CN102866139B - Establishment method based on surface plasma reinforcing energy transferring biosensor - Google Patents
Establishment method based on surface plasma reinforcing energy transferring biosensor Download PDFInfo
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Abstract
The invention provides an establishment method based on a surface plasma reinforcing energy transferring biosensor. The establishment method comprises the following steps of: 1) preparing a fluorescent gold nanoparticle cluster wrapped with protein; 2) preparing the gold nanoparticle modified by mercaptoethylamine; and 3) establishing an experiment system based on the surface plasma reinforcing energy transferring biosensor. The establishment method based on the surface plasma reinforcing energy transferring biosensor provided by the invention has the advantages that the fluorescent gold nanoparticle cluster wrapped with the protein and the gold nanoparticle modified by mercaptoethylamine are introduced to be served as the donor and the receptor for energy transferring; the concept of surface plasma reinforcing energy transfer is utilized to achieve quick, accurate, high-sensitivity and high-selectivity quantifying of the object liquaemin; synthesizing of the nanometer material in the method is simple in steps, removes harsh equipment and condition, is safe, simple and convenient to operate, low in toxicity, and low in cost; and the used synthesizing and detecting instrument and equipment are common equipment; and a reaction condition is simple; and the reaction can be simply carried out at a room temperature of water bath at 37 DEG C.
Description
Technical field
The present invention relates to inorganic nano material preparation and spectrum of use technical field, particularly a kind of construction method that strengthens the biology sensor of energy transfer based on surface plasma.
Background technology
Biology sensor refers to a kind of biochemical analysis device that approaches molecular dimension, can provide live signal when interacting with analyte.It is little that biology sensor has volume, cost is low, the advantage such as do not need pre-service and be not subject to that external electromagnetic field affects, be widely used in recent years life science, the research field such as material science and medical science, scientific-technical progress requires the development trend of biology sensor to be: higher sensitivity and lower detectability, better selectivity and material still less disturb, higher accuracy and better precision, more improve believable morphological analysis, higher analysis speed and automaticity, less sample size is implemented micro-damage or nondestructive analysis, implement original position (in situ), live body (in vivo), (real time) and online (on line) analyzes in real time, analysis device miniaturization, microminiaturized and intelligent etc.
Biology sensor based on FRET (fluorescence resonance energy transfer) (FRET) is widely studied, FRET (fluorescence resonance energy transfer) refer to when a pair of suitable material form an energy donor (donor) and energy acceptor (acceptor) to time, both standoff distances are in 1.0-10.0 nanometer, and the emission spectrum of donor and the absorption spectrum of acceptor can be effectively overlapping, excitation with donor, donor molecule by ground state transition after excited state, due to dipole-dipole interaction, donor molecule excited energy h ν is just likely passed to acceptor molecule with radiationless form effectively, then acceptor molecule by launching photon relaxation, there is fluorescence resonance energy.FRET (fluorescence resonance energy transfer) is as the optics in 1.0-10.0 nanometer distance range " molecule chi ", there is high resolving power, high sensitivity, the advantage such as simple and convenient, is applied in the research of detection of nucleic acids, immunoassay and interaction of biomacromolecules more and more widely.
Noble metal nano cluster refers under the protection of certain molecular layer, by several to the former molecular metastable aggregation of a hundreds of noble metal (golden or silver-colored).At present, utilize template, unimolecular layer Protection Code or part etching method, adopt the noble metal nano cluster that the protective agents such as mercaptan, dendritic, DNA, protein or template can synthesizing water-solubility be good, glow color is adjustable.Noble metal nano cluster diameter is less than 2 nanometers conventionally, between the noble metal of monatomic and nano particle or large volume.This yardstick approaches Fermi's wavelength of electronics, and now, continuous energy state character is split into discrete energy state, and occurs the Size dependence effect of similar molecule.When electronics is from valence band (5d
10) be energized into conduction band (6sp
1) time, noble metal nano cluster can be in visible range to the photoluminescent property that demonstrates Size dependence within the scope of near-infrared region.Compare with fluorescin with little molecular fluorescence dyestuff, fluorescence noble metal nano cluster as fluorescence probe have that photophysical property is good, specific surface area is large, anti-photobleaching, good biocompatibility, the advantage such as surface is easy to modify and photoluminescent property is adjustable, in fields such as compound test, bio-sensing and imaging, optoelectronics and nanosecond medical sciences, there is huge applications prospect.
Although the application of the biology sensor based on FRET (fluorescence resonance energy transfer) is comparatively ripe, but because its distinctive energy shifts coverage (1.0-10.0 nanometer), make to need when the design energy transfering system complicated surface-functionalized and for chemical bond and step between receptor body, cause development of new, biology sensor has been subject to larger restriction efficiently.This method aims to provide a kind of biology sensor construction method that strengthens energy transfer (SPEET) based on surface plasma, the energy transfer mode of this novelty is that the surface plasma volume property by noble metal significantly strengthens in system the energy transfer efficiency between donor and acceptor and expanded the coverage (22 nanometer) that energy shifts, and making to strengthen energy based on surface plasma, to shift the biology sensor of design practical more flexibly.Introduce fluorogold nanocluster and golden nanometer particle respectively as donor and acceptor simultaneously, make the quantivative approach of biology sensor sensitiveer and accurate.Test the maximum excitation wavelength of gold nano cluster of synthetic protein coated in 520 nanometers, maximum emission wavelength is in 690 nanometers, the golden nanometer particle maximum absorption wavelength that mercaptoethylmaine is modified is in 520 nanometers, the maximum excitation wavelength of gold nano cluster and the maximum absorption wavelength of golden nanometer particle overlap, and in buffer medium, because surperficial positive and negative charge generation electrostatic adsorption makes both, distance further, these 2 strengthen as surface plasma the necessary condition that energy shifts.The golden nanometer particle of the gold nano cluster of protein coated and mercaptoethylmaine modification is mixed, there is the transfer of surface plasma enhancing energy and make system fluorescent quenching in both, when having liquaemin to exist, because liquaemin makes the golden nanometer particle generation gathering that the mercaptoethylmaine of positively charged is modified make its maximum absorption wavelength red shift compared with large negative charge density, the molecular configuration of simultaneously liquaemin polymerization long-chain has zoomed out the distance between golden nanometer particle that mercaptoethylmaine modifies and the coated gold nano cluster of albumen, thereby breaking the transfer of surface plasma enhancing energy recovers system fluorescence.
Summary of the invention
The object of the invention is for above-mentioned existing problems, provide a kind of and strengthen based on surface plasma the construction method that energy shifts biology sensor, this construction method is introduced fluorogold nanocluster and golden nanometer particle respectively as donor and acceptor simultaneously, makes the quantivative approach of biology sensor sensitiveer and accurate.
Technical scheme of the present invention:
Based on surface plasma, strengthen the construction method that energy shifts biology sensor, step is as follows:
1) preparation of the fluorogold nanocluster of protein coated:
In reaction vessel, add successively chlorauric acid solution and protein solution, in 37 ° of C water-baths, under lucifuge magnetic agitation, react after 10 minutes, dropwise adding concentration is the sodium hydroxide solution of 1mol/L, the pH of regulation system is 12.0, then continue reaction 36 hours, obtain the crude product of the fluorogold nanocluster of protein coated, by centrifugal 30 minutes of ultra filtration membrane ultrafiltration under the rotating speed of 6000 revs/min for crude product, discard lower clear liquid, the fluorogold nanocluster of the protein coated after separation is dispersed in the ultrapure water of 10 times of volumes again, under 4 ° of C, keep in Dark Place,
2) preparation of the golden nanometer particle that mercaptoethylmaine is modified:
In reaction vessel, add successively high purity water, mercaptoethylmaine solution and chlorauric acid solution, under magnetic agitation, the reaction of room temperature lucifuge is 20 minutes, then add rapidly the sodium borohydride solution of new configuration, vigorous stirring lucifuge reaction 30 minutes, the solution of gold nanoparticles that obtains the mercaptoethylmaine modification of claret keeps in Dark Place under 4 ° of C;
3) based on surface plasma, strengthen the experimental system structure that energy shifts biology sensor:
Phosphoric acid-acetic acid-borate buffer solution that solution of gold nanoparticles, ultrapure water and the pH that adds respectively mercaptoethylmaine to modify in one group of centrifuge tube is 5.0 is as test container, then in each test container, adding respectively concentration is liquaemin standard solution and the solution to be measured of 0.1 – 4.0 μ g/mL, mix rear room temperature reaction 20 minutes, the fluorogold nanocluster solution that adds protein coated in the most backward reaction system, reacts 20 seconds mensuration system fluorescence intensities afterwards.
Described protein is trypsase, human serum albumins, lysozyme, insulin or pepsin.
The concentration of described chlorauric acid solution is 25.4mmol/L, protein solution concentration is 30mg/mL, mercaptoethylmaine solution concentration is 213mmol/L, sodium borohydride solution concentration is 10mmol/L, the volume ratio of chlorauric acid solution and protein solution is 1:1, and the volume ratio of high purity water, mercaptoethylmaine solution, chlorauric acid solution and sodium borohydride solution is 37.5:0.4:2.23:0.01.
Described phosphoric acid-acetic acid-borate buffer solution concentration is 40mmol/L, and the weight ratio of phosphoric acid, acetic acid and boric acid is 98:60:62.
The volume ratio of the fluorogold nanocluster of solution of gold nanoparticles, ultrapure water, phosphoric acid-acetic acid-borate buffer solution and protein coated that described mercaptoethylmaine is modified is 300:1200:500:50.
Advantage of the present invention and effect:
Synthesizing of the nano material that the method is related, step is simple, do not need harsh equipment, condition, simple and safe operation, toxicity is little, cost is low, and used synthesizing with detecting instrument equipment is conventional equipment, and reaction conditions is simple, room temperature or 37 ° of C water-baths, do not have exacting terms requirement.The fluorogold nanocluster of the method introducing protein coated and the golden nanometer particle of mercaptoethylmaine modification are respectively as donor and the acceptor of energy transfer, and the theory realization that utilizes surface plasma enhancing energy to shift is quantitative for quick, the accurate and high sensitivity high selectivity of object liquaemin.
This method strengthens surface plasma the structure that theory that energy shifts is applied to biology sensor first, and given full play to synthesizing simply of noble metal nanometer material, luminescence efficiency is high, the advantages such as the stable and good biocompatibility of photoluminescent property, set up a kind of easy fast and the quantivative approach of high sensitivity high selectivity, overcome the synthetic complexity of nano material in conventional energy transfer method, the shortcomings such as donor acceptor selection condition harshness and energy transfer efficiency are low, can expand energy and shift theory as the application in biology sensor structure, in fields such as biochemical analysis and clinical diagnosises, there is great practice significance.
Accompanying drawing explanation
Fig. 1 is the fluorescence spectrum figure of this biology sensor.
Fig. 2 is the linearity test figure of this biology sensor.
Embodiment
Embodiment 1:
Based on surface plasma, strengthen the construction method that energy shifts biology sensor, step is as follows:
1) preparation of the fluorogold nanocluster of protein coated:
In 50mL round-bottomed flask, adding successively 10mL concentration is the chlorauric acid solution of 25.4mmol/L and the trypsin solution that 10mL concentration is 30mg/mL, in 37 ° of C water-baths, the reaction of lucifuge magnetic agitation is after 10 minutes, dropwise adding 1mL concentration is the sodium hydroxide solution of 1mol/L, the pH of regulation system is 12.0, then continue reaction 36 hours, obtain the crude product of the coated fluorogold nanocluster of trypsase, by centrifugal 30 minutes of ultra filtration membrane ultrafiltration under the rotating speed of 6000 revs/min for crude product, discard lower clear liquid, the fluorogold nanocluster that trypsase after separation is coated is dispersed in ultrapure water again, under 4 ° of C, keep in Dark Place,
2) preparation of the golden nanometer particle that mercaptoethylmaine is modified:
In 50mL round-bottomed flask, add successively the chlorauric acid solution that mercaptoethylmaine solution that 37.5mL high purity water, 400 μ L concentration are 213mmol/L and 2.23mL concentration are 25.4mmol/L, under magnetic agitation, the reaction of room temperature lucifuge is 20 minutes, then the sodium borohydride solution that the concentration that adds rapidly the new configuration of 10 μ L is 10mmol/L, vigorous stirring lucifuge reaction 30 minutes, the solution of gold nanoparticles that obtains the mercaptoethylmaine modification of claret keeps in Dark Place under 4 ° of C;
3) based on surface plasma, strengthen the experimental system structure that energy shifts biology sensor:
The solution of gold nanoparticles that adds respectively 300 μ L mercaptoethylmaines to modify in one group of 5mL centrifuge tube, 1200 μ L ultrapure waters and 500 μ L pH are that 5.0 concentration are that phosphoric acid-acetic acid-borate buffer solution of 40mmol/L is as test container, phosphoric acid in this buffer solution, the weight ratio of acetic acid and boric acid is 98:60:62, then in each test container, adding respectively concentration is liquaemin standard solution and the solution to be measured of 0.1 – 4.0 μ g/mL, mix rear room temperature reaction 20 minutes, in the most backward reaction system, add the coated gold nano cluster solution of 50 μ L trypsase, react 20 seconds mensuration system fluorescence intensities afterwards.
Embodiment 2:
Based on surface plasma, strengthen the construction method that energy shifts biology sensor, step and method is substantially the same manner as Example 1, and difference is that protein is bovine serum albumin(BSA), the corresponding coated fluorogold nanocluster of bovine serum albumin(BSA) that makes.
Embodiment 3:
Based on surface plasma, strengthen the construction method that energy shifts biology sensor, step and method is substantially the same manner as Example 1, and difference is that protein is lysozyme, the corresponding coated fluorogold nanocluster of lysozyme that makes.
Embodiment 4:
Based on surface plasma, strengthen the construction method that energy shifts biology sensor, step and method is substantially the same manner as Example 1, and difference is that protein is insulin, the corresponding coated fluorogold nanocluster of insulin that makes.
Embodiment 5
Based on surface plasma, strengthen the construction method that energy shifts biology sensor, step and method is substantially the same manner as Example 1, and difference is that protein is pepsin, the corresponding coated fluorogold nanocluster of pepsin that makes.
Fig. 1 is this biology sensor fluorescence spectrum figure, in figure, show: along with the increase of concentration of heparin, the fluorescence intensity of system increases, this is because concentration of heparin is higher, the maximum absorption wavelength red shift degree of the golden nanometer particle that mercaptoethylmaine is modified is larger, distance between golden nanometer particle and gold nano cluster is far away simultaneously, so surface plasma enhancing energy transfer efficiency is weakened, and system fluorescence is recovered.
Fig. 2 is the linearity test figure of this biology sensor, in figure, show: the biology sensor that this method strengthens energy transfer foundation based on surface plasma is limited to 0.05 μ g/mL for detecting of liquaemin, and linear detection range is that 0.1 – 4.0 μ g/mL are as Comparison of standards curve.
Claims (3)
1. based on surface plasma, strengthen the construction method that energy shifts biology sensor, it is characterized in that step is as follows:
1) preparation of the fluorogold nanocluster of protein coated:
In reaction vessel, add successively chlorauric acid solution and protein solution, in 37 ℃ of water-baths, under lucifuge magnetic agitation, react after 10 minutes, dropwise adding concentration is the sodium hydroxide solution of 1 mol/L, the pH of regulation system is 12.0, then continue reaction 36 hours, obtain the crude product of the fluorogold nanocluster of protein coated, by centrifugal 30 minutes of ultra filtration membrane ultrafiltration under the rotating speed of 6000 revs/min for crude product, discard lower clear liquid, the fluorogold nanocluster of the protein coated after separation is dispersed in the ultrapure water of 10 times of volumes again, at 4 ℃, keep in Dark Place,
2) preparation of the golden nanometer particle that mercaptoethylmaine is modified:
In reaction vessel, add successively high purity water, mercaptoethylmaine solution and chlorauric acid solution, under magnetic agitation, the reaction of room temperature lucifuge is 20 minutes, then add rapidly the sodium borohydride solution of new configuration, vigorous stirring lucifuge reaction 30 minutes, the solution of gold nanoparticles that obtains the mercaptoethylmaine modification of claret keeps in Dark Place at 4 ℃;
3) based on surface plasma, strengthen the experimental system structure that energy shifts biology sensor:
Phosphoric acid-acetic acid-borate buffer solution that solution of gold nanoparticles, ultrapure water and the pH that adds respectively mercaptoethylmaine to modify in one group of centrifuge tube is 5.0 is as test container, then in each test container, adding respectively concentration is liquaemin standard solution and the solution to be measured of 0.1 – 4.0 μ g/mL, mix rear room temperature reaction 20 minutes, the fluorogold nanocluster solution that adds protein coated in the most backward reaction system, reacts 20 seconds mensuration system fluorescence intensities afterwards;
Described protein is trypsase, human serum albumins, lysozyme, insulin or pepsin.
2. based on surface plasma, strengthen the construction method that energy shifts biology sensor according to claim 1, it is characterized in that: the concentration of described chlorauric acid solution is 25.4 mmol/L, protein solution concentration is 30 mg/mL, mercaptoethylmaine solution concentration is 213 mmol/L, sodium borohydride solution concentration is 10 mmol/L, the volume ratio of chlorauric acid solution and protein solution is 1:1, and the volume ratio of high purity water, mercaptoethylmaine solution, chlorauric acid solution and sodium borohydride solution is 37.5:0.4:2.23:0.01;
Described phosphoric acid-acetic acid-borate buffer solution concentration is 40 mmol/L, and the weight ratio of phosphoric acid, acetic acid and boric acid is 98:60:62.
3. based on surface plasma, strengthen the construction method that energy shifts biology sensor according to claim 2, it is characterized in that: the volume ratio of the fluorogold nanocluster of solution of gold nanoparticles, ultrapure water, phosphoric acid-acetic acid-borate buffer solution and protein coated that described mercaptoethylmaine is modified is 300:1200:500:50.
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| CN104801722A (en) * | 2015-03-13 | 2015-07-29 | 武汉理工大学 | Preparation method of human serum albumin gold nanoclusters |
| CN105537621B (en) * | 2016-01-14 | 2017-09-12 | 南阳师范学院 | A kind of golden nanometer particle preparation method using protein as reducing agent |
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| CN110125437A (en) * | 2019-05-29 | 2019-08-16 | 浙江工业大学 | A kind of preparation method of the size tunable gold nano grain of positive charge modification |
| CN113681020B (en) * | 2021-07-28 | 2023-08-25 | 湖北大学 | Composite material with protein adsorption resistance and photodynamic effect and preparation method thereof |
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