CN104614417A - Electrochemical method for detecting glycoprotein - Google Patents
Electrochemical method for detecting glycoprotein Download PDFInfo
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- CN104614417A CN104614417A CN201410053557.1A CN201410053557A CN104614417A CN 104614417 A CN104614417 A CN 104614417A CN 201410053557 A CN201410053557 A CN 201410053557A CN 104614417 A CN104614417 A CN 104614417A
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Abstract
The invention discloses an electrochemical method for detecting glycoprotein, which belongs to the field of chemistry. The method comprises preparation of a phenylboronic acid-biotin-nanogold compound, preparation of a working electrode of a substance to be detected and an electrochemical test. The method provided in the invention is high in sensitivity of glycoprotein detection, low in cost and good in stability. Compared with a conventional method, an antibody which is high in price and has mutability is not needed.
Description
Technical field
The present invention relates to a kind of detection method of glycoprotein, particularly a kind of electrochemical method detected for glycoprotein, belongs to chemical field.
Background technology
Glycoprotein is the important physiological activator of a class, participates in a lot of vital movement process and Metabolism regulation, plays a part multiple important.The glycosylated change of some protein surfaces usually associates and imply that the generation of some diseases, more such as just find to have certain relevance in recent years with the glycoprotein of related to cancer, contributed to the research and development of life science by analysis and resolution glycoprotein.In the industry cycle also actively finding and opening up efficient, low cost glycoprotein detection method for this reason, electrochemical sensor detection method development in recent years rapidly, is a kind of effective ways detecting protein at glycoprotein context of detection " sandwich " sensor (as enzyme linked immunosorbent assay).In this detection system, target protein can be caught by the antibody of electrode surface (primary antibodie), and then is identified and signal output captured protein by the antibody (two resist) of enzyme labeling.But often price is higher, preparation is more difficult for antibody and enzymic-labelled antibody, and easily sex change.Electrochemical sensor have easy to use, measuring accuracy is high, maintenance is simple and low cost and other advantages, has vast application prospect in bioanalysis.Therefore, it is very necessary for developing a kind of electrochemical sensor for the highly sensitive of glycoprotein and selective enumeration method.
Summary of the invention
The present invention is for providing a kind of electrochemical method detected for glycoprotein.
For the electrochemical method that glycoprotein detects, comprise the following steps:
(A) prepare phenyl boric acid-biotin-nano-Au composite, comprise following sub-step:
A1: the synthesis of nano Au particle, employing presoma is HAuCl
4, reductive agent is sodium citrate, by HAuCl
4solution is heated to boiling, then adds sodium citrate solution fast, continues ebuillition of heated 10-50 minute, then by solution cool to room temperature, namely obtains the nano Au particle solution that citric acid is stable;
A2: the synthesis of phenyl boric acid-biotin-nano-Au composite: take out above-mentioned nano Au particle solution with liquid-transfering gun, then the PBS solution comprising three (2-carboxyethyl) phosphines and polypeptide is added, the pH of PBS solution is 7-8, stirring at room temperature obtains the nm of gold of biotin modification for more than 1 hour, in reaction solution, add 4-mercaptophenyl boronic acid solution again, continue stirring and within more than 0.5 hour, obtain phenyl boric acid-biotin-nano-Au composite;
A3: the purifying of phenyl boric acid-biotin-nano-Au composite: the reaction product centrifuging that A2 is obtained, the unreacted polypeptide in reject upper strata and three (2-carboxyethyl) phosphine, the PBS solution of gained sediment with pH=7-8 is washed, phenyl boric acid-biotin-nano-Au composite PBS solution after the purifying obtained disperseed, Cord blood is for subsequent use;
(B) preparation of working electrode, comprises following sub-step:
B1: at the aptamer of gold electrode surfaces assembling sulfhydrylation;
B2: adopt 6-sulfydryl hexanol and bovine serum albumin to close unreacted gold electrode surfaces;
B3: the determinand containing glycoprotein is modified the electrode surface that B2 obtains;
B4: steps A is prepared phenyl boric acid-biotin-nano-Au composite modify the electrode surface that B3 obtains;
B5: Streptavidin-alkaline phosphatase multienzyme complex is modified the electrode surface that B4 obtains;
B6: the electrode obtained by B5 is soaked in p-APP solution, catalysis produces electroactive material p-aminophenol;
(C) electro-chemical test: the electrode that step (B) prepares is carried out electric performance test as working electrode.
Further, the pH value of steps A 2 and the PBS solution described in A3 is 7.4, and further, the aptamer described in step B1 is: 5'-SH-(CH
2)
6-GATTGAAAGGTCTGTTTTTGGGGTTGGTTTGGGTCAATA, further, in step (C), electro-chemical test adopts three-electrode system, the gold electrode of preparation is as working electrode, saturated Ag/AgCl electrode is contrast electrode, Pt electrode is auxiliary electrode, further, phenyl boric acid-biotin-nano-Au composite Cord blood that steps A 3 obtains is for subsequent use, described storage temperature is 0 DEG C-8 DEG C, further, phenyl boric acid-biotin-nano-Au composite Cord blood that steps A 3 obtains is for subsequent use, and described storage temperature is 4 DEG C.
The Advantageous Effects of a kind of electrochemical method for glycoprotein detection provided by the present invention is: glycoprotein is rich in glycosyl, and glycosyl can form boric acid ester bond with phenyl boric acid interaction, adopt phenyl boric acid to replace two to resist and identify that glycoprotein cost is low specifically, selectivity is high, and the identification accuracy of the present invention to glycoprotein is high, the present invention adopts aptamer to catch glycoprotein, by the effect of boric acid ester bond, adopt phenyl boric acid-biotin-nano-Au composite to carry out derivative thus between being conducive to by biotin-avidin effect to glycoprotein and multiple Streptavidin-alkaline phosphatase complex molecule is fixed on electrode surface, method sensitivity of the present invention is high, detectability is low, selectivity is good, the present invention is expensive without the need to adopting, the antibody of changeableness, cost is lower, good stability.
Accompanying drawing explanation
The test philosophy figure of Fig. 1 to be glycoprotein recombinant human erythropoietin of the present invention be example.
Fig. 2 is the uv-visible absorption spectra figure of the stable nm of gold of citric acid and phenyl boric acid-biotin-nano-Au composite.
Fig. 3 is the transmission electron microscope phenogram of phenyl boric acid-biotin-nano-Au composite.
Fig. 4 is that working electrode is at process and the test result figure without rHuEPO modification step.
Test result figure when Fig. 5 is the rHuEPO modifying variable concentrations.
Fig. 6 is the linear relationship chart of peak current and rHuEPO concentration.
Fig. 7 is the selectivity test figure of sensor of the present invention.
Embodiment
In order to explain enforcement of the present invention more fully, provide embodiment of the present invention.These implement to be only the elaboration to this technique, and do not limit the scope of the invention, illustrate in the present invention by following examples, but be not limited to following embodiment, any change is included in technical scope of the present invention.Material in the present invention representated by various abbreviation is respectively: TCEP: three (2-carboxyethyl) phosphine, CALNNGK (biotin) G: polypeptide, PBS: phosphate buffer, MBA:4-mercaptophenyl boronic acid, rHuEPO: glycoprotein recombinant human erythropoietin, BSA: bovine serum albumin, MCH:6-sulfydryl hexanol, p-APP:4-4-aminophenyl phosphate salt, SA-ALP: Streptavidin-alkaline phosphatase multienzyme complex; MM, μM, pM, be concentration unit, represent 10 respectively
-3mol/L, 10
-6mol/L, 10
-12mol/L, μ L: microlitre, Tris: English name: Tris (hydroxymethyl) aminomethane, Chinese another name: three (methylol) aminomethane; Tromethamine; N-tris hydroxymethyl aminomethane; Trishydroxymethylaminomethane.Following examples are specifically addressed using glycoprotein recombinant human erythropoietin as glycoprotein to be measured.In Fig. 2, curve a is the ultraviolet-ray visible absorbing curve of the stable nm of gold of citric acid, and curve b is the ultraviolet-ray visible absorbing curve of phenyl boric acid-biotin-nano-Au composite.Fig. 3 is the transmission electron microscope phenogram of phenyl boric acid-biotin-nano-Au composite.In Fig. 4, the concentration of rHuEPO is 10 pM, and sweeping speed is 0.1 V/s, and supporting electrolyte is 50 mM Na
2sO
4, arrow is depicted as direction of scanning.In Fig. 5, pulse height is 50 mV, and pulse width is 50 ms, and in Fig. 6, the concentration of rHuEPO is followed successively by 0.02,0.2,0.5,1 and 2 pM.Be electrode pair horseradish peroxidase (1) in Fig. 7, prostate specific antigen (2), metallothionein (3), Streptavidin (4), fibrin ferment (5), rHuEPO(6) and the response condition of 1 ~ 6 potpourri (7).The concentration of rHuEPO is 1 pM, and the concentration of 1 ~ 5 is 10 pM, the aptamer 5'-SH-(CH in the present invention
2)
6-GATTGAAAGGTCTGTTTTTGGGGTTGGTTTGGGTCAATA is known substance, concrete visible list of references " Analyst, 2010,135,2924-2929 ".
Embodiment 1:
(A): the synthesis of phenyl boric acid-biotin-nano-Au composite:
A1: adopt volume ratio to be the HNO of 1:3
3three-neck flask, spherical condensation tube, graduated cylinder clean up by/HCl mixed liquor, dry.The HAuCl of 50 mL 1 mM is added in three-neck flask
4, be heated to boiling, then add the sodium citrate solution of 5 mL 38.8 mM fast, continue ebuillition of heated 30 minutes, then by this red solution cool to room temperature;
A2: take out above-mentioned nano-Au solution 5 mL with liquid-transfering gun, then the PBS solution (pH 7.4) comprising 50 μMs of TCEP and 2 μM CALNNGK (biotin) G of 5 mL 1 mM is added, stirring at room temperature 2 hours, add the 4-mercaptophenyl boronic acid solution (MBA) of 0.1 mL 50 μMs again, continue stirring 2 hours;
A3: reactant is centrifugal under the rotating speed of 13000 rpm/min, reject upper strata unreacted CALNNGK (biotin) G and MBA.The PBS solution (pH 7.4) of the phenyl boric acid-biotin-nano-Au composite of gained with 5 mL 1 mM washed twice, finally this compound 5 mL PBS solution disperseed, 4 DEG C are stored in refrigerator for subsequent use.Synthesized phenyl boric acid-biotin-nano-Au composite adopts ultraviolet-visible spectrophotometer and transmission electron microscope to characterize, and the results are shown in Figure 2, Fig. 3;
(B): the preparation of working electrode;
B1: at the aptamer 5'-SH-(CH of gold electrode surfaces assembling sulfhydrylation
2)
6-GATTGAAAGGTCTGTTTTTGGGGTTGGTTTGGGTCAATA, for catching rHuEPO, being specially diameter is that the gold electrode of 2 mm soaks 12 hours in 1 μM of aptamer solution, then uses distilled water flushing electrode surface;
B2: adopt the unreacted surface of gold electrode that 6-sulfydryl hexanol (MCH) and the closed B1 of bovine serum albumin (BSA) obtains, the electrode obtained by B1 soaks 5 minutes in the MCH solution of 0.1 mM, and then soak 30 minutes in the BSA solution of 1 %, finally use distilled water flushing electrode surface;
B3: the rHuEPO solution of 20 μ L 100 pM is added drop-wise to the electrode surface that B2 obtains, reacts 30 minutes, then use distilled water flushing electrode surface, remove unreacted rHuEPO;
B4: phenyl boric acid-biotin-nano-Au composite prepared by 20 μ L steps A is added drop-wise to the electrode surface that B3 obtains, reacts and uses distilled water flushing electrode after 10 minutes, remove unreacted compound;
B5: 10 μ L, 1 μM of SA-ALP solution is added drop-wise to the electrode surface that B4 obtains, reacts and use distilled water flushing electrode after 10 minutes, remove unreacted SA-ALP;
B6: the electrode surface obtained by B5 is soaked in the p-APP solution of 30 μ L 0.5 mM, reacts and carries out cyclic voltammetry after 20 minutes.P-APP solution comprises the 4-4-aminophenyl phosphate salt of 0.5 mM, the Na of 50 mM
2sO
4, 10 mM trishydroxymethylaminomethane (Tris), pH is 7.4, uses front nitrogen saturated, removing solution in oxygen;
C: electro-chemical test:
Electrochemical Detection adopts three-electrode system, and the gold electrode of modification is as working electrode, and saturated Ag/AgCl electrode is contrast electrode, and Pt electrode is that auxiliary electrode carries out electro-chemical test.
Working electrode through rHuEPO and without rHuEPO modify embodiment and test result:
Adopt the working electrode that the Overall Steps preparation of embodiment 1 is modified through rHuEPO, adopt the working electrode that other step preparation in embodiment 1 except step B3 is modified without rHuEPO, test result as shown in Figure 4, curve a1 in Fig. 4, a2 group be working electrode through rHuEPO modify cyclic voltammetry result, curve a1 in Fig. 4, a pair redox peak on a2 derives from the redox of p-aminophenol, curve b1, b2 is the result of working electrode without the cyclic voltammetry of B3 step, comparison curves two groups can be learnt: the method may be used for the detection of glycoprotein.
Adopt the embodiment of different rHuEPO concentration:
Adopt other step identical, the concentration of rHuEPO in step B3 is changed, method of testing in step B6 is Differential Pulse Voltammetry, other step condition does not change, test result is as Fig. 5, shown in Fig. 6, the rHuEPO concentration (pM) that in Fig. 5, each curve adopts from top to bottom is respectively 0.02, 0.2, 0.5, 1, 2, 3, 10, as can be seen from Figure 5, oxidation current increases along with the increase of rHuEPO concentration, illustrate that the generation quantity of p-aminophenol depends on the concentration of glycoprotein, the rHuEPO concentration (pM) that in Fig. 6, each point adopts is respectively 0.02, 0.2, 0.5, 1, 2, as can be seen from Figure 6, strength of current linearly increases with the increase of rHuEPO concentration (0.02 ~ 2 pM), show that the method may be used for the quantitative detection of glycoprotein.
Response comparative example to other oroteins:
RHuEPO in step B3 is changed into the protein described in following 1,2,3,4,5,6 still adopt rHuEPO, 7 adopt be 1 ~ 6 potpourri, other step is identical, prepares each working electrode and tests, and the method for testing in step B6 is Differential Pulse Voltammetry, the concentration adopting rHuEPO is 1 pM, the concentration of other oroteins is 10 pM, and other step condition does not change, and test result corresponding to each sample as shown in Figure 7.In figure, 1,2,3,4,5 protein adopted are: 1: horseradish peroxidase, 2: prostate specific antigen, 3: metallothionein, 4: Streptavidin, 5: fibrin ferment, 6 is rHuEPO, the potpourri of 7:1 ~ 6, the concentration of rHuEPO is 1 pM, and the concentration of 1 ~ 5 is 10 pM.As can be seen from Figure 7, the electrochemical signals adopting 1 ~ 5 protein to produce is negligible, and in addition, in comparison diagram 76 and 7 can learn, the mensuration of protein 1 ~ 5 pair of glycoprotein is not disturbed, and illustrate that method of the present invention can selective enumeration method glycoprotein.
After detailed description embodiments of the present invention, the personage being familiar with this technology can be well understood to, do not departing under above-mentioned claim and spirit and can carry out various change and amendment, all above embodiment is done according to technical spirit of the present invention any simple modification, equivalent variations and modification, all belong to the scope of technical solution of the present invention, and the present invention is not also limited to the embodiment of example in instructions.
Claims (6)
1., for the electrochemical method that glycoprotein detects, it is characterized in that: comprise the following steps:
(A) prepare phenyl boric acid-biotin-nano-Au composite, comprise following sub-step:
A1: the synthesis of nano Au particle, employing presoma is HAuCl
4, reductive agent is sodium citrate, by HAuCl
4solution is heated to boiling, then adds sodium citrate solution fast, continues ebuillition of heated 10-50 minute, then by solution cool to room temperature, namely obtains the nano Au particle solution that citric acid is stable;
A2: the synthesis of phenyl boric acid-biotin-nano-Au composite: take out above-mentioned nano Au particle solution with liquid-transfering gun, then the PBS solution comprising three (2-carboxyethyl) phosphines and polypeptide is added, the pH of PBS solution is 7-8, stirring at room temperature obtains the nm of gold of biotin modification for more than 1 hour, in reaction solution, add 4-mercaptophenyl boronic acid solution again, continue stirring and within more than 0.5 hour, obtain phenyl boric acid-biotin-nano-Au composite;
A3: the purifying of phenyl boric acid-biotin-nano-Au composite: the reaction product centrifuging that A2 is obtained, the unreacted polypeptide in reject upper strata and three (2-carboxyethyl) phosphine, the PBS solution of gained sediment with pH=7-8 is washed, boric acid-biotin-nano-Au composite PBS solution after the purifying obtained disperseed, Cord blood is for subsequent use;
(B) preparation of working electrode, comprises following sub-step:
B1: at the aptamer of gold electrode surfaces assembling sulfhydrylation;
B2: adopt 6-sulfydryl hexanol and bovine serum albumin to close unreacted gold electrode surfaces;
B3: the determinand containing glycoprotein is modified the electrode surface that B2 obtains;
B4: phenyl boric acid-biotin-nano-Au composite steps A prepared modifies the electrode surface that B3 obtains;
B5: Streptavidin-alkaline phosphatase multienzyme complex is modified the electrode surface that B4 obtains;
B6: the electrode obtained by B5 is soaked in p-APP solution, catalysis produces electroactive material p-aminophenol;
(C) electro-chemical test: the electrode that step (B) prepares is carried out electric performance test as working electrode.
2. a kind of electrochemical method detected for glycoprotein according to claim 1, is characterized in that: the pH value of steps A 2 and the PBS solution described in A3 is 7.4.
3. a kind of electrochemical method detected for glycoprotein according to claim 1, is characterized in that: the aptamer described in step B1 is: 5'-SH-(CH
2)
6-GATTGAAAGGTCTGTTTTTGGGGTTGGTTTGGGTCAATA.
4. a kind of electrochemical method detected for glycoprotein according to claim 1, it is characterized in that: in step (C), electro-chemical test adopts three-electrode system, the gold electrode of preparation is as working electrode, and saturated Ag/AgCl electrode is contrast electrode, and Pt electrode is auxiliary electrode.
5. a kind of electrochemical method detected for glycoprotein according to claim 1, is characterized in that: phenyl boric acid-biotin-nano-Au composite Cord blood that steps A 3 obtains is for subsequent use, and described storage temperature is 0 DEG C-8 DEG C.
6. a kind of electrochemical method detected for glycoprotein according to claim 1, is characterized in that: phenyl boric acid-biotin-nano-Au composite Cord blood that steps A 3 obtains is for subsequent use, and described storage temperature is 4 DEG C.
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