CN104020194A - Melanoma biomarker detection method based on polypeptide - Google Patents
Melanoma biomarker detection method based on polypeptide Download PDFInfo
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- CN104020194A CN104020194A CN201410117664.6A CN201410117664A CN104020194A CN 104020194 A CN104020194 A CN 104020194A CN 201410117664 A CN201410117664 A CN 201410117664A CN 104020194 A CN104020194 A CN 104020194A
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Abstract
The invention discloses a melanoma biomarker detection method based on polypeptide. The method comprises the following steps: (1) pretreating a gold electrode: sanding the gold electrode with the diameter of 3mm sequentially with P3000 carborundum sandpaper and P5000 carborundum sandpaper, polishing the gold electrode with alumina powder, and carrying out ultrasonic cleaning for 5 minutes respectively with anhydrous ethanol and double-distilled water; (2) incubating the pretreated gold electrode and melanoma biomarkers S100B or biological samples of different concentrations for 2.5 hours at the temperature of 30 DEG C, meanwhile, incubating a 2.5micronM signal peptide solution and copper chloride at the temperature of 30 DEG C according to the concentration ratio of 1: 1, so as to form a signal peptide-copper ion complex, flushing the gold electrode, which is incubated with target protein or a biological sample, with double-distilled water, then, incubating the gold electrode and a signal peptide-copper ion complex solution for 2.5 hours at the temperature of 30 DEG C, and then, recording copper ion output signals. The method has good sensitivity and specificity and has the advantages of simplicity, low consumption and the like.
Description
Technical field
The present invention relates to a kind of detection method of label, particularly relate to a kind of detection method of the melanoma biomarker based on polypeptide.
Background technology
As a kind of tumour that has invasiveness and high mortality, melanomatous feature is mainly manifested in the unpredictability of branch mode and recurrence and the mortality risk of height, even after operative treatment, patient's recurrence and dead risk still exist.Therefore, monitor in time the progress of tumour and recur extremely important.Result of study in recent years shows, S100B (target protein) is independently melanoma biomarker of one, it not only with tumour by stages, the progress of disease and recur closely relatedly, and the prognosis information of melanoma patients can also be provided and predict corresponding result for the treatment of.Based on these unique biological significances of melanoma biomarker S100B, at present existing pertinent literature report: as melanomatous biomarker, S100B is obviously better than lactate dehydrogenase L DH, although lactic dehydrogenase is current unique label that is applied to melanoma clinical practice.
The content that detects melanoma patients peripheral blood S100B is the basis of realizing above-mentioned biological significance.Current existing detection method comprises enzyme linked immunosorbent assay (ELISA) and immunoluminescence analytic approach (LIA) etc., and the antibody that these methods all depend on high-affinity removes to identify target protein S100B.Rely on antibody recognition target protein and there is certain limitation, be mainly reflected in: (1) antibody, as a kind of albumen, has more complicated chemical constitution, thereby can not be by artificial synthetic realization.(2) need of production cell line or the animal of antibody, production run complexity and production cycle are longer; (3) the relative valency of antibody is expensive and unstable.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of detection method of the melanoma biomarker based on polypeptide, and it has good sensitivity and specificity, and has the advantages such as simple low consumption.
The present invention solves above-mentioned technical matters by following technical proposals: a kind of detection method of the melanoma biomarker based on polypeptide, it is characterized in that, and it comprises the following steps:
Step 1, the pre-service of gold electrode, the gold electrode of 3mm diameter is polished through the carborundum paper of P3000, P5000 successively, the alumina powder polishing of 1 μ m, 0.3 μ m, the ultrasonic cleaning of absolute ethyl alcohol and the each 5min of distilled water; Subsequently electrode is immersed in to the nitric acid 30min of Piranha solution 5min and 50%; Finally activate gold electrode by the sulfuric acid galvanochemistry of 0.5M; First with under 4 DEG C of conditions of 50 μ L signal peptide solution gold electrode through above-mentioned processing hatches 16 hours, under room temperature condition, electrode is immersed in subsequently to the occupy-place in 3 hours of 100 μ MMNH solution, with the non-specific adsorption on blocking electrode surface;
Step 2, by the melanoma biomarker S100B of pretreated gold electrode and variable concentrations or 30 DEG C of incubations of biological sample 2.5 hours; To under 2.5 μ M signal peptide solution and 30 DEG C of conditions of cupric chloride, press 1: 1 incubation of concentration ratio to form signal peptide-copper ion compound simultaneously; Rinse through distilled water with the gold electrode after target protein or biological sample incubation, subsequently with 30 DEG C of conditions of signal peptide-copper ion complex solution under incubation 2.5 hours, can record subsequently the output signal of copper ion.
Preferably, described gold electrode is immersed in 50 DEG C of water-bath 30min in the substrate solution that 1ml contains excessive o-phenylenediamine and 1ul HCL; Can in substrate reactions solution, record afterwards the output signal of the Diammonium phosphate (DAP) of tool electrochemical activity.
Positive progressive effect of the present invention is: the bio-identification of S100B and signal output all realize by simple polypeptide.At present increasing document thinks that polypeptide has lot of advantages, and these advantages make in some cases, and polypeptide is that one is more suitably selected than antibody.First, a lot of polypeptide can with the specific combination of corresponding target protein, this combination has the affinity of height, thereby can realize the bio-identification to target protein; Secondly, the chemical constitution of polypeptide is simply clear and definite, thereby can direct labor synthesize; Again, because polypeptide has simple chemical constitution, thereby we can be according to different need to further modifying polypeptide; Finally, by being combined with some metallic ion, polypeptide can be used as the carrier of signal output, and this compares with the mode signal output of relative traditional detection method, more simple.We with the polypeptide of target protein specific binding on modified a GHK motif, this motif can be used as the sequestrant of copper ion, copper ion has electrochemical activity, thereby chelating the polypeptide of copper ion bio-identification can be converted to electrical output signal.In addition, copper ion can also be served as the catalyzer of o-phenylenediamine oxidation, and reaction generates the diaminophenazine equally with electrochemical activity.The electrochemical signals amplification strategy being based upon on catalytic base obviously improves the detection sensitivity of this method.
Brief description of the drawings
Fig. 1 is detection principle schematic of the present invention.
Fig. 2 a be of the present invention catch electrode that peptide modifies respectively through variable concentrations S100B, 2.5 μ M complexings the SWV signal graph that obtains after hatching of the signal peptide of copper ion.Fig. 2 b is that 3nM S100B group and blank group scan respectively the CV signal curve that obtains DAP.
Fig. 3 a is the SWV signal graph that detects the DAP that variable concentrations S100B obtains.What Fig. 3 b showed is that the signal peak of SWV and the logarithm value of S100B concentration are linear dependence
Fig. 4 is that the SWV signal graph of DAP is in order to show the selectivity of this method to S100B, BSA and blank.
Fig. 5 detects the SWV signal graph of the DAP that perioperatively melanoma patients blood sample originally obtains.
Fig. 6 a catches peptide and 3nM S100B hatches the SWV signal curve of the DAP that different time obtains.What Fig. 6 b figure showed is the signal peak that different incubation times obtain.
Fig. 7 a combine the electrode of S100B and complexing the signal peptide of copper ion hatch the SWV signal curve of the DAP that different time obtains.Remaining experimental procedure and condition are all with Fig. 6 a.What Fig. 7 b figure showed is the signal peak that different incubation times obtain.
Fig. 8 a represents the SWV signal curve of the DAP that on electrode, copper ion catalysis obtains containing the substrate solution of variable concentrations OPD.Remaining experimental procedure and condition are all with Fig. 6 a.What Fig. 8 b showed is the signal peak that different OPD concentration obtains.Error bar has represented mean standard deviation (n=3).
Embodiment
Provide preferred embodiment of the present invention below in conjunction with Fig. 1 to Fig. 8, to describe technical scheme of the present invention in detail.
Identification polypeptide [k (MUA)-KRLRRSAHARKETEFLRLKRTRLGLE, freeze-dried powder, purity > 95%] and signal peptide [D (GHK)-KRLRRSAHARKETEFLRLKRTRLGLE, freeze-dried powder, purity > 95%] order in Shanghai Ke Tai biotech company, recombined human S100B (purity > 95%) is purchased from Yi Qiao Divine Land biotech firm, TCEP, MNH and BSA are purchased from sigma company, Spectra/Pro7 standard level regenerated cellulose dialysis membrane is purchased from spectrum company, it is pure that other reagent is analysis, without extra process, identification polypeptide and signal peptide freeze-dried powder are dissolved in 20mM Tris-Hcl (pH7.5).Target protein S100B freeze-dried powder is dissolved in the 20mM Tris-Hcl solution containing 1mM TCEP (pH7.5), then need to be configured to variable concentrations according to experiment.All solution all forms with distilled water configuration, and distilled water is to produce by the purification system of a Milli-Q, and its specificity impedance is greater than 18M Ω cm.
The detection method that the present invention is based on the melanoma biomarker of polypeptide comprises the following steps:
Step 1, the pre-service of gold electrode, the gold electrode of 3mm diameter is polished through the carborundum paper of P3000, P5000 successively, the alumina powder polishing of 1 μ m, 0.3 μ m, the ultrasonic cleaning of absolute ethyl alcohol and the each 5min of distilled water; Subsequently electrode is immersed in to the Piranha solution (concentrated sulphuric acid: 30%H2O2=3: 1) 5min and 50% nitric acid 30min; Finally activate gold electrode by the sulfuric acid galvanochemistry of 0.5M; First with under 4 DEG C of conditions of 50 μ L signal peptide solution gold electrode through above-mentioned processing hatches (containing 2.5 μ M signal peptides and 5mM TCEP, pH7.5) 16 hours, under room temperature condition, electrode is immersed in subsequently to the occupy-place in 3 hours of 100 μ M MNH solution (pH7.5), with the non-specific adsorption on blocking electrode surface;
Step 2, by the melanoma biomarker S100B of pretreated gold electrode and variable concentrations or 30 DEG C of incubations of biological sample 2.5 hours (should add lime chloride before hatching in incubation system is 5mM to final concentration, so that the combination of target protein and polypeptide); To under 2.5 μ M signal peptide solution and 30 DEG C of conditions of cupric chloride, press 1: 1 incubation of concentration ratio to form signal peptide-copper ion compound simultaneously; Rinse through distilled water with the gold electrode after target protein or biological sample incubation, subsequently with 30 DEG C of conditions of signal peptide-copper ion complex solution under incubation 2.5 hours, can record subsequently the output signal of copper ion.For signal is amplified, improve the sensitivity detecting, the gold electrode of above-mentioned processing is immersed in to 50 DEG C of water-bath 30min in the substrate solution that 1ml contains excessive OPD (o-phenylenediamine, 2.5mg) and 1ul HCL (hydrochloric acid); Can in substrate reactions solution, record afterwards the output signal of the DAP (Diammonium phosphate (DAP)) of tool electrochemical activity.
What all electrochemical gagings used is all CHI660D electrochemical workstation and traditional three-electrode system, comprising: saturated calomel electrode is contrast electrode, and platinum electrode is to electrode, and gold electrode is working electrode.For avoiding the interference of oxygen reduction, the buffer solution using before detecting is all through nitrogen stream deoxygenation 10min.The SWV signal of complex copper ion is to scan and obtain in 20mM Tris-Hcl damping fluid (pH7.5).Experiment parameter is respectively: sweep limit: 0.4～-0.1V, step electromotive force: 4mV, frequency, 15Hz, amplitude, 25mV.CV and the SWV signal of the diaminophenazine generating are to scan and obtain in substrate reactions solution, and experiment parameter is as follows: CV, sweep limit ,-0.24～-0.6V, speed, 100mV/s.SWV, sweep limit :-0.2～-0.6V, step electromotive force: 4mV, frequency, 15Hz, amplitude, 25mV.
Fig. 1 has explained the principle taking polypeptide as basic S100B detection scheme.S100B can be in 1: 2 mode in conjunction with 2 specific peptides.We are an end modified MUA of polypeptide therein, is fixed in electrode surface with identification target protein S100B, and at the end modified GHK motif of another polypeptide in order to export electrochemical signals.These two peptides are by difference called after identification polypeptide and signal peptide.In the situation that target protein S100B exists, the identification specific identification of Toplink in conjunction with target protein.Introduce subsequently and the signal peptide of copper ion incubation, the target protein that has been fixed on electrode assembling aspect can be further combined with the signal peptide of introducing, thereby forms the compound of a polypeptide-albumen-polypeptide.On signal peptide amino terminal, the copper ion of GHK motif complexing can be exported corresponding electrochemical signals, and this signal is proportional to the concentration of S100B.In order to improve the sensitivity of this detection method, the copper ion of complexing generates the product diaminophenazine with electrochemical activity as the catalyzer of o-phenylenediamine oxidation reaction, thereby realizes the object of amplifying as basic signal taking catalysis.Because the amount of complex copper ion is proportional to the amount of target protein S100B, the amount of the diaminophenazine of generation is also proportional to the amount of S100B, and in sum, we can construct a kind of S100B based on polypeptide and detect strategy.
Adopt square wave voltammetry (SWV) and cyclic voltammetry (CV) to verify the feasibility of this detection method.First we verified the electrochemical signals of complex copper ion, result as shown in Figure 2 a: in the situation that there is no target protein, in the sweep limit from 0.4V to-0.1V, there is not any signal.Along with the increase of target protein concentration, also increase successively the formation of the compound of prompting polypeptide-albumen-polypeptide from the typical SWV signal peak of complex copper ion.Subsequently we by the copper ion of complexing as the catalyzer of o-phenylenediamine oxidation reaction, result as shown in Figure 2 b: we have obtained the CV signal of the diaminophenazine that reaction generates.In addition we also replace blank target protein to repeat experiment, and result does not demonstrate any CV signal peak.This is because identification polypeptide is not in conjunction with target protein, naturally also there is no the follow-up chemical event such as target protein binding signal peptide.Our experimental result confirms that our detection method is feasible.
Part experiment condition is optimized.First, we have studied the incubative time of identification polypeptide and target protein, and the incubative time of 2.5 hours is best.Secondly, we also grope the incubative time of target protein and signal peptide, target protein and the abundant combination of signal Toplink under 2.5 hours conditions, finally, for the catalytic capability that makes copper ion performs to the best, we assess the amount of substrate o-phenylenediamine, and in order to ensure the abundant conversion of OPD, we have selected excessive OPD (2.5mg/ml) in experiment subsequently
Quantitatively detect the feasibility of target protein S100B in order to assess this detection method, we have introduced the target protein of variable concentrations and have recorded corresponding electrochemical signals, result is as shown in Figure 3 a: with the continuous increase of S100B concentration, the signal peak of typical SWV also increases gradually.There is a positive correlation clearly in both, detecting lower limit is 0.1nM within the scope of 0.1-25.6nM.For every kind of target protein concentration, we are independent experiment at least in triplicate, and average relative standard deviation < 5% shows that the quantified detection method of this S100B has good repeatability.
In order to verify the specificity of this method, we have carried out a series of control test, study the signal data obtaining in BSA and blank situation, result shows: compared with the SWV peak value of typical S100B, although the concentration of BSA (300nM) is far longer than the concentration (3nM) of target protein, BSA group demonstrates insignificant background signal, and blank group does not almost demonstrate signal reaction, and the specificity of prompting body series is good.
For melanoma patients, S100B is a very valuable label, and the detection that our method is applied to melanoma patients serum S100B level is very crucial.For this reason, we gathered melanoma patients before and after treatment blood sample this, and the content of S100B is wherein analyzed.The results are shown in Figure 4: the blood sample of pre-operative patients originally demonstrates an obvious signal peak, in prompting serum, there is the S100B of higher concentration, through effectively treatment, the obvious decline of the serum display peak value of postoperative patient.These data show that our detection method is feasible in clinical sample.
In sum, we have designed a kind of S100B detection method, and people can be according to different need to modifying simple polypeptide accordingly, and we have utilized this point fully, on signal peptide, modify a copper ion complexing motif, thereby realized generation and the amplification of signal.This detection method has good susceptibility, selectivity, and the simplicity of operation.To the blood sample of clinical patients originally, there is too good feasibility.
Those skilled in the art can carry out various remodeling and change to the present invention.Therefore, the present invention has covered various remodeling and the change in the scope that falls into appending claims and equivalent thereof.
Claims (2)
1. a detection method for the melanoma biomarker based on polypeptide, is characterized in that, it comprises the following steps:
Step 1, the pre-service of gold electrode, the gold electrode of 3mm diameter is polished through the carborundum paper of P3000, P5000 successively, the alumina powder polishing of 1 μ m, 0.3 μ m, the ultrasonic cleaning of absolute ethyl alcohol and the each 5min of distilled water; Subsequently electrode is immersed in to the nitric acid 30min of Piranha solution 5min and 50%; Finally activate gold electrode by the sulfuric acid galvanochemistry of 0.5M; First with under 4 DEG C of conditions of 50 μ L signal peptide solution gold electrode through above-mentioned processing hatches 16 hours, under room temperature condition, electrode is immersed in subsequently to the occupy-place in 3 hours of 100 μ MMNH solution, with the non-specific adsorption on blocking electrode surface;
Step 2, by the melanoma biomarker S100B of pretreated gold electrode and variable concentrations or 30 DEG C of incubations of biological sample 2.5 hours; To under 2.5 μ M signal peptide solution and 30 DEG C of conditions of cupric chloride, press 1: 1 incubation of concentration ratio to form signal peptide-copper ion compound simultaneously; Rinse through distilled water with the gold electrode after target protein or biological sample incubation, subsequently with 30 DEG C of conditions of signal peptide-copper ion complex solution under incubation 2.5 hours, can record subsequently the output signal of copper ion.
2. the detection method of the melanoma biomarker based on polypeptide as claimed in claim 1, is characterized in that, described gold electrode is immersed in 50 DEG C of water-bath 30min in the substrate solution that 1ml contains excessive o-phenylenediamine and 1ul HCL; Can in substrate reactions solution, record afterwards the output signal of the Diammonium phosphate (DAP) of tool electrochemical activity.
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| CN117147640A (en) * | 2023-11-01 | 2023-12-01 | 广东盈峰科技有限公司 | Method, system and use for producing microbial electrochemical sensors |
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| CN117147640A (en) * | 2023-11-01 | 2023-12-01 | 广东盈峰科技有限公司 | Method, system and use for producing microbial electrochemical sensors |
| CN117147640B (en) * | 2023-11-01 | 2024-02-20 | 广东盈峰科技有限公司 | Method, system and use for producing microbial electrochemical sensors |
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Application publication date: 20140903 |