CN102866139A - 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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- CN102866139A CN102866139A CN2012103535589A CN201210353558A CN102866139A CN 102866139 A CN102866139 A CN 102866139A CN 2012103535589 A CN2012103535589 A CN 2012103535589A CN 201210353558 A CN201210353558 A CN 201210353558A CN 102866139 A CN102866139 A CN 102866139A
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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 can provide near molecular dimension, when interacting with analyte a kind of biochemical analysis device of live signal.It is little that biology sensor has a volume, cost is low, the advantage such as do not need pre-service and be not subjected 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 require 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 little damage or nondestructive analysis, implement original position (in situ), live body (in vivo), (real time) and online (on line) analyzes in real time, the 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 consist of an energy donor (donor) and energy acceptor (acceptor) to the time, both standoff distances are in the 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, because dipole-dipole interaction, donor molecule excited energy h ν just might be passed to acceptor molecule effectively with radiationless form, then fluorescence resonance energy namely occurs to acceptor molecule in relaxation by launching photon.FRET (fluorescence resonance energy transfer) is as the optics in the 1.0-10.0 nanometer distance range " molecule chi ", have 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.
The 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, but adopt the protective agent such as mercaptan, dendritic, DNA, protein or the template synthesizing water-solubility is good, glow color is adjustable noble metal nano cluster.Noble metal nano cluster diameter is usually less than 2 nanometers, between the noble metal of monatomic and nano particle or large volume.This yardstick is near Fermi's wavelength of electronics, and at this moment, continuous energy state character is split into discrete energy state, and the Size dependence effect of similar molecule occurs.When electronics from valence band (5d
10) be energized into conduction band (6sp
1) time, the noble metal nano cluster can demonstrate in the visible range photoluminescent property of Size dependence in 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 the surface is easy to modify and photoluminescent property is adjustable, have the huge applications prospect in fields such as compound test, bio-sensing and imaging, optoelectronics and nanosecond medical sciences.
Although the application based on the biology sensor of FRET (fluorescence resonance energy transfer) is comparatively ripe, but shift coverage (1.0-10.0 nanometer) so that when the design energy transfering system, need the surface-functionalized of complexity and for chemical bond and step between the receptor body, cause development of new, efficient biology sensor to be subject to larger restriction owing to its distinctive energy.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 the system energy transfer efficiency between the donor and acceptor and enlarged the coverage (22 nanometer) that energy shifts, and to shift the biology sensor of design practical more flexibly so that strengthen energy based on surface plasma.Introduce simultaneously fluorogold nanocluster and golden nanometer particle respectively as donor and acceptor, so that the quantivative approach of biology sensor is sensitiveer and accurate.The maximum excitation wavelength of the gold nano cluster of the protein coated that experiment is synthetic is 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 since surperficial positive and negative charge generation electrostatic adsorption so that both distances further, these 2 namely strengthen the necessary conditions that energy shifts as surface plasma.The golden nanometer particle that gold nano cluster and the mercaptoethylmaine of protein coated are modified mixes, surface plasma occurs and strengthens the energy transfer so that system fluorescent quenching in both, when having liquaemin to exist, so that occuring to assemble, the golden nanometer particle that the mercaptoethylmaine of positively charged is modified makes its maximum absorption wavelength red shift owing to the larger negative charge density of liquaemin, the molecular configuration of simultaneously liquaemin polymerization long-chain has zoomed out the distance between the gold nano cluster that golden nanometer particle that mercaptoethylmaine modifies and albumen coats, and strengthens that energy shifts so that system fluorescence recovers thereby break surface plasma.
Summary of the invention
The objective of the invention is for above-mentioned existing problems, provide a kind of and strengthen the construction method that energy shifts biology sensor based on surface plasma, this construction method is introduced fluorogold nanocluster and golden nanometer particle simultaneously respectively as donor and acceptor, so that the quantivative approach of biology sensor is sensitiveer and accurate.
Technical scheme of the present invention:
A kind of construction method based on surface plasma enhancing energy transfer 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, react after 10 minutes under the lucifuge magnetic agitation, dropwise add the sodium hydroxide solution that concentration is 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, crude product is used centrifugal 30 minutes of ultra filtration membrane ultrafiltration under 6000 rev/mins rotating speed, discard lower clear liquid, the fluorogold nanocluster of the protein coated after separating is dispersed in the ultrapure water of 10 times of volumes again, under 4 ° of C, keeps in Dark Place;
2) preparation of the golden nanometer particle of mercaptoethylmaine modification:
In reaction vessel, add successively high purity water, mercaptoethylmaine solution and chlorauric acid solution, the reaction of room temperature lucifuge is 20 minutes under the magnetic agitation, then the sodium borohydride solution that adds rapidly new configuration, vigorous stirring lucifuge reaction 30 minutes, namely obtain the solution of gold nanoparticles of the mercaptoethylmaine modification of claret, under 4 ° of C, keep in Dark Place;
3) strengthen the experimental system structure that energy shifts biology sensor based on surface plasma:
Solution of gold nanoparticles, ultrapure water and the pH that adds respectively the mercaptoethylmaine modification in one group of centrifuge tube is that phosphoric acid-acetic acid of 5.0-borate buffer solution is as test container, then adding respectively concentration in each test container 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, add the fluorogold nanocluster solution of protein coated in the most backward reaction system, react 20 seconds afterwards mensuration system fluorescence intensities.
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, the 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 get final product, and do not have the 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 the 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 the biology sensor structure, have great practice significance in fields such as biochemical analysis and clinical diagnosises.
Description of drawings
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:
A kind of construction method based on surface plasma enhancing energy transfer biology sensor, step is as follows:
1) preparation of the fluorogold nanocluster of protein coated:
Adding successively chlorauric acid solution and the 10mL concentration that 10mL concentration is 25.4mmol/L in the 50mL round-bottomed flask is the trypsin solution of 30mg/mL, the reaction of lucifuge magnetic agitation is after 10 minutes in 37 ° of C water-baths, dropwise add the sodium hydroxide solution that 1mL concentration is 1mol/L, the pH of regulation system is 12.0, then continue reaction 36 hours, obtain the crude product of the fluorogold nanocluster of trypsase coating, crude product is used centrifugal 30 minutes of ultra filtration membrane ultrafiltration under 6000 rev/mins rotating speed, discard lower clear liquid, the fluorogold nanocluster that trypsase after separating is coated is dispersed in the ultrapure water again, keeps in Dark Place under 4 ° of C;
2) preparation of the golden nanometer particle of mercaptoethylmaine modification:
The mercaptoethylmaine solution and the 2.23mL concentration that add successively 37.5mL high purity water, 400 μ L concentration and be 213mmol/L in the 50mL round-bottomed flask are the chlorauric acid solution of 25.4mmol/L, the reaction of room temperature lucifuge is 20 minutes under the magnetic agitation, then the concentration that adds rapidly the new configuration of 10 μ L is the sodium borohydride solution of 10mmol/L, vigorous stirring lucifuge reaction 30 minutes, namely obtain the solution of gold nanoparticles of the mercaptoethylmaine modification of claret, under 4 ° of C, keep in Dark Place;
3) strengthen the experimental system structure that energy shifts biology sensor based on surface plasma:
In one group of 5mL centrifuge tube, add respectively the solution of gold nanoparticles that 300 μ L mercaptoethylmaines are modified, 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 adding respectively concentration in each test container 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, add the gold nano cluster solution that 50 μ L trypsase coat in the most backward reaction system, react 20 seconds afterwards mensuration system fluorescence intensities.
Embodiment 2:
A kind of construction method based on surface plasma enhancing energy transfer 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 fluorogold nanocluster that makes the bovine serum albumin(BSA) coating.
Embodiment 3:
A kind of construction method based on surface plasma enhancing energy transfer biology sensor, step and method is substantially the same manner as Example 1, and difference is that protein is lysozyme, the corresponding fluorogold nanocluster that makes the lysozyme coating.
Embodiment 4:
A kind of construction method based on surface plasma enhancing energy transfer biology sensor, step and method is substantially the same manner as Example 1, and difference is that protein is insulin, the corresponding fluorogold nanocluster that makes the insulin coating.
Embodiment 5
A kind of construction method based on surface plasma enhancing energy transfer biology sensor, step and method is substantially the same manner as Example 1, and difference is that protein is pepsin, the corresponding fluorogold nanocluster that makes the pepsin coating.
Fig. 1 is this biology sensor fluorescence spectrum figure, show among the figure: 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 the gold nano cluster is far away simultaneously, so surface plasma enhancing energy transfer efficiency is weakened, so that system fluorescence recovers.
Fig. 2 is the linearity test figure of this biology sensor, show among the figure: this method is limited to 0.05 μ g/mL based on the biology sensor that surface plasma strengthens energy transfer foundation for detecting of liquaemin, and linear detection range is that 0.1 –, 4.0 μ g/mL are as the Comparison of standards curve.
Claims (5)
1. one kind strengthens the construction method that energy shifts biology sensor based on surface plasma, 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 C water-baths, react after 10 minutes under the lucifuge magnetic agitation, dropwise add the sodium hydroxide solution that concentration is 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, crude product is used centrifugal 30 minutes of ultra filtration membrane ultrafiltration under 6000 rev/mins rotating speed, discard lower clear liquid, the fluorogold nanocluster of the protein coated after separating is dispersed in the ultrapure water of 10 times of volumes again, under 4 ° of C, keeps in Dark Place;
2) preparation of the golden nanometer particle of mercaptoethylmaine modification:
In reaction vessel, add successively high purity water, mercaptoethylmaine solution and chlorauric acid solution, the reaction of room temperature lucifuge is 20 minutes under the magnetic agitation, then the sodium borohydride solution that adds rapidly new configuration, vigorous stirring lucifuge reaction 30 minutes, namely obtain the solution of gold nanoparticles of the mercaptoethylmaine modification of claret, under 4 ° of C, keep in Dark Place;
3) strengthen the experimental system structure that energy shifts biology sensor based on surface plasma:
Solution of gold nanoparticles, ultrapure water and the pH that adds respectively the mercaptoethylmaine modification in one group of centrifuge tube is that phosphoric acid-acetic acid of 5.0-borate buffer solution is as test container, then in each test container, add respectively liquaemin standard solution and solution to be measured that concentration is 0.1-4.0 μ g/mL, mix rear room temperature reaction 20 minutes, add the fluorogold nanocluster solution of protein coated in the most backward reaction system, react 20 seconds afterwards mensuration system fluorescence intensities.
2. describedly according to claim 1 strengthen the construction method that energy shifts biology sensor based on surface plasma, it is characterized in that: described protein is trypsase, human serum albumins, lysozyme, insulin or pepsin.
3. describedly according to claim 1 strengthen the construction method that energy shifts biology sensor based on surface plasma, it is characterized in that: the concentration of described chlorauric acid solution is 25.4mmol/L, protein solution concentration is 30mg/mL, the 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.
4. describedly according to claim 1 strengthen the construction method that energy shifts biology sensor based on surface plasma, it is characterized in that: 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.
5. describedly according to claim 1 strengthen the construction method that energy shifts biology sensor based on surface plasma, 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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