CN107543924A - Biology sensor detection myeloperoxidase based on copper palladium platinum mesh nanometer material - Google Patents
Biology sensor detection myeloperoxidase based on copper palladium platinum mesh nanometer material Download PDFInfo
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Abstract
It is used to detect myeloperoxidase (MPO) present invention introduces the new unmarked electrochemical immunosensor based on copper palladium Pt nanowires Web materials (CuPdPt NNWs).Locate good catalytic activity because copper palladium Pt nanowires network structure has excellent electronic conductivity and hydrogen peroxide is showed, it is considered as a kind of new effective elctro-catalyst.Meanwhile network structure provides the high surface area available for deposited Au nano particle (AuNP).AuNPs not only increases the conduction of electronics, and can pass through covalent bond sessile antibody.As a result, the unmarked immunosensor of invention shows the more sensitive detection to MPO.Under optimum experimental condition, the immunosensor invented is shown from 100fg mL1To 50ng mL1The wide range of linearity, lowest detection is limited to 33.3fg mL‑1(S/N=3).In addition, unmarked immunosensor is used to determining MPO in human serum, its rate of recovery is 99.8% 103.6%, shows that electrochemical immunosensor can apply to the actually detected of MPO.Simultaneously, it can also be used to the detection of other biological mark in serum.
Description
Technical field：
The present invention relates to a kind of preparation side for the electrochemical immunosensor for clinically quantitatively detecting myeloperoxidase
Method and application, it is based especially on copper palladium Pt nanowires netty compound material and builds immunosensor as signal direct method, use
In detection myeloperoxidase, belong to field of electrochemical detection.
Background technology：
Coronary heart disease is most common disease in cardiovascular system, and acute coronary syndrome (ACS) is Severe hat
Worry.Because ACS has high incidence and poor prognosis, therefore it is extremely important to early diagnose ACS.Myeloperoxidase (MPO)
It is a kind of abundant phagocyte hemalbumin in mammal body.Biochemistry and genetics research show containing for MPO in blood
Measure popular closely related with angiocardiopathy.It is cardiovascular to be proved to predictable ACS patient for increased MPO contents in blood
The risk that event occurs.In addition, MPO can be used for identifying the potential patient having a heart disease and predict at 30 days and 6 months
The risk of bad the events of heart attack occurs for period.Therefore, the exploitation of new MPO biology sensors in ACS patient's early detection and
There are very big potentiality in risk profile.
Include radioimmunology (RIA) and enzyme linked immunosorbent assay (ELISA) for the conventional method that MPO is quantitatively detected.
Although these methods show high sensitivity and selectivity, these traditional method of immunity still have some places
Need to improve, for example, it is time-consuming, and detection device is expensive.As the alternative of routine immunization assay method, based on immunologic opsonin
The electrochemical immunosensor of reaction is widely used to clinical diagnosis.Compared with routine immunization assay method, electro-chemistry immunity
Sensor has pretreatment simply, analyte small volume, the advantages that quickly analysis.
In recent years, using electrochemical immunosensor of the various nano materials for building MPO detections, such as nanometer
Gold/ceria -1- butyl -3- methylimidazoles hexafluorophosphate/Cys composite membrane, 3,3 ', 5,5 '-tetramethyl biphenyl
Amine and ionic liquid composite membrane.However, the synthesis hell to pay of these nano composite materials and time-consuming.In recent years, due to nano wire
Its fabulous catalytic performance of Web materials and simple synthetic method are considered as the effective elctro-catalyst of new class.There is research table
It is bright when nano material contains transition metal, nano material will pass with excellent hydrogen peroxide catalyzed active and excellent electronics
The property led.In materials synthesis preliminary process, the nano material rich in palladium and platinum shows good hydrogen peroxide and urged for we
Change activity, meanwhile, copper also has the ability of regulation electrocatalysis characteristic.Therefore we select our former material of these three metal functions
Material.By simply synthesizing, these three elements can form ultrathin nanometer line network structure.At present, copper palladium Pt nanowires are netted
Material does not use in electrochemical field.In order to realize specific detection, more fixed MPO antibody, we select gold nano
Particle (AuNPs) adsorbs antibody and keeps the bioactivity of antibody securely.Therefore, it is netted to combine copper palladium Pt nanowires for we
Material and golden nanometer particle CuPdPt NNWs/AuNPs are used for amplified signal to detect MPO as the basis of sessile antibody.
We have proposed a kind of unmarked electrochemical immunosensor of innovation, for quantitatively detecting MPO, depend on
CuPdPt NNWs/AuNPs modified glassy carbon electrodes are as conductive and signal material.CuPdPt NNWs/AuNPs compare surface due to it
Product is big, good conductivity, can not only improve electron transfer efficiency, and can improve the amount of sessile antibody.When for determining MPO
During amount detection, the immunosensor proposed shows high sensitivity, reappearance and good stability.Meanwhile structure is exempted from
Epidemic disease sensor can also be used for the detection of other biological sample in serum.
The project, which establishes a simple, quick detection method, realizes special, super sensitivity detection to MPO.For ACS
Patient's early detection and risk profile provide foundation.
The content of the invention：
1. the purpose of the present invention is for detecting the preparation method of the electrochemical immunosensor of myeloperoxidase with answering
With providing foundation for the early detection and risk profile of clinically ACS patient, its feature comprises the following steps：
(1) CuPdPt NNWs nano materials are prepared；
(2) electrochemical immunosensor is established, detects MPO, draws standard curve.
2. the preparation process of CuPdPt NNWs nano materials of the present invention specifically includes following steps, its feature includes
Following steps：
By 6.00mg NaBH4It is dissolved in 3mL ultra-pure waters, 2mL is then contained into 1.94mg Na2PdCl4, 3.32mg
CuCl2With 93.32mL H2PtCl6(5%) mixed solution is added rapidly to above-mentioned NaBH under magnetic agitation (300rpm)4It is molten
In liquid.React 2 minutes at ambient temperature.Reaction solution is quickly transferred in EP pipes (2mL) afterwards, passes through centrifugation
(10000rpm) collects solid product, is finally placed in dried for standby in vacuum drying chamber with milli-Q water three times.
3. according to claim 1 establish electrochemical immunosensor, myeloperoxidase is detected, it is bent to draw standard
Line, it is characterised in that comprise the following steps：
(1) respectively with 0.3 and 0.05 μm of Al2O3Powder by polishing electrode into minute surface, then respectively by ultra-pure water, anhydrous
Order each 5min of ultrasound electrode of ethanol, ultra-pure water, drying at room temperature are standby；
(2) 10 μ L electrode modified material copper palladium Pt nanowires Web materials (CuPdPt NNWs) are added dropwise in electrode surface,
Dry at ambient temperature.
(3) electrode after above-mentioned modification is immersed into 3mL HAuCl4 (1%) solution, reacted under -0.25V voltage conditions
30 seconds deposited Au nano particles.
(4) electrode washing is totally added dropwise to 8 μ L myeloperoxidase specific antibodies, 4 DEG C of incubation 12h afterwards with ultra-pure water.
(5) electrode washing after incubation is totally added dropwise to 8 μ L, 1% BSA solution incubation at room temperature 30min afterwards with ultra-pure water.
(6) electrode after above-mentioned BSA is closed is clean with ultrapure water and dries at ambient temperature.
(7) the myeloperoxidase antigen of various concentrations is added dropwise and 37 DEG C of incubation 2h is placed on electrode.
(8) by the electrode after incubation with ultrapure water it is clean after be placed in room temperature condition dry.
(9) electrode is placed in 5mL, 0.1M PBS (0.1M Na2HPO4, 0.1M KH2PO4, 0.1M KCl) in carry out table
Sign, 20 μ L, 1.4mM H are added every 50s2O2, measure its chrono-amperometric variable-current value.
(10) it is linear according to gained current variation value and myeloperoxidase antigen concentration, drawing curve.
Compared with prior art, a kind of preparation method of electrochemical immunosensor for quantitatively detecting MPO of the invention with
Using, its protrude the characteristics of be：
(1) copper palladium Pt nanowires netty compound material will be based on as signal material, it will be combined with golden nanometer particle
It is incorporated into the preparation of electrochemical immunosensor, not only effectively raises the catalytic performance of material, and improves biology
The supported quantity of molecule, and then improve sensitivity and the detection range of electrochemical immunosensor；
(2) electrochemical immunosensor prepared by this method can provide foundation for clinic early diagnosis ACS patient, and
It can be used for the risk for predicting that cardiovascular event occurs.In addition, the method is easy, quickly, commercialization is easy to implement, so as to promote
The development of accurate medical science.
(3) use identical nano material and method of modifying, can in the immobilized different antibody of electrode surface, so as to
Realize to being detected while various biomolecules, more comprehensive foundation is provided for the diagnosis of disease.
Brief description of the drawings：
Fig. 1 is the structure schematic diagram of the electrochemical immunosensor of the present invention.
Fig. 2 is the copper palladium platinum nano material scanning electron microscope (SEM) photograph of the present invention, XPS figures and EDS figures.
Fig. 3 is the chrono-amperometric variable-current and concentration that the electrochemical immunosensor of the present invention obtains when detecting MPO
Linear relationship, and the stability of sensor, reappearance and specificity.
Embodiment：
The present invention is further elaborated with reference to specific embodiment, it should be appreciated that these embodiments are merely to illustrate
The present invention rather than limitation the scope of the present invention.
Embodiment 1
Step 1. is by 6.00mg NaBH4It is dissolved in 3mL ultra-pure waters, 2mL is then contained into 1.94mg Na2PdCl4,
3.32mg CuCl2With 93.32mL H2PtCl6(5%) mixed solution is added rapidly to above-mentioned under magnetic agitation (300rpm)
NaBH4In solution.React 2 minutes at ambient temperature.Afterwards by reaction solution be quickly transferred to EP pipe (2mL) in, by from
The heart (10000rpm) collects solid product, is finally placed in dried for standby in vacuum drying chamber with milli-Q water three times.
Step 2. is respectively with 0.3 and 0.05 μm of Al2O3Polishing electrode into minute surface, is then pressed ultra-pure water, nothing by powder respectively
Order each 5min of ultrasound electrode of water-ethanol, ultra-pure water, drying at room temperature are standby；
10 μ L, electrode modified material copper palladium Pt nanowires Web materials (CuPdPt NNWs) are added dropwise in electrode step 3.
Surface, drying at room temperature；
Electrode after above-mentioned modification is immersed 3mL HAuCl4 (1%) solution by step 4., under -0.25V voltage conditions
React 30 seconds deposited Au nano particles；
Step 5. electrode washing is totally added dropwise afterwards with ultra-pure water 8 μ L myeloperoxidase specific antibodies, 4 DEG C of incubations
12h；
Step 6. electrode washing after incubation is totally added dropwise afterwards with ultra-pure water 8 μ L, 1% BSA solution incubation at room temperature
30min；
Step 7. dries totally and at ambient temperature the electrode after above-mentioned BSA closings with ultrapure water；
The myeloperoxidase antigen of various concentrations is added dropwise step 8. is placed in 37 DEG C of incubation 2h on electrode；
Step 9. by the electrode after incubation with ultrapure water it is clean after be placed in room temperature condition dry；
Electrode is placed in 5mL, 0.1M PBS (0.1M Na by step 10.2HPO4, 0.1M KH2PO4, 0.1M KCl) in carry out
Characterize, 20 μ L, 1.4mM H are added every 50s2O2, measure its chrono-amperometric variable-current value；
Step 11. is linear according to gained current variation value and myeloperoxidase antigen concentration, and drawing is bent
Line；
Step 12. is linear according to gained current variation value and MPO concentration, drawing curve；Measurement result table
Bright MPO concentration is linear in the range of 100fM-50nM, linearly dependent coefficient 0.998, and detection is limited to 33fM.
Step 13. is by the sensor of the present invention in 4 DEG C of preservations, discontinuity detection sensor current response, after storing 28 days
Current-responsive is still the 86.09% of initial current, represents that sensor has good stability；
Step 14. present invention takes immunosensor 5 prepared by same batch, and 1pM MPO is distinguished under the same conditions
It is measured, each determination of electrode 3 times, as a result the relative standard deviation of response current is 1.55%, illustrates the sensing of structure
Difference is small in device batch, and sensor reappearance is good.
Step 15. will detect MPO under the conditions of existing for the sensor of the present invention in blood other biological molecule, as a result
The presence of other biological molecule does not influence the change of MPO electric currents, illustrates the specific good of sensor, can distinguish target point very well
Son.
Described above is only the preferred embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, under the precondition for not departing from the principle of the invention, some improvements and modifications can also be made, these improve and
Retouching also should be regarded as protection scope of the present invention.
Claims (3)
1. the biology sensor detection myeloperoxidase based on copper palladium platinum mesh nanometer material, it is characterised in that including following step
Suddenly：
(1) preparation of copper palladium Pt nanowires Web materials；
(2) electrochemical immunosensor is established, detects myeloperoxidase, draws standard curve.
2. the preparation process of copper palladium Pt nanowires Web materials, its feature comprise the following steps according to claim 1：
By 6.00mg NaBH4It is dissolved in 3mL ultra-pure waters, 2mL is then contained into 1.94mg Na2PdCl4, 3.32mg CuCl2
With 93.32mL H2PtCl6(5%) mixed solution is added rapidly to above-mentioned NaBH under magnetic agitation (300rpm)4In solution.
React 2 minutes at ambient temperature.Reaction solution is quickly transferred in EP pipes (2mL) afterwards, by centrifuging (10000rpm)
Solid product is collected, is finally placed in dried for standby in vacuum drying chamber with milli-Q water three times.
3. according to claim 1 establish electrochemical immunosensor, myeloperoxidase is detected, draws standard curve,
It is characterized in that comprise the following steps：
(1) respectively with 0.3 and 0.05 μm of Al2O3Powder by polishing electrode into minute surface, then respectively by ultra-pure water, absolute ethyl alcohol,
Order each 5min of ultrasound electrode of ultra-pure water, drying at room temperature are standby；
(2) 10 μ L electrode modified material copper palladium Pt nanowires Web materials (CuPdPt NNWs) are added dropwise in electrode surface, in room
Dried under the conditions of temperature.
(3) electrode after above-mentioned modification is immersed into 3mL HAuCl4 (1%) solution, reacted 30 seconds under -0.25V voltage conditions
Deposited Au nano particle.
(4) electrode washing is totally added dropwise to 8 μ L myeloperoxidase specific antibodies, 4 DEG C of incubation 12h afterwards with ultra-pure water.
(5) electrode washing after incubation is totally added dropwise to 8 μ L, 1% BSA solution incubation at room temperature 30min afterwards with ultra-pure water.
(6) electrode after above-mentioned BSA is closed is clean with ultrapure water and dries at ambient temperature.
(7) the myeloperoxidase antigen of various concentrations is added dropwise and 37 DEG C of incubation 2h is placed on electrode.
(8) by the electrode after incubation with ultrapure water it is clean after be placed in room temperature condition dry.
(10) electrode is placed in 5mL, 0.1M PBS (0.1M Na2HPO4, 0.1M KH2PO4, 0.1M KCl) in characterized, often
20 μ L, 1.4mM H are added every 50s2O2, measure its chrono-amperometric variable-current value.
(11) it is linear according to gained current variation value and myeloperoxidase antigen concentration, drawing curve.
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Application publication date: 20180105 |
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