CN108802390A - A kind of preparation of the pancreatic tumour marker immunosensor based on graphene-gold-palladium nanocomposite - Google Patents
A kind of preparation of the pancreatic tumour marker immunosensor based on graphene-gold-palladium nanocomposite Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
Abstract
The present invention provides a kind of novel unmarked electrochemical immunosensor of graphene-gold based on reduction-palladium nanocomposite modification for detecting tumor markers CA242, the dopant of selective oxidation graphene (GO) and polyvinylpyrrolidone as the reproducibility of synthesizing water-solubility and stabilization graphene oxide-gold-palladium (rGO-Au-Pd) nanocomposite.The material has excellent electrochemical redox activity and electron transfer capacity.In order to optimize the performance of immunosensor, the present invention has probed into synthetic method, material concentration, the influence of reduction cycle and pH, make the CA242 immunosensors of preparation in linear detection range, detection limit, sensitivity, excellent performance is shown in terms of stability and reproducibility, and there is reproducibility and reliability.The present invention provides a platform for the new bio measurement that early-stage cancer diagnoses, and promotes application of the biosensor technique in medical domain.
Description
Technical field
Nano-function thin films and biosensor technology field, more particularly to it is a kind of for detecting tumor markers
The novel unmarked electrochemical immunosensor of graphene-gold based on reduction of CA242-palladium nanocomposite modification.
Background technology
Cancer is still the chief threat to mankind's publilc health.Currently, tumor in digestive tract is that population in the world ten is big pernicious
One of tumour, the lethal of these cancers are that breaking-out is attacked since its is hidden, and early stage shifts, and undercut removes success rate and fast-growth.By
In lacking sensitive and special diagnostic method, patient is usually in final stage with tumor in digestive tract.Due to knubble biological flag
Object is to the specificity of cancer cell, so Tumor biomarkers provide a kind of new hand for the early detection of cancer onset and recurrence
Section, can carry out therapy intervention in time.Carbohydrate Antigens 24-2 (CA242) is the tumor-marker of pernicious tumor in digestive tract
Object has higher specificity compared with CA199 and CA50 to cancer of pancreas and colorectal cancer.Therefore, exploitation is sensitive and reliable
Method these malignant tumours will provide tool for early detection to detect CA242.
Currently, many immunosensors, including ampere meter, fluorescence, colorimetric, potentiometer, piezoelectricity and radiation sensor etc.,
It is used for sensitive detection tumor markers.All these measurement both depend on the reaction between antibody and application specific antigen.
In these methods, unmarked electrochemical immunosensor due to its relatively high sensitivity, cost-effectiveness, quickly detection and
It does not need secondary antibody and receives significant attention.
The sensitivity of electrochemical immunosensor is by the specific surface area of modified electrode and significantly affecting for conductivity.Packet at present
Include metal nanoparticle, the various nano materials of carbon material, metal oxide and its derivative have been used for changing electrode to put
The performance of big signal and booster immunization sensor.The electricity of enhancing, optics, magnetics and chemical property are multiple better than monocomponent nanocomposite
It closes, in these nano materials, hybridized nano composite material has very high attraction due to its long-time stability and material
Power.About the unmarked immunosensor of detection CA242, patent CN105158313A one kind being based on molybdenum disulfide/Ni-Pd alloy
The preparation method and a kind of electrochemistry stomaches of 104502593 B of patent CN of the unmarked immunosensor of nanocomposite structure
The preparation method of the unmarked immunosensor of cancerous swelling tumor markers discloses different unmarked immunosensor detection CA242,
But its sensitivity and linear measurement range is relatively low, and specificity is also to be improved.
Gold-palladium (Au-Pd) nanocomposite is a kind of bimetallic material, due to its high catalytic capability, big surface body
Product ratio and biocompatibility, are widely studied in bio-sensing field.However, bimetallic material is easy on nano-scale
Aggregation, this is the reason of hindering its broader applications.The graphene being made of single layer of carbon atom is since its is unique mechanically and electrically
It learns property and is widely used in and prepares electrochemical sensor, but also there is highly soluble and environment friendly.It there is no base at present
In the report that the CA242 immunosensors of modified rGO-Au-Pd nanometer combined electrodes measure.It would therefore be highly desirable to develop a kind of linear
Detection range, detection limit, sensitivity, stability and reproducibility aspect show the sensor of excellent performance, are used for for exploitation
The new bio measurement of early-stage cancer diagnosis provides a platform.
Invention content
The purpose of the present invention is to provide it is a kind of prepare simple, high sensitivity, detection quickly, high specificity can be used for swelling
The preparation method of the unmarked electrochemical immunosensor of tumor markers CA242 detections, prepared sensor can be used for tumour
Quick, the Sensitive Detection of marker CA242.Based on this purpose, the present invention prepares a kind of reproducibility graphene oxide-gold-palladium
(rGO-Au-Pd) nanocomposite, and electrode is modified using it.Selective oxidation graphene (GO) of the present invention and poly- second
Alkene pyrrolidone (protective agent) is used as dopant, and formic acid is as reducing agent, the reproducibility graphite oxide of synthesizing water-solubility and stabilization
Alkene-gold-palladium (rGO-Au-Pd) nanocomposite, obtained rGO-Au-Pd/GCE can due to bimetallic synergistic effect
Show best chemical property.The present invention is by being optimized rGO-Au-Pd/GCE experiment conditions, i.e., to synthesis side
The influence of method, concentration, reduction cycle and pH value to sensor performance is studied, and the specific surface of electrode is effectively raised
Product, increases the adsorbance of antibody, and enhance the electronic response speed of electrode and the electro-catalysis signal to test fluid, then
By suction-operated, fixed tumor markers CA242 antibody utilizes the specific quantification of antibody and antigen when being detected
In conjunction with so that electrode gradually reduces the current responsing signal of the potassium ferricyanide, to keep the CA242 immunosensors of preparation online
Property detection range, detection limit, sensitivity, specificity, stability, reliability and reproducibility etc. show excellent performance.
The technical solution adopted by the present invention is as follows：
The present invention provides a kind of for detecting being modified based on rGO-Au-Pd nanocomposites for tumor markers CA242
Novel unmarked electrochemical immunosensor preparation method, step is：
(1) preparation of rGO-Au-Pd composite materials solution
PVP and GO are dissolved in distilled water and stirred；By PdCl2、HAuCl4It is added in above-mentioned solution with HCOOH, 90
It is vigorously stirred at~100 DEG C until color becomes black from brown, centrifugation is washed using the mixture of ethyl alcohol and distilled water, is done
It is dry to get rGO-Au-Pd compounds, be suspended in buffer solution and rGO-Au-Pd solution be made；
(2) modification of glass-carbon electrode (GCE)
The cleaning of 2.1GCE：Alumina powder carefully grinds the surfaces GCE to remove oxide layer, after polishing using distilled water and
EtOH Sonicate is cleaned to remove any other physical absorption substance, dry.
The preparation of the anti-CA242/GCE of 2.2rGO-Au-Pd-：
GCE is placed in 6~10 μ L rGO-Au-Pd suspension obtained by step (1), it is dry, existed using cyclic voltammetry
N2It is scanned from -1.5V to 0V in saturation PBS so that rGO-Au-Pd is restored completely by electrochemistry；The drop coating 8 on the electrode obtained surface
The CA242 antibody-solutions of~12 μ L are incubated, and obtain the anti-CA242/GCE of rGO-Au-Pd-；Then rGO-Au-Pd- is anti-
CA242/GCE and 0.1% bovine serum albumin(BSA) (BSA) are incubated with to close other remaining sites, are preserved to get for detecting
The novel unmarked electrochemical immunosensor based on the modification of rGO-Au-Pd nanocomposites of tumor markers CA242.
Preferably, in step (1), the mass ratio (mg/mg) of the PVP and GO are (70~90)：(10~30), PVP and
GO mixings time in distilled water are 10~14h；
Preferably, in step (1), a concentration of 0.5mg/mL of the GO；
Preferably, in step (1), the PdCl2、HAuCl4It is (248~252) with HCOOH volume ratios (μ L/ μ L/ μ L)：
(280~284)：(348~352), wherein PdCl2A concentration of 56.4mM, HAuCl4A concentration of 50mM, wherein Au and Pd
Molar ratio is 1:1；
Preferably, in step (1), preferably 95 ± 2 DEG C of the reaction；
Preferably, in step (1), the centrifugation is 10,000rpm；The washing times are 3~4 times；
Preferably, in step (1), the buffer solution is the PBS of 2mg/mL；
Preferably, a concentration of 0.5~2mg/mL of rGO-Au-Pd solution described in step (1).
Further, it is preferred that a concentration of 2mg/mL of rGO-Au-Pd solution described in step (1).
Preferably, in the step 2.1, alumina powder is 0.3 and 0.05 μm；Scavenging period is 15~25min；It is described
Dry is dry in nitrogen；The glass-carbon electrode (GCE) is working electrode, a diameter of 3mm.
Preferably, in step 2.2, a concentration of 60 μ g/mL of CA242 antibody-solutions；Described be incubated was incubated for 4 DEG C
Night；It is preserved in the PBS for saving as pH=4~9 under the conditions of 4 DEG C.
Further, it is preferable that preserved in the PBS for saving as the pH=7 under the conditions of 4 DEG C.
Preferably, in step 2.2, the cyclic voltammetry scans 1~12 cycle；
Further, it is preferred that in step 2.2, the cyclic voltammetry scans 10 cycles；
The novel unmarked electrochemistry immuno-sensing for the rGO-Au-Pd nanocomposites modification that above-mentioned preparation method obtains
Device is also within protection scope of the present invention.
The present invention also provides the application of above-mentioned unmarked electrochemical immunosensor, the application is for tumor-marker
Quick, the Sensitive Detection of object CA242.
Beneficial effects of the present invention
1) graphene-gold-palladium based on reduction of the present invention for detecting tumor markers CA242 is nano combined
The novel unmarked electrochemical immunosensor of material modification is prepared simply, easy to operate, is realized to the quick, clever of sample
Quick, highly selective detection has market development foreground.
2) present invention is prepared for a kind of rGO-Au-Pd nanocomposites, and its first Application is passed in electro-chemistry immunity
In the preparation of sensor, using the bigger serface of rGO-Au-Pd, to increase the adsorbance of antibody, assisted by Au and Pd bimetallics
Same-action, high catalytic activity and high electron transport rate significantly improve response speed and reproduction of the electrode to the potassium ferricyanide
Property, the detection sensitivity of electrochemical sensor is finally substantially increased, there is important scientific meaning and application value.
3) in order to optimize the performance of immunosensor, the present invention has probed into the optimization of rGO-Au-Pd/GCE experiment conditions, i.e.,
Influence to synthetic method, concentration, reduction cycle and pH value to sensor performance is studied, and the CA242 of preparation is made to exempt from
Epidemic disease sensor shows excellent performance at linear detection range, detection limit, sensitivity, stability and reproducibility aspect, heavier
It wants, immunosensor, which detects the CA242 in human serum, has similar reproducibility and reliability.
Description of the drawings
Fig. 1 is the phenogram of composite material, and Figure 1A is the transmission electron microscope shape appearance figure of GO, and Figure 1B is composite material graphene-
The transmission electron microscope shape appearance figure of gold-palladium (rGO-Au-Pd), Fig. 1 C are GO, rGO-Pd, rGO-Au-Pd that x-ray diffraction method measures
Diffraction pattern, Fig. 1 D be differential pulse voltammetry test CA242 immunosensors preparation process, wherein a indicate test it is naked
GCE, b indicate test GO-Au-Pd, and c indicates test rGO-Au-Pd, after d indicates that BSA is incubated.
Fig. 2 is that the electro catalytic activity of naked GCE, rGO/GCE, rGO-Pd/GCE and rGO-Au-Pd/GCE electrode characterizes.
Fig. 3 is influence of the sweep speed of cyclic voltammetry to modified electrode, and wherein Fig. 3 A indicate that rGO-Au-Pd/GCE exists
Cyclic voltammetry scan curve in 10mM potassium ferricyanide solutions, for sweep speed from 10 to 100mV/s, Fig. 3 B indicate oxidation peak current
It is worth (Ipa), reduction peak current value (Ipc) with the square root (v of sweep speed1/2) linear fit curve.
Fig. 4 is the optimization for preparing CA242 immunosensor experiment conditions, and wherein Fig. 4 A indicate to prepare using different reducing agents
Electrode cyclic voltammetry curve, Fig. 4 B indicate the cyclic voltammetry curve of electrode prepared by the concentration of different modifying material, Fig. 4 C
Indicate that cyclic voltammetry curve of the electrode of different reduction number of turns preparations in the PBS full of nitrogen, Fig. 4 D indicate CA242 sensors
To the response current of 5U/mL CA242 in different pH solution.
Fig. 5 is that the immunosensor prepared probes into various concentration CA242 detections in PBS, and wherein Fig. 5 A are indicated should
To the current-responsive of the CA242 in 0.001-10000U/mL ranges in PBS, Fig. 5 B are indicated in the sensor PBS sensor
Detect the linear fit curve of CA242.Error bar=± relative standard deviation, n=5.
Fig. 6 is that the immunosensor prepared probes into various concentration CA242 detections in serum, and wherein Fig. 6 A are indicated should
Sensor in serum to the current-responsive of the CA242 in 0.001-100U/mL ranges, during Fig. 6 B are indicated in the sensor serum
Detect the linear fit curve of CA242.Error bar=± relative standard deviation, n=3.
Fig. 7 is that the selectivity of CA242 sensors for preparing, repeatability, stability are probed into.Wherein a, b, c, d curve point
It Wei not be to pure blood be clear, detects 5U/mL five realities of CA242 in 0.15mM ascorbic acid, 0.5mM uric acid and 0.15mM dopamines
Test the statistical analysis of result.
Fig. 8 is the novel unmarked electrochemistry modified based on rGO-Au-Pd nanocomposites for detecting tumor markers CA242
The preparation process of immunosensor.
Specific implementation mode
Feature of present invention and other correlated characteristics are described in further detail by the following examples, in order to the same industry
The understanding of technical staff：
1. chemicals and reagent
Graphene is bought from Xian Feng Nono-material Science & Technology Ltd. (Nanjing of China).Carbohydrate Antigens 24-2
(CA242) it is commercially available from Fitzgerald Industries International with anti-CA242 antibody.Polyvinyl pyrrole
Alkanone (PVP), palladium bichloride (PdCl2), gold chloride (HAuCl4), formic acid (HCOOH), ascorbic acid, uric acid and dopamine are purchased from
Sigma-Aldrich.All aqueous solutions are prepared using distilled water.
2. electrochemical measurement
Electrochemical measurement use equipped with M270 softwares EG&G283 potentiostats-current measurement electrochemical workstation into
Row.The electrochemical cell for being connected with standard three electrode system is used for all electrochemical measurements.Use the modification glass carbon electricity of diameter 3mm
Pole (GCE) is used as working electrode.Ag/AgCl (saturation KCl) electrode and platinum filament (1mm diameters) are used separately as reference electrode and right
Electrode.All electrochemistry experiments carry out at room temperature.
3. characterizing the nanocomposite prepared
Use Quanta-200 field emission microscope,s (FEI；Hillsboro, OR, USA) obtain transmission electron microscope figure
Picture.Using the Rigaku D/max-rA with Cu K α radiations (λ=1.5418) in diffractometer (Rigaku；Japan) on record X-
X ray diffration pattern x.
Embodiment 1
1, the preparation of rGO-Au-Pd composite materials solution
80mg PVP and 20mg GO (0.5mg/mL) are dissolved in distilled water 12 hours with continuous stirring.At room temperature
(molar ratio of Au and Pd are 1:1) by the 56.4mM PdCl of 250 μ L2, the 50mM HAuCl of 282 μ L4Add with the HCOOH of 350 μ L
Enter into the solution and is vigorously stirred.Mixture is set to be reacted at 95 DEG C until color becomes black from brown.By with 10,
000rpm centrifugal clarification black even solution, then at least three times using the washing of the mixture of ethyl alcohol and distilled water.It then will be molten
Liquid is dried in drying box to obtain rGO-Au-Pd compounds.The phosphate that compound is resuspended in groove type ultrasonic wave instrument is delayed
It rushes in brine (PBS, 2mg/mL).Simultaneously rGO and rGO-Pd is prepared using similar method.
2, the characterization of above-mentioned material and composite material
The pattern and structure of GO and rGO-Au-Pd nanocomposites respectively characterize it with transmission electron microscope.It is pure
Transparent membrane is presented in GO, shows that it is by ultrasonic uniform (Figure 1A) in aqueous solution.It modifies on graphene oxide film
Gold nano grain (AuNPs) and palladium nano-particles (PdNPs) average diameter be 5 nanometers (Figure 1B).
The crystal structure that nanocomposite is determined with x-ray diffraction method obtains spreading out for GO, rGO-Pd, rGO-Au-Pd
Penetrate pattern (Fig. 1 C).In the x-ray diffraction collection of illustrative plates of GO, (0 0 2) characteristic peak of GO is apparent that at 2 θ=10.6 °
Value.After HCOOH and electrochemical reduction processing, GO can be proved in the emerging wide diffraction maximum in 2 θ=22.7 ° by observing
It is reduced.This characteristic peak peak also appears in the X ray diffracting spectrum of rGO-Pd and rGO-Au-Pd, the above result shows that, it should
GO is successfully reduced to rGO by experimental method.The characteristic peak of rGO is wider, mainly due to that can be attributed to relatively short definition
The rGO laminations in domain and chip size.In the X-ray diffraction pattern of rGO-Pd, be located at 2 θ=40.118 °, 46.685 °, and
Diffraction maximum at 68.119 ° is generated by palladium nano-particles (111), (200), (220) corresponding lattice plane respectively.rGO-
Characteristic peak in the diffracting spectrum of Au-Pd simultaneously present in 2 θ=38.2 °, 38.2 °, 40.1 ° and 46.7 ° is alloy category
The characteristic peak of formation.These results indicate that this method is successfully prepared for rGO-Au-Pd nanocomposites.
In order to probe into the preparation process of CA242 immunosensors, inventor is in 10mMK3[Fe(CN)6] carry out difference arteries and veins
Rush volt-ampere test (Fig. 1 D).The current signal of GO-Au-Pd (b) and rGO-Au-Pd (c) is noticeably greater than naked GCE (a), and CA242 is anti-
After body is coated on modified electrode, current peak further decreases, and shows that antibody is successfully adsorbed in electrode surface (d).In order to hinder
Disconnected non-specific sites absorption, We conducted BSA incubations, electric current further decreases (e).Result above is mainly due to albumen
The obstruction of confrontation electronics transfer.These the result shows that CA242 immunosensors successful preparation.
Embodiment 2
1, electrode modification
(glass-carbon electrode) surfaces GCE are carefully ground using alumina powder (0.3 and 0.05 μm) first to remove oxide layer.
After polishing, GCE is cleaned 20 minutes to remove any other physical absorption substance using distilled water and EtOH Sonicate.Electrode is immediately
It dries under a nitrogen.Then it by the way that 8 μ LrGO-Au-Pd suspension (2mg/mL) are placed on the surfaces GCE, then does at room temperature
It is dry, with rGO-Au-Pd film process electrodes.Using cyclic voltammetry in N2Scan that (10 are followed to 0V from -1.5V in saturation PBS
Ring), rGO-Au-Pd is restored completely by electrochemistry.In order to obtain the anti-CA242/GCE of rGO-Au-Pd-, the 60 μ g/ of 10 μ L are added dropwise
The anti-CA242 of mL, and solution is incubated overnight at 4 DEG C.Then by the anti-CA242/GCE of rGO-Au-Pd- and 0.1% bovine serum albumin
(BSA) is incubated with to close other remaining sites in vain, generates the anti-CA242-BSA/GCE of rGO-Au-Pd-. rGO-Au-Pd-
Anti- CA242-BSA/GCE electrodes are stored in 4 DEG C when not in use.
2, the electro catalytic activity characterization of modified electrode
Cyclic voltammetry research naked GCE, rGO/GCE, rGO-Pd/GCE and rGO-Au- are used in iron cyanide system
Pd/GCE's is electroactive.In cyclic voltammetry experiment, each electrode at 280 and 170mV there are two specific redox peaks,
What this can be attributed to ferricyanide ions will definitely inverse Redox behavior (Fig. 2).Naked GCE, rGO/GCE, rGO-Pd/GCE and
The cathodic peak current value of rGO-Au-Pd/GCE is 82.0,100.9,133.3 and 142.6 μ A respectively.RGO-Au-Pd/GCE can
Best chemical property is shown mainly due to bimetallic synergistic effect.The microcosmic electroactive area of rGO-Au-Pd/GCE
Area is the 1.74 of blank GCE, rGO/GCE and rGO-Pd/GCE, 1.41 and 1.07 times respectively.The increase of electroactive area can
Can be the good conductivity due to rGO-Au-Pd nanocomposites, surface area causes greatly.Result above also shows rGO-Au-
Pd nanocomposites are suitble to prepare biosensor.
The calculating of microcosmic electro-active region
The microcosmic electroactive area of each composite material is calculated according to Randles-Sevcik formula：Ip=2.69 ×
105AD1/2n3/2γ1/2C, wherein Ip indicate peak point current, and A corresponds to the electro-active region of modified electrode, in D expressions and system
The relevant diffusion coefficient of molecule (about 6.7 × 10-6×10-6cm2s-1), n is electronics transfer quantity (constant), and γ is sweep speed
(50mV/s), C are the concentration (10mM) of probe molecule.
In addition, inventor has carried out power by studying influence of the sweep speed of cyclic voltammetry to modified electrode to it
Learn research.The electrochemical behavior of rGO-Au-Pd/GCE is in 10mM K3[Fe(CN)6] be scanned in solution, sweep speed from
10mV/s to 100mV/s changes, the results showed that the peak point current of redox reaction is linearly increased with the increase of sweep speed.
In addition, anode and cathode peak is respectively to more positively and negatively moving (Fig. 3 A).Based on these as a result, anode can be respectively obtained
(Ipa) and linear fit curve (Fig. 3 B) of cathode (Ipc) peak point current relative to the square root (v1/2) of sweep speed.?
The linear equation arrived is Ipa=13.79v1/2(mV/s)-4.20(R2=0.99675), Ipc=-18.09v1/2(mV/s)-
11.08(R2=0.99991).These result of calculations show:Modified electrode is a kind of controllable diffusion into the surface reaction, by surface material
Expect itself caused electrochemical behavior.
Embodiment 3 prepares the optimization of rGO-Au-Pd/GCE experiment conditions
Experiment condition can influence the chemical property of modified electrode, so as to cause the change final sensor of catalytic performance
Function.In order to optimize the detection result of CA242, the present invention is to the method for materials synthesis, concentration, reduction cycle and pH value to sensing
The influence of device performance is studied.
The influence of synthetic method, the present invention by with cyclic voltammetry in 5mM H2O2To rGO-Au-Pd/GCE's in solution
Chemical property is detected.The experimental results showed that H2O2Cathode peak occur at 100mV, and modified electrode exists
When HCOOH is as reducing agent more preferably catalytic performance (figure is shown as reducing agent than potassium iodide and hexadecylpyridinium chloride
4A)。
The influence of the concentration of material, the present invention are sent out by being analyzed it (Fig. 4 B) using cyclic voltammetry in PBS
Existing electrode surface is modified using 2mg/mL materials, best results.
In addition, influence of the reduction number of turns to the chemical property of rGO-Au-Pd/GCE, the present invention are existed using cyclic voltammetry
It is measured in 0.1M PBS.Resulting collection of illustrative plates shows that an apparent cathode occurs in (- 1.5-0V) in first lap curve
Peak, this is the result (Fig. 4 C) of reduction, this shows that rGO-Au-Pd is completely reduced.The redox peaks of rGO-Au-Pd/GCE
It tends towards stability after four reduction cycles, because, in order to ensure that complete and stable reduction rGO-Au-Pd/GCE each adopt by experiment
With 10 reduction cycles.
The pH value of solution plays a crucial role protein structure and function, and pH value of the present invention is to 5U/mL
The influence of the response current of CA242 is measured in 0.1M PBS.Response current with detection solution PBS pH value from 4.0 to
7.0 increase and increase, pH be 7.0 when reach maximum value, when pH value is higher reduce (Fig. 4 D).In pH 7.0,
CA242 antibody may have more activity, it is easier to interact with CA242.Therefore, to reach optimum efficiency, PBS is in pH
7.0 times for subsequent experimental.
Immunosensor performance is probed into CA242 detections in embodiment 4PBS
Under the experiment condition of optimization, in 10mM K3[Fe(CN)6] in the CA242 solution of various concentration is detected,
To probe into the performance without enzyme electrochemical immunosensor.Electro-chemistry immunity prepared by the test display of differential pulse voltammetry
The typical peak-current signal of transducer flex, and with the increase of CA242 concentration reduce (Fig. 5 A).Under this electric current
The phenomenon that drop is the electron transfer capacity that the protein gradually increased due to electrode surface inhibits electrode, and electric current is made gradually to drop
It is low.In 10mMK3[Fe(CN)6] in solution, electrode shows good electrochemical redox activity at 200mV, to make
To detect the current signal of CA242.The immunosensor of preparation has the extensive range of linearity (0.001-10000U/mL), inspection
Survey is limited to 0.067mU/mL (signal-to-noise ratio 3).In the range of linearity of CA242 detectable concentrations, concentration is measured with poor Pulse Voltammetry
Linear relationship, equation of linear regression I=-4.24log is presented in the electric current arrived10CCA242+ 52.874, Pearson's coefficient be-
0.992 (Fig. 5 B).Inventor is to the immunosensor range of linearity, detectable limit and sensitivity and the immune biography reported in the past
Sensor performance compares, and wherein linear detection range obviously expands (table 1).
Table 1 prepares the control of the electrochemical sensor for detecting tumor markers
Immunosensor performance is probed into CA242 detections in 5 human serum of embodiment
In order to evaluate the reliability of CA242 immunosensors, the present invention carries out various concentration CA242 in human serum sample
Detection, serum are diluted (pH 7.4) with 0.1M PBS, are analyzed using Differential Pulse Voltammetry (Fig. 6 A).Human serum is to each
Sample is detected three times, is as a result shown in 0.001-100U/mL ranges and linear change is presented.Calibration value is arrived 1.8%
In 2.7% range (n=3, Pearson's coefficient=- 0.995)；, the result shows that in human serum sample, preparation is used to examine for these
The immunosensor for surveying CA242 still has good reliability and reproducibility (Fig. 6 B).
Embodiment 6CA242 immunosensors have high reproducibility, highly selective and high stability
By five parallel detections to 5U/mL CA242, obtained current-responsive value analyzes CA242 immunosensors
Reproducibility.As a result show that detection relative standard deviation is 2.3%, this result illustrates the repeatability for preparing electrode very well
(Fig. 7 A).Ascorbic acid, uric acid and dopamine are most active electroactive components in blood of human body, are easy to Electrochemical Detection point
Analysis interferes.Therefore, in immunosensor, the present invention selects ascorbic acid, uric acid and dopamine to evaluate the sensor
Selectivity.0.15mM ascorbic acid (Fig. 7 B), 0.5mM uric acid (Fig. 7 C), opposite mark caused by 0.15mM dopamines (Fig. 7 D)
Quasi- deviation is respectively 4.4%, 2.0% and 3.8%.These results indicate that the immunosensor has good specificity.It prepares
Sensor when not in use, be stored in the PBS at 4 DEG C (pH value 7.0).It after storage one month, detects, remains current again
The 80.6% of response illustrates that prepared sensor has good stability.
Finally it should be noted that the foregoing is only a preferred embodiment of the present invention, it is not limited to this hair
It is bright, although the present invention is described in detail referring to the foregoing embodiments, for those skilled in the art, still
It can modify to the technical solution recorded in previous embodiment, or equivalent replacement is carried out to which part.It is all in this hair
Within bright spirit and principle, any modification, equivalent replacement, improvement and so on should be included in protection scope of the present invention
Within.Above-mentioned, although the foregoing specific embodiments of the present invention is described with reference to the accompanying drawings, not to the scope of the present invention
Limitation, those skilled in the art should understand that, based on the technical solutions of the present invention, those skilled in the art are not required to
Make the creative labor the various modifications or changes that can be made still within protection scope of the present invention.
Claims (10)
1. a kind of novel unmarked for detecting being modified based on rGO-Au-Pd nanocomposites for tumor markers CA242
The preparation method of electrochemical immunosensor, which is characterized in that including following preparation process：
(1) preparation of rGO-Au-Pd composite materials solution
PVP and GO are dissolved in distilled water and stirred；By PdCl2、HAuCl4It is added in above-mentioned solution with HCOOH, 90~100
It is vigorously stirred at DEG C until color becomes black from brown, centrifugation is washed using the mixture of ethyl alcohol and distilled water, dry, i.e.,
RGO-Au-Pd compounds are obtained, is suspended in buffer solution and rGO-Au-Pd solution is made；
(2) modification of glass-carbon electrode (GCE)
The cleaning of 2.1GCE：Alumina powder carefully grinds the surfaces GCE, then is cleaned with distilled water and EtOH Sonicate, dry；
The preparation of the anti-CA242/GCE of 2.2rGO-Au-Pd-：
GCE is placed in the rGO-Au-Pd suspension obtained by step (1), it is dry, using cyclic voltammetry in N2It is saturated in PBS
It is scanned from -1.5V to 0V so that rGO-Au-Pd is restored completely by electrochemistry；8~12 μ LCA242 of drop coating on the electrode obtained surface
Antibody-solutions are incubated, and obtain the anti-CA242/GCE of rGO-Au-Pd-；Then in the anti-CA242/GCE tables of the rGO-Au-Pd- of gained
Face drop coating 0.1%BSA solution, preserve to get for detect tumor markers CA242 based on the nano combined materials of rGO-Au-Pd
Expect the novel unmarked electrochemical immunosensor of modification；
A concentration of 2mg/mL of the rGO-Au-Pd solution；The cyclic voltammetry scans 10 cycles.
2. preparation method according to claim 1, which is characterized in that in step (1), the mass ratio of the PVP and GO
(mg/mg) it is (70~90)：(10~30), PVP and the GO mixing time in distilled water are 10~14h；The GO's is a concentration of
0.5mg/mL。
3. preparation method according to claim 1, which is characterized in that in step (1), the PdCl2、HAuCl4And HCOOH
Volume ratio (μ L/ μ L/ μ L) is (248~252)：(280~284)：(348~352), wherein PdCl2A concentration of 56.4~
57mM, HAuCl4A concentration of 50~51mM, Au and Pd molar ratio be 1:1.
4. preparation method according to claim 1, which is characterized in that in step (1), the reaction is 95 ± 2 DEG C；It is described
Centrifugation is 10,000rpm；The washing times are 3~4 times.
5. preparation method according to claim 1, which is characterized in that in step (1), the buffer solution be 1.5~
The PBS of 2.5mg/mL.
6. preparation method according to claim 1, which is characterized in that in step 2.1, the alumina powder is 0.3 He
0.05μm；Scavenging period is 15~25min；The drying is dry in nitrogen；The glass-carbon electrode (GCE) is work electricity
Pole, a diameter of 3mm.
7. preparation method according to claim 1, which is characterized in that in step 2.2, the rGO-Au-Pd suspension
Volume is 6~10 μ L.
8. preparation method according to claim 1, which is characterized in that in step 2.2, the CA242 antibody-solutions concentration
For 60~70 μ g/mL；Preferably, a concentration of 60 μ g/mL of the CA242 antibody-solutions；It is described to be incubated as 4 DEG C of overnight incubations；It is excellent
Selection of land preserves in the PBS for saving as the pH=7.0 under the conditions of 4 DEG C.
9. any preparation method of claim 1~8 obtain for detect tumor markers CA242 based on rGO-Au-
The novel unmarked electrochemical immunosensor of Pd nanocomposites modification.
10. the application of the novel unmarked electrochemical immunosensor described in claim 9, which is characterized in that the application is
Quick, Sensitive Detection for tumor markers CA242.
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| CN110632142A (en) * | 2019-08-28 | 2019-12-31 | 江南大学 | Preparation method and application of electrochemical biosensor based on gold palladium-graphene quantum dot composite material |
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| CN113655101A (en) * | 2021-07-23 | 2021-11-16 | 山东师范大学 | Electrochemical biosensor and preparation method and application thereof |
| CN117269288A (en) * | 2023-08-07 | 2023-12-22 | 山东师范大学 | Electrochemical biosensor for GLUT1 detection and preparation method thereof |
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|---|---|---|---|---|
| CN110632142A (en) * | 2019-08-28 | 2019-12-31 | 江南大学 | Preparation method and application of electrochemical biosensor based on gold palladium-graphene quantum dot composite material |
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| CN113655101A (en) * | 2021-07-23 | 2021-11-16 | 山东师范大学 | Electrochemical biosensor and preparation method and application thereof |
| CN117269288A (en) * | 2023-08-07 | 2023-12-22 | 山东师范大学 | Electrochemical biosensor for GLUT1 detection and preparation method thereof |
| CN117269288B (en) * | 2023-08-07 | 2024-04-19 | 山东师范大学 | Electrochemical biosensor for GLUT1 detection and preparation method thereof |
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