CN109470757A - Electrochemical immunosensor electric signal marker and detection method for prostate specific antigen detection - Google Patents
Electrochemical immunosensor electric signal marker and detection method for prostate specific antigen detection Download PDFInfo
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Abstract
Electrochemical immunosensor electric signal marker for prostate specific antigen detection, electric signal marker is protease, the catalysis substrate of protease is a kind of polypeptide, and the sequence signature of polypeptide is: the third amino acid in cleavage site toward carbon teminal direction is histidine H.Included the following steps, A: secondary antibody/carbon nanotube/proteinase complex preparation for the detection method of prostate antigen detection using the above-mentioned electrochemical immunosensor electric signal marker for prostate specific antigen detection；B: the preparation of working electrode；C: working electrode is impregnated 2 hours in the PBS solution containing copper ion and polypeptide substrate, electro-chemical test is then carried out using linear sweep voltammetry.This electric signal marker electric signal is obvious, can meet the detection of prostate specific antigen concentration in human serum, and does not have to biphenyl substance in catalytic substrate, to staff and environmentally friendly.
Description
Technical field
The present invention relates to prostate specific antigen detection methods, in particular to the electrification for prostate specific antigen detection
Immunosensor signal tracer and detection method are learned, technological field of biochemistry is belonged to.
Background technique
Prostate specific antigen is a kind of biomarker of prostate class disease, it in serum the height of concentration with it is preceding
The generation of column gland cancer is closely related.For example, the concentration of prostate specific antigen is lower than 4 ng/mL in normal human serum, but work as it
Concentration is greater than 10 ng/mL, and prostate patient very likely suffers from prostate cancer.Therefore, the detection of prostate specific antigen exists
Have great importance in terms of the early diagnosis of prostate class disease.Method currently used for prostate specific antigen clinical detection
Mainly enzyme linked immunosorbent assay, but this method is needed using special instrument, and expensive, detection program is complicated, and
Testing cost is higher, and need to be using biphenyl compound as detection substrate, and biphenyl compound has certain carcinogenic work
With therefore, developing the novel method for prostate specific antigen detection and have a very important significance.
Electrochemical immunosensor has many advantages, such as that easy to use, measurement accuracy is high, maintenance is simple and at low cost, is detection
One of effective ways of protein have vast application prospect in bioanalysis.In this detection architecture, target protein
Matter can be by the antibody 1(primary antibody of electrode surface) capture, the antibody 2(secondary antibody then marked again by enzyme) to captured albumen
Matter carries out identification and signal output.Enzyme is the common electric signal marker of electrochemical immunosensor signal output, main at present
It to include catalase, glucose oxidase, alkaline phosphomonoesterase.However, these native enzymes are with some potential scarce
Point, for example, stability is poor, at high cost, preparation is more difficult.In addition, the metal catalytic center of catalase, glucose oxidase
It is buried among protein, is not easy that electron transmission directly occurs in electrode surface, and need using special enzymatic substrate
(such as hydrogen peroxide, glucose)；The catalysate oxidation resistance of alkaline phosphomonoesterase is poor, in air it is unstable,
Electric signal is weaker, polymer easy to form and passivated electrodes.These factors seriously limit electrochemical immunosensor in reality
In application.
Summary of the invention
It is an object of the invention to overcome the above problem present in current prostate specific antigen detection, one kind is provided
Electrochemical immunosensor electric signal marker and detection method for prostate specific antigen detection.
For the electrochemical immunosensor electric signal marker of prostate specific antigen detection, the telecommunications labelled notation
Object is protease, and the catalysis substrate of protease is a kind of polypeptide, and polypeptide can be cut by the protease；The sequence of polypeptide is special
Sign is: the third amino acid in cleavage site toward carbon teminal direction is histidine H；Further；The protease and prostate
The corresponding secondary antibody of specific antigen is combined to formation secondary antibody/carbon nanotube/proteinase complex in carbon nanotube.
For the detection method of prostate specific antigen detection, using the above-mentioned electricity for prostate specific antigen detection
Chemo-immunity sensor electric signal marker, comprising the following steps:
A: secondary antibody/carbon nanotube/proteinase complex preparation, including following sub-step:
A1: the pretreatment of carbon nanotube；
The nitric acid of effective 2 M of multi-wall carbon nano-tube is boiled 12 hours, it is cooling after centrifuge washing three times, then the multi wall that will be centrifuged
Carbon nanotube heats 12 hours in nitric/sulfuric acid mixed liquor, and heating temperature is 60 ~ 100 DEG C, then centrifuge washing is multiple, directly
To pH close to 7, shorter carbon nanotube is arrived into last centrifugation object vacuum drying；
A2: secondary antibody/carbon nanotube/proteinase complex preparation；
The carboxyl of the obtained carbon nano tube surface of step A1 is activated with EDC/NHS, centrifuge separation removes extra EDC and NHS,
Then secondary antibody is added, concussion reaction 1 as a child, adds protease and continues concussion 12 hours, by gained sediment secondary water
For centrifuge washing three times to get secondary antibody/carbon nanotube/proteinase complex is arrived, cryo-conservation is spare；
B: the preparation of working electrode
B1: modifying carboxylated graphene in glassy carbon electrode surface: carboxylated graphene solution be added drop-wise to glassy carbon electrode surface, from
So dry；
B2: the preparation of primary antibody modified electrode activates the obtained electrode of step B1 with EDC/NHS, then rinses electrode with secondary water
Primary antibody is added drop-wise to electrode surface by surface, rinses electrode with secondary water after 2 hours；
B3: the capture of prostate specific antigen soaks the obtained electrode of step B2 in the PBS solution containing prostate specific antigen
Bubble, then with the clean electrode surface of distilled water flushing, dry；
B4: secondary antibody/carbon nanotube/proteinase complex capture, the electrode that step B3 is obtained containing secondary antibody/carbon nanotube/
It impregnates in the PBS solution of proteinase complex, then with the clean electrode surface of distilled water flushing, dries；
C: the electrode that step B4 is obtained impregnates 2 hours in the PBS solution containing copper ion and polypeptide substrate, then uses line
Property scanning voltammetry carry out electro-chemical test.
Further, secondary antibody/carbon nanotube/spare, described guarantor of proteinase complex cryo-conservation that step A3 is obtained
Depositing temperature is 4 DEG C.
Further, the glass-carbon electrode diameter in step B1 is 3 mm.
Further, electro-chemical test uses three-electrode system in step C, and the electrode that step B4 is prepared is as work
Electrode.
Positive advantageous effects of the invention are: compared with current commercialized enzyme linked immunosorbent assay, the present invention
Method eliminate the detecting instrument of Large expensive.Due to the instrument and equipment small volume of electrochemical sensor, sensing electrode can
To be made as small-sized electrod-array, so the present invention is easy to detect, so that carrying out the solid door of prostate specific antigen detection
Sill substantially reduce, and can widely carry out this detection project, with report at present electric signal enzyme marker catalase, Portugal
Grape carbohydrate oxidase, alkaline phosphomonoesterase etc. are compared, and the catalysate electric signal of this electric signal marker is obvious, stability,
Sensitivity and detection limit etc. have all shown certain superiority, can meet prostate specific antigen in human serum completely
The detection of concentration, and biphenyl substance is not used in catalytic substrate, to staff and environmentally friendly.
Detailed description of the invention
Fig. 1 is using protease as the detection principle diagram of the electrochemical immunosensor of signal tracer.
Fig. 2 is that the product that secondary antibody/carbon nanotube/trypsase complex catalysts substrate polypeptide hydrolysis generates be not complexed
Mass spectrogram when object copper ion.
Fig. 3 is secondary antibody/carbon nanotube/trypsase complex catalysts substrate polypeptide hydrolysis generation product in complex compound copper
Mass spectrogram when ion.
Fig. 4 is primary antibody modified electrode in the prostate specific antigen and secondary antibody/carbon nanotube/protease by various concentration
After compound modification step, then impregnate in substrate solution 2 hours linear sweep voltammetry figures.The concentration of prostate specific antigen
It is followed successively by 0,0.01,0.1,0.5,1,2,5,10,20 ng/mL.
Fig. 5 is the peak current of sensor and the linear relationship chart of prostate specific antigen concentration.
Fig. 6 is the linear sweep voltammetry figure of sensor measurement different proteins.
Fig. 7 is the linear sweep voltammetry figure of prostate specific antigen in sensor measurement serum.
Specific embodiment
In order to more fully explain implementation of the invention, embodiment of the invention is provided.These embodiments are only
Elaboration to the technique, does not limit the scope of the invention, and is illustrated in the present invention with following embodiment, but be not limited to following implementations
Example, any change are included in technical scope of the invention.
Heretofore described electric signal marker is protease, and the protease is corresponding with prostate specific antigen
Secondary antibody/carbon nanotube/proteinase complex is formed in secondary antibody modification to carbon nanotube, the substrate of protease is a kind of polypeptide, should
The sequence signature of polypeptide is: the third amino acid in cleavage site toward carbon teminal direction is histidine H.Various abbreviation institutes in the present invention
The substance of representative is respectively as follows: EDC:(1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride)；NHS:N- hydroxyl amber
Amber acid imide；MES:(2- (N- morpholine) ethanesulfonic acid monohydrate)；PBS: phosphate buffer solution；PSA: prostate-specific is anti-
It is former.
Embodiment 1: secondary antibody/carbon nanotube/proteinase complex preparation
A1: the pretreatment of carbon nanotube；
The nitric acid of effective 50 mL, 2 M of 20 mg multi-wall carbon nano-tubes is boiled 12 hours, three times, centrifugation turns centrifuge washing after cooling
Speed is 14000 rpm, and each centrifugation time is 5 minutes.Again by the multi-walled carbon nanotube being centrifuged in 50 mL nitric/sulfuric acids
(volume ratio 1:3) mixed liquor heats 12 hours, and heating temperature is 60 ~ 120 DEG C, then centrifuge washing is multiple, until pH is close
7, finally centrifugation object is dried in vacuo to get shorter carbon nanotube is arrived；
A2: secondary antibody/carbon nanotube/proteinase complex preparation；
The carbon nanotube (1.5 mg) that A1 is obtained is added in 1 mL MES buffer solution, then ultrasound 30 minutes is added 1
ML contains 7.4,10 mM of MES(pH of 0.4 M EDC and 0.1 M NHS) in buffer solution, the extra EDC of centrifuge separation reject
And NHS, the secondary antibody of 150 μ L, 0.1 μ g/mL is added in centrifugal sediment thereto, concussion reaction 1 as a child, adds 150
The trypsase of 1 mg/mL of μ L continues concussion 12 hours, by gained sediment with secondary water centrifuge washing three times to get to two
Anti-/carbon nanotube/proteinase complex, is dispersed with 1 mL PBS buffer solution, and cryo-conservation is spare；
Embodiment 2: secondary antibody/carbon nanotube/proteinase complex activity analysis
By secondary antibody/carbon nanotube/proteinase complex obtained in 5 μ L step A2 and 100 containing 2 mM polypeptide GARGGH
μ L PBS buffer solution (7.4,0.2 M of pH) mixing, after room temperature reaction 1 hour, by reaction solution with or without 0.5 mM copper
The PBS of ion is diluted to 500 μ L, then is centrifuged under the revolving speed of 14000 rpm, and supernatant liquor carries out mass spectrometric measurement, mass spectrographic test
Model is negative ion model.Figure it is seen that synthesized secondary antibody/carbon nanotube/proteinase complex can be with catalytic polypeptide
GARGGH hydrolysis, the mass spectra peak (Fig. 2) that the main peak at 268.1035 Da is hydrolysate GGH, this shows modification to carbon nanometer
The trypsase that pipe shows can be hydrolyzed with catalytic polypeptide GARGGH；After copper ion is added, occur newly at 330.0253 Da
Mass spectra peak (Fig. 3) corresponds to GGH-Cu (II) complex compound, this shows that hydrolysate GGH and Cu (II) form complex compound.
Embodiment 3: response of the sensor to prostate specific antigen
B: the preparation of working electrode
B1: carboxylated graphene is modified in glassy carbon electrode surface, i.e., is dripped the carboxylated graphene solution of 5 μ L, 0.5 mg/mL
It is added to the glassy carbon electrode surface that diameter is 3 mm, naturally dry；
B2: the preparation of primary antibody modified electrode, i.e., the electrode obtained B1 are molten in the PBS buffering containing 0.4 M EDC and 0.1 M NHS
It is impregnated 15 minutes in liquid, electrode surface is rinsed well with secondary water, is dried with nitrogen；Then 10 μ L primary antibodies are dripped into electrode table
Face rinses electrode with secondary water after reaction 2 hours, then electrode is impregnated 30 in the PBS solution containing 0.1% BSA (w/v)
Minute, electrode is taken out, electrode surface is rinsed with secondary water, is dried with nitrogen；
B3: detecting the capture of object, i.e., the 10 μ L PBS solution for including a certain concentration prostate specific antigen is dripped to B2 and obtained
The electrode surface arrived rinses electrode with secondary water after reaction 30 minutes, is dried with nitrogen；
B4: secondary antibody/carbon nanotube/proteinase complex capture drips 10 μ L secondary antibodies/carbon nanotube/proteinase complex
The electrode surface obtained to B3 is dried with nitrogen with the clean electrode surface of distilled water flushing after reaction 30 minutes；
C: electro-chemical test；Electrode that B4 is obtained is contained into 0.5 mM copper ion and 0.5 mM polypeptide GARGGH in 30 μ L
It is impregnated in PBS solution 2 hours, electro-chemical test is then carried out using linear sweep voltammetry;
Electrochemical Detection uses three-electrode system, and the electrode that B4 is obtained is working electrode, and saturation Ag/AgCl electrode is reference electricity
Pole, Pt electrode are auxiliary electrode.Test results are shown in figure 4.Curve is that cyclic voltammetric of the working electrode Jing Guo B1-B3 step is surveyed
Test result, the oxidation peak in figure are generated by GGH-Cu (II) complex compound electro-catalysis water oxygen that enzymatic generates.From figure
In as can be seen that catalysis peak current increase with the increase of prostate specific antigen concentration, illustrate the GGH-Cu finally generated
(II) quantity of complex compound depends on the concentration of prostate specific antigen, and Fig. 5 is oxidation current and prostate specific antigen concentration
Relationship.From fig. 5, it can be seen that current strength is in line with the increase of prostate specific antigen concentration (0.01 ~ 2 ng/mL)
Property increase, show that this method can be used for the quantitative detection of prostate specific antigen.Detection is limited to 0.01 ng/mL.Result above
Show that the secondary antibody/carbon nanotube/proteinase complex may be used as the electric signal marker of electrochemical sensor.
To the response implementation example of different albumen:
Change prostate specific antigen in step B3 into other protein to be tested, the condition of other steps does not change, experiment
As a result as shown in Figure 6.From fig. 6 it can be seen that other oroteins do not generate catalysis peak.Therefore, this method property of can choose
Detect prostate specific antigen.Curve 1 ~ 5 in Fig. 6 is corresponding to be: IgG(immunoglobulin G), AFP (first tire egg
It is white), Thrombin(fibrin ferment), HSA(human albumin) and, PSA(prostate specific antigen).The concentration of prostate specific antigen
For 5 ng/mL, the concentration of other oroteins is 100 ng/mL.
Embodiment 4: the detection of prostate specific antigen in serum
Prostate specific antigen in step B3 is changed into human serum sample, with the prostate specific antigen containing different number when test
Serum is diluted 10 times by standard items, and the condition of other steps does not change, and experimental result is as shown in Figure 7.It can be seen from figure 7 that
When the concentration of the prostate specific antigen standard sample of addition is 0 ng/mL, blood serum sample produces a small catalysis peak,
Contain prostate specific antigen in the blood serum sample of surface, with the increase of the prostate specific antigen standard sample concentration of addition,
The catalysis peak-to-peak signal is remarkably reinforced.Surface this method can be used for the detection of prostate specific antigen in serum.According to the mark of Fig. 5
0.11 ng/mL of concentration of prostate specific antigen in the serum sample after diluting is calculated in directrix curve.
Trypsase is used into papain, corresponding polypeptide substrate still uses GARGGH, or by trypsase
Change dipeptidyl peptidase-IV into, corresponding polypeptide substrate is GPGGH, can obtain similar result.Using other protease and
Corresponding polypeptide, polypeptide can be cut by the protease, and the third amino acid in cleavage site toward carbon teminal direction is a group ammonia
Sour H should can be realized technical solution of the present invention.It is cheap and easy to get in view of trypsase, it is easier to clinically shape
At large-scale application, the application preferentially selects trypsase.
After the embodiment that the present invention will be described in detail, one of ordinary skilled in the art is clearly understood that, is not being taken off
It is lower from above-mentioned claim and spirit to carry out various change and modify, it is all according to the technical essence of the invention to the above reality
Any simple modification, equivalent change and modification made by example are applied, belong to the range of technical solution of the present invention, and the present invention is also not
It is limited to the embodiment of example in specification.
Sequence table
<110>Anyang Teachers College
<120>the electrochemical immunosensor electric signal marker and detection method for prostate specific antigen detection
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
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Gly Ala Arg Gly Gly His
1 5
<210> 2
<211> 5
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
Gly Pro Gly Gly His
1 5
Claims (6)
1. the electrochemical immunosensor electric signal marker for prostate specific antigen detection, it is characterised in that: described
Electric signal marker is protease, and the catalysis substrate of protease is a kind of polypeptide, and polypeptide can be cut by the protease, more
The sequence signature of peptide is: the third amino acid in cleavage site toward carbon teminal direction is histidine H.
2. the electrochemical immunosensor electric signal marker according to right 1 for prostate specific antigen detection,
Be characterized in that: protease secondary antibody corresponding with prostate specific antigen is combined to formation secondary antibody/carbon in carbon nanotube and receives
Mitron/proteinase complex.
3. being used for the detection method of prostate specific antigen, examined using of any of claims 1 or 2 for prostate specific antigen
The electrochemical immunosensor electric signal marker of survey, it is characterised in that the following steps are included:
A: secondary antibody/carbon nanotube/proteinase complex preparation, including following sub-step:
A1: the pretreatment of carbon nanotube；
The nitric acid of effective 2 M of multi-wall carbon nano-tube is boiled 12 hours, it is cooling after centrifuge washing three times, then the multi wall that will be centrifuged
Carbon nanotube heats 12 hours in nitric/sulfuric acid mixed liquor, and heating temperature is 60 ~ 100 DEG C, then centrifuge washing is multiple, directly
To pH close to 7, shorter carbon nanotube is arrived into last centrifugation object vacuum drying；
A2: secondary antibody/carbon nanotube/proteinase complex preparation；
The carboxyl of the obtained carbon nano tube surface of step A1 is activated with EDC/NHS, centrifuge separation removes extra EDC and NHS,
Then secondary antibody is added, concussion reaction 1 as a child, adds protease and continues concussion 12 hours, by gained sediment secondary water
For centrifuge washing three times to get secondary antibody/carbon nanotube/proteinase complex is arrived, cryo-conservation is spare；
B: the preparation of working electrode
B1: modifying carboxylated graphene in glassy carbon electrode surface: carboxylated graphene solution be added drop-wise to glassy carbon electrode surface, from
So dry；
B2: the preparation of primary antibody modified electrode activates the obtained electrode of step B1 with EDC/NHS, then rinses electrode with secondary water
Primary antibody is added drop-wise to electrode surface by surface, rinses electrode with secondary water after 2 hours；
B3: the capture of prostate specific antigen soaks the obtained electrode of step B2 in the PBS solution containing prostate specific antigen
Bubble, then with the clean electrode surface of distilled water flushing, dry；
B4: secondary antibody/carbon nanotube/proteinase complex capture, the electrode that step B3 is obtained containing secondary antibody/carbon nanotube/
It impregnates in the PBS solution of proteinase complex, then with the clean electrode surface of distilled water flushing, dries；
C: the electrode that step B4 is obtained impregnates 2 hours in the PBS solution containing copper ion and polypeptide substrate, then uses line
Property scanning voltammetry carry out electro-chemical test.
4. the detection method according to claim 3 for prostate specific antigen, it is characterised in that: step A3 is obtained
Secondary antibody/carbon nanotube/proteinase complex cryo-conservation is spare, and the storage temperature is 4 DEG C.
5. the detection method according to claim 3 for prostate specific antigen, it is characterised in that: the glass in step B1
Carbon electrode diameter is 3 mm.
6. the detection method according to claim 3 for prostate specific antigen, it is characterised in that: electrochemical in step C
It learns test and uses three-electrode system, the electrode that step B4 is prepared is as working electrode.
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