CN112067679A - Preparation method of nanogold modified electrode and application of nanogold modified electrode in hemoglobin biomolecule determination - Google Patents
Preparation method of nanogold modified electrode and application of nanogold modified electrode in hemoglobin biomolecule determination Download PDFInfo
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Abstract
The invention provides a preparation method of a nano-gold modified electrode and application of the nano-gold modified electrode in hemoglobin biomolecule determination. Gold nanoparticles are prepared by a sol method, and the gold nanoparticles are modified on the surface of a gold electrode to prepare a nano gold modified electrode which can be used for measuring bovine hemoglobin. The nanogold modified electrode has good biological compatibility, the adsorption of hemoglobin on the surface of the electrode is enhanced due to the large specific surface area of the nanogold, the electrochemical response of the hemoglobin is improved, the sensitivity of the hemoglobin is greatly improved, and the nanogold modified electrode can be used for preparing a specific biosensor.
Description
Technical Field
The invention belongs to the field of biomolecule detection sensors, and relates to a preparation method of a nano-gold modified electrode and application of the nano-gold modified electrode in hemoglobin biomolecule determination.
Background
Nanoparticles have quantum size effects and functionalization with high specific surface area, and have exceptional optical, electrical, magnetic and structural properties. When the biological conjugate is combined on the surface of a biological molecule, the generated biological conjugate species is similar to biological macromolecules due to size dependence and dimension, and is very suitable to be used as a large-circulation carrier for active magnetic resonance imaging and drug release/transportation and a structural framework for tissue engineering. And can be used in molecular recognition and labeling, DNA sensors and biochips. The gold nanoparticles become a hotspot for research due to good stability, small size effect, surface effect, optical effect and special biological affinity effect. In bioassays, gold nanoparticles have been used for DNA detection, immunodetection, and the like. Meanwhile, the biosensor prepared by assembling gold nanoparticles with different sizes on the surface of a solid matrix in a specific mode by adopting a self-assembly technology has great application potential.
Hemoglobin (Hb) is respiratory protein in vertebrate red blood cells, is the main substance for transporting oxygen in blood, and plays a role in transporting oxygen and decomposing H in organisms2O2The important activities related to oxygen and energy metabolism, such as electron transfer, play a key role in all life activities. Research on hemoglobin mostly focuses on its structure, oxygen transport function, and electron transfer process and mechanism between it and an electrode. The determination of the hemoglobin content in human serum is an important content of clinical detection, and the hemoglobin content in normal human serum is within the range of 100-200 g/L. If the hemoglobin content is reduced, anemia may be caused by various reasons, and the relative increase in the hemoglobin content is mainly caused by the loss of a large amount of body fluid and plasma, so that accurate measurement of the hemoglobin content is of great practical significance.
The electrochemical work on hemoglobin is realized by the action of a mediator or a promoter to catalyze the reaction on an electrode. The mediator is some electroactive substances modified on the electrode, mainly takes dye molecules as main components, and mainly plays a role in transferring electrons between the electrode and hemoglobin molecules. Promoters are a class of non-electroactive organic substances that do not themselves react at the electrode, but which can adsorb, the presence of which serves to accelerate the rate at which Hb exchanges electrons at the electrode.
In recent years, protein electrochemistry has been rapidly developed due to intensive studies on biological systems. The study of protein electrochemistry has also attracted the attention of scientists. For proteins with relatively small molecular weight and simple molecular structure, such as cytochrome C, hemoglobin, myoglobin and the like, the current response obtained on the electrode becomes simple. Therefore, it has become possible to detect proteins by electrochemical analysis. However, most of proteins have huge molecular structures, so that electroactive centers are embedded and strongly adsorbed and denatured on a conventional electrode, so that good electrochemical response is difficult to obtain on a common electrode. Therefore, electron mediators and promoters are continuously sought to obtain the effective electrochemical response of redox proteins. The chemically modified electrode with a special structure can accelerate the electron transfer reaction between the protein active center and the electrode surface, and can obtain better electrochemical response so as to improve the selectivity and sensitivity of analysis.
In conclusion, the invention provides a preparation method of a novel nano-gold modified electrode, and the nano-gold modified electrode is applied to the measurement of hemoglobin biomolecules.
Disclosure of Invention
The invention aims to provide a preparation method of a nano-gold modified electrode.
The method specifically comprises the following steps:
s1, preprocessing the gold electrode;
s2, mixing HAuCl4Heating the solution to boil, rapidly adding sodium citrate solution under vigorous stirring, stirring and reacting for 15min under boiling state, turning off the heat source, and continuously stirring to room temperature to obtain bright red nanogold solution;
s3, blowing the pretreated gold electrode to dry with nitrogen, dripping 1% aminoethanethiol solution to uniformly cover the gold electrode, placing the gold electrode in a drying oven at 35-50 ℃ for 10 hours, washing with secondary water, soaking the gold electrode in the nano-gold solution, and naturally drying the gold electrode to obtain the nano-gold modified electrode.
Further, in step S1, the preprocessing method specifically includes: polishing the gold electrode by using metallographic abrasive paper, and then sequentially using 1.0 and 0.3 mu m of Al2O3Polishing the slurry to a mirror surface, moving the mirror surface into an ultrasonic water bath for cleaning, and finally sequentially using 1: 1 ethanol and 1: 1HNO3And distilled water, and then 0.5mol/L H is put in2SO4Activating the solution by cyclic voltammetry, scanning the solution within the range of 1.0 to-1.0V, and repeatedly scanning the solution until a stable cyclic voltammogram is obtained.
Further, in the step S2, the HAuCl4And the mass ratio of the sodium citrate is 2: 7.
Tests show that the peak current of hemoglobin is gradually increased and the peak shape is good with the increase of the electrode modification time, but the peak current reaches a stable value and hardly increases when the modification time reaches 12 h. Because after more than 12h, a stable monomolecular film can be formed on the surface of the bare gold electrode. If the modification time is too short, the amount of the nanogold adsorbed on the surface of the electrode does not reach saturation, and the electrode is unstable; however, if the modification time is too long, the density of the film may increase, and the peak current may decrease. Therefore, the electrode modification time is preferably 12h in the invention.
Further, in the step S3, the soaking time is 11 to 12 hours, preferably 12 hours.
The invention also aims to provide an application of the nano-gold modified electrode in biomolecule determination, in particular to hemoglobin biomolecules.
Specifically, 0.2mol/L NaAc-HAc is used as a buffer solution, the pH value of the solution is adjusted to be 4.6, the scanning speed is set to be 100mV/s, the scanning range is 1.0 to-1.0V, and cyclic voltammetry detection is carried out.
FIG. 1 is a plot of cyclic voltammetry for hemoglobin on (a) gold electrodes, (b) thiol-modified electrodes, and (c) nanogold electrodes. Specifically, a gold electrode, a thiol modified electrode and a nano-gold modified electrode are arranged at 1 multiplied by 10-5Cyclic voltammetric scans were performed in mol/L hemoglobin solution (pH 4.6). As can be seen, hemoglobin has a very small reduction peak at the gold electrode, and the oxidation peak is not apparent. On the thiol modified electrode, the electrochemical reaction is almost completely inhibited, and the charges can not be transmitted through the thiol layer on the surface of the gold electrode. On the nano-gold modified electrode, hemoglobin with the same concentration has a pair of obvious redox peaks corresponding to heme prosthetic group Fe in hemoglobin3+/Fe2+Oxidation-reduction reaction of (1). Compared with a bare electrode, the peak current of the electrode is obviously increased, which shows that the nanogold can promote the electron transfer process between hemoglobin and the electrode, and that the nanogold modified electrode prepared by the method has stronger response capability in hemoglobin measurement.
FIG. 2 is a line of oxidation peak current and concentration of hemoglobin on the nano-gold modified electrodeAnd (4) a sexual relationship diagram. As can be seen from the graph, the hemoglobin concentration is 5.0X 10-5～5.0×10-3In the mol/L range, the oxidation peak current of the hemoglobin has a good linear relation with the concentration.
In conclusion, the nano-gold particles have good biocompatibility, hemoglobin has a strong adsorption effect on the nano-gold, and the nano-gold modified electrode has good catalytic action and enrichment effect on the hemoglobin. Hemoglobin can be effectively and stably transferred by direct electrons on the surface of the nano-gold modified electrode, a cyclic voltammetry curve shows a pair of redox peaks, and the electrode stability is good, so that nano-gold particles have a good detection effect on hemoglobin.
Meanwhile, the gold nanoparticles have good biocompatibility, do not damage the activity of enzymes and proteins in organisms, and provide a new way for the in-vivo detection of neurotransmitters. After the gold nanoparticles are self-assembled through thiol modification, the adsorption speed and the surface state of the particles are obviously influenced, so that the modified electrode has a better detection effect.
The gold nanoparticles are prepared by a sol method, and the gold nanoparticles are modified on the surface of a gold electrode to prepare a nano gold modified electrode, and the electrode can be used for measuring bovine hemoglobin. The nanogold modified electrode has good biological compatibility, the adsorption of hemoglobin on the surface of the electrode is enhanced due to the large specific surface area of the nanogold, the electrochemical response of the hemoglobin is improved, the sensitivity of the hemoglobin is greatly improved, and the nanogold modified electrode can be used for preparing a specific biosensor.
The invention has the beneficial effects that:
(1) the gold nanoparticles are prepared by a sol method and are prepared into the nano gold modified electrode, so that the sensitivity and the response speed are greatly improved.
(2) The nano-gold modified electrode prepared by the invention can be applied to the detection of hemoglobin biomolecules and can be used in the field of biomedical sensing.
(3) The preparation process is simple, the detection method is easy to operate, the nano material electrode can be controllably, repeatedly and conveniently processed, and the industrialization is facilitated.
Drawings
The invention is further illustrated by means of the attached drawings, but the embodiments in the drawings do not constitute any limitation to the invention, and for a person skilled in the art, other drawings can be obtained on the basis of the following drawings without inventive effort.
FIG. 1 is a plot of cyclic voltammograms of hemoglobin on (a) a gold electrode, (b) a thiol-modified electrode, and (c) a nanogold electrode;
FIG. 2 is a linear relationship graph of oxidation peak current and concentration of hemoglobin on the nano-gold modified electrode.
Detailed Description
In order that the objects, aspects and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the following detailed description and the accompanying drawings.
Example 1
Preparing a nano-gold modified electrode:
s1, polishing the gold electrode with metallographic abrasive paper, and sequentially using 1.0 and 0.3 mu m Al2O3Polishing the slurry to a mirror surface, moving the mirror surface into an ultrasonic water bath for cleaning, and finally sequentially using 1: 1 ethanol and 1: 1HNO3And distilled water, and then 0.5mol/L H is put in2SO4Activating the solution by cyclic voltammetry, scanning the solution within the range of 1.0 to-1.0 v, and repeatedly scanning the solution until a stable cyclic voltammogram is obtained;
s2, 150mL of 0.01% (m/v) HAuCl4Heating the solution to boiling, rapidly adding 5.25mL of 1% (m/v) sodium citrate solution under vigorous stirring, stirring and reacting for 15min under boiling state, turning off the heat source, and continuously stirring to room temperature to obtain bright red nano gold solution;
s3, blowing the pretreated gold electrode to dry with nitrogen, dripping 1% aminoethanethiol solution to uniformly cover the gold electrode, placing the gold electrode in a drying oven at 35-50 ℃ for 10 hours, washing with secondary water, placing the gold electrode in the nano-gold solution to soak for 12 hours, and naturally drying the gold electrode to obtain the nano-gold modified electrode.
Example 2
Preparing a nano-gold modified electrode:
s1, polishing the gold electrode with metallographic abrasive paper, and sequentially using 1.0 and 0.3 mu m Al2O3Polishing the slurry to a mirror surface, moving the mirror surface into an ultrasonic water bath for cleaning, and finally sequentially using 1: 1 ethanol and 1: 1HNO3And distilled water, and then 0.5mol/L H is put in2SO4Activating the solution by cyclic voltammetry, scanning the solution within the range of 1.0 to-1.0V, and repeatedly scanning the solution until a stable cyclic voltammogram is obtained;
s2, 150mL of 0.01% (m/v) HAuCl4Heating the solution to boiling, rapidly adding 5.25mL of 1% (m/v) sodium citrate solution under vigorous stirring, stirring and reacting for 15min under boiling state, turning off the heat source, and continuously stirring to room temperature to obtain bright red nano gold solution;
s3, blowing the pretreated gold electrode to dry with nitrogen, dripping 1% aminoethanethiol solution to uniformly cover the gold electrode, placing the gold electrode in a drying oven at 35-50 ℃ for 10 hours, washing with secondary water, placing the gold electrode in the nano-gold solution to soak for 11 hours, and naturally drying the gold electrode to obtain the nano-gold modified electrode.
Example 3
Preparing a nano-gold modified electrode:
s1, polishing the gold electrode with metallographic abrasive paper, and sequentially using 1.0 and 0.3 mu m Al2O3Polishing the slurry to a mirror surface, moving the mirror surface into an ultrasonic water bath for cleaning, and finally sequentially using 1: 1 ethanol and 1: 1HNO3And distilled water, and then 0.5mol/L H is put in2SO4Activating the solution by cyclic voltammetry, scanning the solution within the range of 1.0 to-1.0V, and repeatedly scanning the solution until a stable cyclic voltammogram is obtained;
s2, mixing 100mL of 0.01% (m/v) HAuCl4Heating the solution to boiling, rapidly adding 3.50mL of 1% (m/v) sodium citrate solution under vigorous stirring, stirring and reacting for 15min under boiling state, turning off the heat source, and continuously stirring to room temperature to obtain bright red nano gold solution;
s3, blowing the pretreated gold electrode to dry with nitrogen, dripping 1% aminoethanethiol solution to uniformly cover the gold electrode, placing the gold electrode in a drying oven at 35-50 ℃ for 10 hours, washing with secondary water, placing the gold electrode in the nano-gold solution to soak for 12 hours, and naturally drying the gold electrode to obtain the nano-gold modified electrode.
Example 4
Regeneration and reproducibility test of the nano-gold modified electrode:
0.2mol/L NaAc-HAc is used as buffer solution, the pH value is adjusted to 4.6, and 1 × 10 is added-5mol/L hemoglobin was juxtaposed to the nanogold-modified electrode prepared in example 1, and cyclic voltammetric scanning was performed at a scanning rate of 100mV/s in a potential range of-0.2V to 0.6V. The nanogold modified electrode prepared in example 1 was sampled and measured in parallel to have a concentration of 1X 10-5The Relative Standard Deviation (RSD) of the peak current value of the hemoglobin with mol/L is measured 8 times in parallel, and the Relative Standard Deviation (RSD) is 3.7 percent, which shows that the modified electrode has good reproducibility.
After the used electrode was washed with water and placed in a HAc-NaAc buffer solution having a pH of 4.6, the current response to hemoglobin was 98.7% of the initial current after one week, indicating that the electrode had good stability.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single technical solution, and such description is for clarity only, and those skilled in the art should take the description as a whole, and the technical solutions in the embodiments may be combined appropriately to form other embodiments that those skilled in the art can understand. The technical details not described in detail in the present invention can be implemented by any of the prior arts in the field. In particular, all technical features of the invention which are not described in detail can be achieved by any prior art.
Claims (7)
1. A preparation method of a nano-gold modified electrode is characterized by comprising the following steps:
s1, preprocessing the gold electrode;
s2, mixing HAuCl4Heating the solution to boil, rapidly adding sodium citrate solution under vigorous stirring, stirring and reacting for 15min under boiling state, turning off the heat source, and continuously stirring to room temperature to obtain bright red nanogold solution;
s3, blowing the pretreated gold electrode to dry with nitrogen, dripping 1% aminoethanethiol solution to uniformly cover the gold electrode, placing the gold electrode in a drying oven at 35-50 ℃ for 10 hours, washing with secondary water, soaking the gold electrode in the nano-gold solution, and naturally drying the gold electrode to obtain the nano-gold modified electrode.
2. The method for preparing the nano-gold modified electrode according to claim 1, wherein in the step S1, the pretreatment method specifically comprises: polishing the gold electrode by using metallographic abrasive paper, and then sequentially using 1.0 and 0.3 mu m of Al2O3Polishing the slurry to a mirror surface, moving the mirror surface into an ultrasonic water bath for cleaning, and finally sequentially using 1: 1 ethanol and 1: 1HNO3And distilled water, and then 0.5mol/L H is put in2SO4Activating the solution by cyclic voltammetry, scanning the solution within the range of 1.0 to-1.0V, and repeatedly scanning the solution until a stable cyclic voltammogram is obtained.
3. The method as claimed in claim 1, wherein in step S2, the HAuCl is added to the solution4And the mass ratio of the sodium citrate is 2: 7.
4. The method for preparing the nano-gold modified electrode according to claim 1, wherein in the step S3, the soaking time is 11-12 h.
5. The method for preparing the nano-gold modified electrode according to claim 4, wherein the soaking time is preferably 12 h.
6. Use of the nanogold-modified electrode according to claim 1 in a biomolecular assay.
7. The use of the nanogold-modified electrode according to claim 6, wherein the nanogold-modified electrode can be used for measurement of hemoglobin.
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