% bovine serum albumin solution that 6 μ L, mass fraction is added dropwise Avtive spot, dries in 4 °C of refrigerators, is cleaned up with ultra-pure water；(5) 6 μ L, 10 are added dropwise-5A series of prostate-specific antigen standard of various concentrations of the ng/mL of ng/mL ~ 100 is molten Liquid, dries in 4 °C of refrigerators, is cleaned up with ultra-pure water；(6) 6 μ L are added dropwise, the detection antibody-solutions that the cuprous oxide doping Pd nano particle of 1 ~ 3 mg/mL marks, are placed in 4 ° Dried in C refrigerators, that is, a kind of immunosensor based on cuprous oxide doping Pd nano particle mark is made.
- A kind of 2. preparation side of immunosensor based on cuprous oxide doping Pd nano particle mark as claimed in claim 1 Method, the preparation of the detection antibody-solutions of the cuprous oxide doping Pd nano particle mark, it is characterised in that step is as follows：(1) preparation of cuprous oxide doping Pd nano particleIn beaker, 0.100 ~ 0.300 g copper acetates are dissolved in 20 ~ 60 mL ultra-pure waters, magnetic agitation, is clarified Solution；The N of 4.0 ~ 12.0 mL, 0.1 mol/L2H4·H2The above-mentioned clarification that O solution is added rapidly under being vigorously stirred is molten Liquid, color change to glassy yellow from blueness immediately；Gained mixture continues stirring 30 minutes, centrifugation, respectively with ultra-pure water and anhydrous Ethanol is washed several times, and in 60 °C of baking ovens it is dry 4 it is small when；0.5 ~ 4.0 mL, the palladium chloride solution of 0.05 mol/L are added, Magnetic agitation 30 minutes, centrifuge washing；(2) preparation of cuprous oxide doping Pd nano particle mark detection antibody-solutionsBy 1 mL of preparation, concentration be 1 ~ 3 mg/mL cuprous oxide/Pd nano particle solution and 1 mL, concentration be 0.5 ~ The detection antibody mixing of 1.5 μ g/mL, when concussion 12 is small, is added dropwise the ox blood that 0.5 ~ 1.5 mL mass fractions are 1.0 ~ 3.0% Pure protein solution, continue concussion 12 it is small when, centrifugation, with pH be 7.4 phosphate buffer solution wash, obtain cuprous oxide and mix The detection antibody incubation content of miscellaneous Pd nano particle mark, is scattered in the phosphate buffer solution that 1 mL, pH is 7.4, system The detection antibody-solutions of cuprous oxide doping Pd nano particle mark are obtained, are stored for future use in 4 °C of refrigerators.
- 3. the detection method of immunosensor prepared by preparation method as claimed in claim 1, it is characterised in that step is such as Under：(1) tested using electrochemical workstation with three-electrode system, platinum electrode is auxiliary electrode, and saturated calomel electrode is Reference electrode, prepared modified electrode are working electrode, are surveyed in the phosphate buffer solution that 10 mL, pH are 7.4 Examination；(2) selection chronoamperometry is detected prostate antigen, input voltage is arranged to -0.4 V, sampling interval is set For 0.1 second, run time was arranged to 200 seconds；(3) after background current tends towards stability, 10 μ L were injected into phosphate buffer solution, concentration is 5 mol/L every 50 seconds Hydrogenperoxide steam generator, then record current change with time, drawing curve；(4) testing sample solution is detected instead of prostate-specific antigen standard solution.
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