CN107101977A - Determine Quercetin and the surface plasma body resonant vibration analysis method of protein binding level - Google Patents
Determine Quercetin and the surface plasma body resonant vibration analysis method of protein binding level Download PDFInfo
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Abstract
Quercetin and surface plasma body resonant vibration (SPR) analysis method of protein binding level are determined the invention discloses a kind of, using the Interaction System of Quercetin and albumen as analysis target, the equilibrium constant that can be reacted in the hope of the interaction, i.e., with reference to level.In this method, diluted using pyridinium dissolution Quercetin and with the cushioning liquid containing pyridine, to solve Quercetin, solubility is not enough in aqueous, the problem of caused SPR analysis difficulties, the dilute solution of same composition but the pyridine without Quercetin is prepared analyze simultaneously during correspondingly in the Quercetin solution of prepared and diluted, for characterizing influence of the pyridine to spr signal, accurately to obtain the interaction signal of Quercetin generation.
Description
Technical field
The present invention relates to the application process of Applications of surface plasmon resonance in analytical chemistry, and in particular to one kind is used to survey
Determine the surface plasma body resonant vibration analysis method of Quercetin and protein interaction combination level.
Background technology
Surface plasmon resonance, referred to as SPR (surface plasmon resonance), are a kind of use extensively
In analysis, the analysis method of the intermolecular interaction of detection material, it can be used in macromolecular, small molecule, interionic phase interaction
Research.SPR general principle excites incident light first to utilize incident light irradiation SPR sensorgram chip under suitable conditions
The evanescent wave produced in incidence point and the resonance with the surface plasma-wave of censorchip surface thin metal layer so that output intensity
Weaken；Hereafter interaction process is occurred in censorchip surface, cause the change of SOLUTION PROPERTIES, cause the change of resonance condition
Change, and then outgoing light property changes, you can realize the monitoring of Thermodynamic parameters reaction.In a typical SPR experiment,
A reactant for participating in interaction reaction is often attached to SPR by modes such as covalent bond, electrostatic interaction, hydrophobic effects
Detection chip surface, the solution of another (or several) reaction partner is flowed through the detection chip surface during analysis.
When occurring interaction reaction, the surface of detection chip and the property of near surface solution change, and finally produce instrument
The response of signal.SPR is particularly suitable for use in determining the physical-chemical datas such as speed constant, the equilibrium constant of interaction reaction, can
It is measured with being not required to the system of mark ground in real time, quickly to unstable balance.
Quercetin (quercetin) also known as quercetin or Quercetin, are one kind in Flavonoid substances, its structural formula is such as
Under：
Quercetin is the glucoside member in various other Flavonoid substances glucoside structures such as rutin, is lived with a variety of physiological and pharmacologicals
Property.It has antioxidation activity, can be used anti-aging effects；It can significantly inhibit the growth of in vitro cancer cell, can be with
It is used as cancer therapy drug component；It can suppress platelet aggregation and suppress the effect of serotonin release, thus in cardiovascular and cerebrovascular
There is also application in the treatment of disease.When as medicine in use, Quercetin enter human body after need by with multiple proteins
Generation interaction is to realize its physiology, pharmacological function, for example, Quercetin transporting dependent on the people in it and blood in vivo
The Reversible binding of seralbumin (human serum albumin, HSA) with, its efficacy exertion also with itself and receptor protein
Reversible binding is relevant.Therefore, research Quercetin process relevant with protein interaction, particularly tries to achieve itself and multiple protein
The equilibrium constant of association reaction occurs for matter, often the basic data of the research such as its pharmacology, metabolism.
SPR methods are a kind of methods for being suitable for analyzing molecules interphase interaction, are particularly suitable between albumen-Quercetin
Quick analysis in real time.But Quercetin is practically insoluble in water and is soluble in organic solvent, therefore analysis presence of the SPR to Quercetin is tired
It is difficult.SPR analysis processes rely on the signal produced when analyte solution is flowed through into sensing chip, and its signal intensity depends on sensing
The size of the quality of (in about 50 nanometers) measured object near chip surface, therefore when analyte is the small-molecule substances such as Quercetin
When, it must have sufficiently large solubility in water environment, to form sufficiently strong signal.In addition, SPR analyses process often makes
Sample solution is prepared with the cushioning liquid containing inorganic salts to simulate physiological environment in organism, this has been further exacerbated by quercitrin
The problem of plain solubility is low.Had no in current patented method using SPR method research Quercetins and protein interaction method
Report, reason is Quercetin solubility in aqueous.Therefore, it is necessary to by the improvement of analytical procedure, improve Mongolian oak
Solubility of the Pi Su in water environment, and further develop the fast of the Quercetin based on SPR-protein interaction combination level
Fast analysis determining method.
The content of the invention
In order to make up the deficiency of existing method, Surface Plasmon Resonance Technology is based on it is an object of the invention to provide one kind
New interaction analyzing method, by adding organic solvent in a solvent to improve the solubility of Quercetin, realize to Mongolian oak
Pi Su and protein interaction combination level (i.e. the equilibrium constant of association reaction) measure.
Analysis method of the present invention based on surface plasma body resonant vibration (SPR) technology includes the cleaning of sensing chip, chip
The carboxylated modification on surface, protein solution pH screening, the activation of chip surface carboxyl, the covalent bond of protein, quercitrin
The steps such as the dissolving and dilution, the measure of protein and Quercetin interaction process, data analysis of element.Detailed process is as follows,
Each step must be carried out successively.
1st, the cleaning of sensing chip.
When SPR sensorgram chip (being usually gold-plated sheet glass) is Demountable, sensing chip is integrally immersed in
By concentrated ammonia liquor, 30% hydrogen peroxide, water by volume 1:1:In 5 solution mixed, it is placed in 60-95 DEG C of environment at least
10 minutes, then cleaned, used after drying with water.When sensing chip is that (i.e. metal level is directly plated on rib to non-dismountable formula structure
Mirror surface) when, above-mentioned solution is added dropwise in chip surface detection window position, is placed in less than 90 DEG C but as high as possible (does not damage
The structures such as prism) at a temperature of at least 30 minutes, surface is then cleaned with water and used after drying.Brand-new sensing can also be used
Chip.
It is preferred that, each analysis process uses brand-new sensing chip, to be coated with the sheet glass of exposed golden film, disposably makes
With being discarded after the completion of analysis.
2nd, the carboxylated modification of chip surface.
The sensing chip of cleaning is installed after SPR instruments, the solution for meeting following condition is flowed through chip surface：
Solute is the straight-chain carboxylic acid that a kind of sulfydryl replaces, and wherein sulfydryl is located at another end of carbochain away from carboxyl；Carbon chain lengths are
Containing three to 20 carbon atoms, i.e. mercaptopropionic acid, mercaptobutyric acid until one of sulfydryl arachic acid；Solute concentration is 2mmol/L
To 50mmol/L；Solvent is water and/or ethanol, depending on concentration needed for solute can be dissolved to by whichever.In the process of circulation in real time
Monitor spr signal, the currency, but at least 1 minute, solution flow rate was after sample introduction untill spr signal no longer changes
0.1 mul/min to 1 ml/min, depending on flow cell volume.Whole pipeline and core are rinsed with water after the completion of above step
Piece surface.Chip area temperature is 0 DEG C to 60 DEG C during circulation.
It is preferred that, using the 5mmol/L 3- mercaptopropionic acids aqueous solution with 5 mul/min of flow rate 30 minutes, chip
37 DEG C of controlling temperature with region, after the completion of with water flushing line and chip surface.
3rd, the pH screenings of protein solution.
The targeted target protein of this method is water soluble protein, and its isoelectric point is more than 4.Monitoring spr signal, makes in real time
Chip surface is flowed through with the solution for meeting following condition：Protein concentration is 1 nanograms/milliliter into 50 mg/mls
Some value；Solvent is acetic acid-sodium acetate buffer solution, and Na ion concentration is a value no more than 50mmol/L；PH is 2.5
At least three value into 6.0, scope must cover at least two pH units, and interval is at least 0.2, is at most 1 pH unit.That is,
The solution of circulation is several ankyrin species solution different with concentration, fixed buffer concentration but pH.Each solution circulation
The identical time, between 5 seconds and 60 minutes, flow velocity is identical, between 0.1 mul/min between 1 ml/min.Chip
Regional temperature is a value between 0 DEG C to 60 DEG C.The SPR when protein solution for comparing each different pH flows through the chip of carboxyl modified
The change of signal.If in the several pH value attempted, under the used currency, each solution makes spr signal reach surely
It is fixed, then select signal after stabilization to differ the pH of maximum solution with baseline as subsequent analysis；If under the currency used
Each solution does not make signal reach stabilization, then selection signal changes the pH of most fast solution as subsequent analysis；If part reaches
Steady component is not up to stabilization, then selection circulation completes time-ofday signals and the pH of maximum solution is differed with baseline as subsequent analysis.
After the completion of this step whole pipeline and chip are rinsed with water.
It is preferred that, protein is dissolved separately in Na ion concentration for 10mmol/L, pH is respectively 4.0,4.5,5.0,5.5
Acetic acid-sodium acetate buffer solution in, with 5 mul/min of flow velocity respectively circulation 5 minutes, 37 DEG C of temperature control, signal at the end of selection
Change the pH of maximum solution.
4th, the activation of chip surface carboxyl.
The carboxyl for the mercaptan carboxylic acid that one of following two methods modify chip surface is selected to be activated.
First, making the solution for meeting following condition flow through chip surface：Solute is EDC, i.e. N- (3- dimethylaminos third
Base)-N '-ethyl-carbodiimide hydrochloride and NHS, i.e. N- hydroxysuccinimides, both concentration are 0.0001 to 0.5 gram/
Milliliter, and EDC concentration is not less than NHS concentration, flow velocity between 0.1 mul/min between 1 ml/min, the currency with
Spr signal, which no longer changes, to be defined, but at least 1 minute.Chip area temperature is a value between 0 DEG C to 60 DEG C.
Second, successively making EDC solution and NHS solution flow through chip surface respectively, respective concentration range, flow velocity are same
On, the currency is no longer changed by respective spr signal to be defined, but is each at least 1 minute.Chip area temperature is 0
DEG C to a value between 60 DEG C.
It is preferred that, circulated 30 minutes with EDC and NHS mixed solution, both concentration are respectively 0.01 and 0.002 gram/milli
Rise, flow velocity is 5 mul/min.Temperature control is 37 DEG C.
5th, the covalent bond of protein.
By target protein with the buffer solution determined in the 3rd step, protein concentration be 1 nanograms/milliliter to 50 milligrams/
Between milliliter, then it is allowed to flow through chip surface, chip area temperature is a value between 0 DEG C to 60 DEG C.Flow velocity between
0.1 mul/min is defined between 1 ml/min, the currency is no longer changed by spr signal, but at least 1 minute.Complete
Afterwards with water flushing line and chip.
It is preferred that, using 0.01 grams per milliliter, the protein solution that pH is 5.0, with 5 mul/min of flow rates 20 minutes,
37 DEG C of temperature control.
6th, the dissolving and dilution of Quercetin.
Select phosphate buffer solution (PBS), Tris cushioning liquid (TRIS buffer, TBS) or
One of HEPES cushioning liquid (N-2- hydroxyethyl piperazine -2 '-ethanesulfonic acid buffers of-N ', HBS) solution, pH is 6.6 at 25 DEG C
To 8.5,3% to 15% pyridine (volume fraction) is added thereto.Hereafter other appropriate additives, such as ethylenediamine tetrem are added
Sour sodium (EDTA) and surfactant P20 etc., total mass fraction are no more than 5%.The retarder thinner of Quercetin is used as using this solvent.
Quercetin is dissolved in pure pyridine, concentration is a value in 1-50mmol/L.This is diluted with above-mentioned retarder thinner
The pyridine solution of Quercetin, at least obtains at least differing 20% between 3 various concentrations, and each concentration.Each dilution water contains
During the pyridine of Quercetin, the pure pyridine of a undissolved Quercetin is diluted with same ratio, the pyridine solution of various concentrations is obtained,
Its pyridine content and the solution containing Quercetin of above-mentioned dilution are same.Quercetin concentration is designated as c respectively in each solution1、c2、…
ci。
It is preferred that, 10mmol/LHEPES, 0.15mol/L sodium chloride, 3mmol/L (are contained with HBS-EP+ buffer solutions
EDTA, the surfactant P20 of 0.005% volume fraction, pH is the molten of 7.4) 8% (volume fraction) pyridine of middle addition at 25 DEG C
Liquid is diluent, and Quercetin is dissolved in into pure pyridine to 10mmol/L, be then diluted to 0.06 with this diluent, 0.10,0.15,
0.20th, 0.25,0.30mmol/L, dilutes the pure pyridine solution without Quercetin during dilution with same ratio every time.
7th, the measure of protein and Quercetin interaction process.
Circulation solution in SPR instrument pipelines is replaced with to Quercetin retarder thinner used in the 6th step.By in the 6th step
After the dilution of the Quercetin of each obtained concentration solution with it is corresponding respectively without Quercetin dilution pyridine solution according to by
The order of Quercetin is sequentially sent to SPR instruments and analyzed after dilute to dense, first pyridine.Sample flow rate between 0.1 mul/min extremely
Between 1 ml/min, between 0 DEG C to 60 DEG C of chip area temperature control.Tracer signal is until no longer change after each sample introduction, and
At least maintain 1 minute.Each sample introduction is until after the close stabilization of signal, stop sample introduction, with ten between mass fraction 1% to 10%
Sodium dialkyl sulfate (SDS) solution flushing line and chip are until hereafter signal again replaces with solution in pipeline close to stable
Retarder thinner used, is analyzed next time in 6th step.Each concentration is recorded for ciQuercetin solution sample introduction and signal
Signal value after stable, is designated as Ri, each and ciSame dilution, but be free from after the solution sample introduction of Quercetin and signal stabilization
Signal value be ri。
It is preferred that, 37 DEG C of temperature control, sample flow rate is 5 mul/min during analysis every time, and the SDS mass fractions used are
5%.
8th, data analysis.
Protein used is tried to achieve according to following steps, and the equilibrium constant of association reaction occurs with Quercetin.
A, as described in step 7, record each ciWhen RiAnd ri；
B, calculate each 1/ciWith 1/ (Ri-ri), and with 1/ (Ri-ri) to 1/ciMapping, 1/ciFor abscissa, 1/ (Ri-ri)
For ordinate.
C, linear fit is carried out to data point in figure, the intercept of gained equation is designated as b, and slope is designated as a, then association reaction
The equilibrium constant is b/a, the inverse of unit concentration unit used in experiment.
In the analysis method of the present invention, diluted using pyridinium dissolution Quercetin and with the cushioning liquid containing pyridine, to solve
Certainly solubility is not enough in aqueous for Quercetin, the problem of caused SPR analysis difficulties, while in prepared and diluted during analysis
Quercetin solution when prepared correspondingly same composition but without Quercetin pyridine dilute solution, for characterizing
Influence of the pyridine to spr signal, accurately to obtain the interaction signal of Quercetin generation.
Brief description of the drawings
Fig. 1：The present invention determines Quercetin and the analysis process of protein binding level using Applications of surface plasmon resonance
Schematic diagram.
Fig. 2：7th step in embodiments of the invention, HSA protein interacts with solution after each dilution containing Quercetin
SPR testing result figures, for trying to achieve each Ri。
Fig. 3：7th step in embodiments of the invention, HSA protein and each pyridine dilute solution for not containing Quercetin are mutual
The SPR testing result figures of effect, for trying to achieve each ri。
Fig. 4：8th step in embodiments of the invention, maps and linear fit result.
Embodiment
Below in conjunction with accompanying drawing, by embodiment, technical scheme is expanded on further, but the protection model of the application
Enclose and do not limited by the actual conditions of these embodiments.
Embodiment：Quercetin is determined using the analysis method in the present invention and human serum albumins (HSA) interaction is anti-
The equilibrium constant answered.Comprise the following steps that.
1st, using brand-new sensing chip, to be coated with the sheet glass of exposed golden film.It is installed into SPR instruments, starts instrument
Device, chip area temperature control maintains 37 DEG C.
2nd, using the 5mmol/L 3- mercaptopropionic acids aqueous solution with 5 mul/min of flow rate 30 minutes, chip area
37 DEG C of temperature control, after the completion of with water flushing line and chip surface.
3rd, HSA is dissolved in Na ion concentration for 10mmol/L, pH is respectively 4.0,4.5,5.0,5.5 acetic acid-acetic acid
In sodium cushioning liquid, respectively circulated 5 minutes with 5 mul/min of flow velocity.The pH of the maximum solution of signal intensity is at the end of circulation
5.5, so also using this pH cushioning liquid during rear protein covalent bond.
4th, circulated 30 minutes with EDC and NHS mixed solution, both concentration are respectively 0.01 and 0.002 grams per milliliter, stream
Speed is 5 mul/min.
5th, using 0.01 grams per milliliter, the HSA solution that pH is 5.0, with 5 mul/min of flow rates 20 minutes.
6th, with HBS-EP+ buffer solutions (containing 10mmol/LHEPES, 0.15mol/L sodium chloride, 3mmol/L EDTA,
PH is for the solution of 8% (volume fraction) pyridine of addition in 7.4) during P20,25 DEG C of the surfactant of 0.005% volume fraction
Diluent, pure pyridine is dissolved in 10mmol/L by Quercetin, be then diluted to 0.06 with this diluent, 0.10,0.15,
0.20th, 0.25,0.30mmol/L, dilutes the pure pyridine solution without Quercetin during dilution with same ratio every time.
7th, the measure of protein and Quercetin interaction process.
Circulation solution in SPR instrument pipelines is replaced with to Quercetin diluent used in the 6th step.It will be obtained in 6th step
After the dilution of the Quercetin of each concentration arrived solution with it is corresponding respectively without Quercetin dilution pyridine solution according to by dilute
Order to Quercetin after dense, first pyridine is sequentially sent to SPR instruments and analyzed.Sample flow rate is 5 mul/min, after sample introduction
Maintain 5 minutes, hereafter with the SDS solution flushing lines of mass fraction 5%, then solution in pipeline replaced with the 6th step used
Retarder thinner, analyzed next time.HSA interacts gained spr signal such as with each sample solution containing Quercetin
Shown in Fig. 2, HSA and spr signal obtained by the interaction of each pyridine dilute solution without Quercetin are as shown in Figure 3.This reality
Apply in example, c1=0.04mmol/L, c2=0.06mmol/L, c3=0.10mmol/L, c4=0.20mmol/L, c5=
0.30mmol/L；R1=66.2RU, R2=97.2RU, R3=148RU, R4=308RU, R5=434RU；r1=8.97RU, r2=
12.4RU, r3=17.9RU, r4=21.4RU, r5=17.2RU.
8th, data analysis.
Protein used is tried to achieve according to following steps, and the equilibrium constant of association reaction occurs with Quercetin.
A, record each ciWhen RiAnd ri；Described in the 7th step；
B, with each 1/ (Ri-ri) to 1/ciMapping, 1/ciFor abscissa, 1/ (Ri-ri) it is ordinate, as shown in Figure 4；
C, in Fig. 4 each data point carry out linear fit, gained equation be y=6.95 × 10-4x+2.35×10-4, line
Property coefficient is 0.99.With intercept divided by slope, it is 0.34mmol to obtain Quercetin with the binding constant that HSA interacts-1L,
I.e. 3.4 × 102mol-1·L。
Claims (10)
1. a kind of determine Quercetin and the surface plasma body resonant vibration analysis method of protein binding level, comprise the following steps：
1) surface carboxyl groups modification is carried out to clean sensing chip；
2) pH value of the buffer solution for solubilized target albumen is screened；
3) carboxyl that censorchip surface is modified is activated；
4) target protein is dissolved in step 2) in selected pH buffer solution, pass through step 3) chip surface of activation, will
Target protein is covalently bound on chip；
5) it is c to prepare quercetin concentration1、c2、…、ciSerial solution, wherein i be natural number；
6) by concentration by dilute to dense order by step 5) the Quercetin solution prepared sent into surface plasma body resonant vibration instrument, logical
Cross step 4) sensing chip after processing, each concentration is recorded for ciQuercetin solution sample introduction and signal stabilization after signal
Value Ri, meanwhile, record does not contain the signal value r after Quercetin but other compositions identical contrast solution sample introduction and signal stabilizationi；
7) data analysis：Calculate each 1/ciWith 1/ (Ri-ri), and with 1/ (Ri-ri) to 1/ciMapping, 1/ciFor abscissa, 1/
(Ri-ri) it is ordinate；Linear fit is carried out to data point in figure, the intercept of gained equation is designated as b, and slope is designated as a, then combined
The equilibrium constant of reaction is b/a, the inverse of unit concentration unit used in experiment.
2. analysis method as claimed in claim 1, it is characterised in that in step 1) in, the sensing chip is gold-plated glass
Piece, using by concentrated ammonia liquor, 30% hydrogen peroxide, water by volume 1:1:5 solution mixed are cleaned, then with washing
It is net and dry.
3. analysis method as claimed in claim 1, it is characterised in that in step 1) surface carboxyl groups modification is carried out to chip
Method is to install chip into surface plasma body resonant vibration instrument, then flows through chip list using the solution for meeting following condition
Face：Solute is the straight-chain carboxylic acid that a kind of sulfydryl replaces, and wherein sulfydryl is located at another end of carbochain away from carboxyl, carbon chain lengths
For containing three to 20 carbon atoms, solute concentration is 2mmol/L to 50mmol/L, solvent is water and/or ethanol；In the process of circulation
Monitoring spr signal in real time, the currency after sample introduction untill spr signal no longer changes, but at least 1 minute, solution stream
Speed is 0.1 mul/min to 1 ml/min.
4. analysis method as claimed in claim 1, it is characterised in that in step 2) relatively more different pH protein solution flows through carboxylic
The change of spr signal during the chip of base modification：If in the several pH value attempted, under the used currency, each solution
Spr signal is set to reach stabilization, then signal differs the pH of maximum solution with baseline after selection is stable；If in the circulation used
Between under each solution signal is reached stabilization, then the pH of selection signal change most fast solution；If part reaches steady component
Not up to stable, then circulation is selected to complete the pH that time-ofday signals differ maximum solution with baseline.
5. analysis method as claimed in claim 1, it is characterised in that step 3) following method I or II are used to chip surface
The carboxyl of modification is activated：
Method I：The solution for meeting following condition is set to flow through chip surface：Solute is N- (3- dimethylamino-propyls)-N '-second
Base carbodiimide hydrochloride and N- hydroxysuccinimides, both concentration are 0.0001 to 0.5 grams per milliliter, and the former concentration
It is not less than the latter's concentration, flow velocity is between 0.1 mul/min between 1 ml/min, and the currency is no longer changed with spr signal
It is defined, but at least 1 minute, chip area temperature is a value between 0 DEG C to 60 DEG C；
Method II：Successively make N- (3- dimethylamino-propyls)-N '-ethyl-carbodiimide hydrochloride solution and N- maloyls sub-
Amine aqueous solution flows through chip surface respectively, and respective concentration is 0.0001 to 0.5 grams per milliliter, and flow velocity is between 0.1 mul/min
It is defined, but is each at least 1 minute, core between 1 ml/min, the currency is no longer changed by respective spr signal
Panel region temperature is a value between 0 DEG C to 60 DEG C.
6. analysis method as claimed in claim 1, it is characterised in that step 4) by the target protein second that pH is 4 to 5.5
Acid-sodium acetate buffer dissolving, Na ion concentration is not higher than 50mmol/L, and target protein concentration is 1 nanograms/milliliter to 50 millis
Between grams per milliliter, be allowed to flow through chip surface, chip area temperature is a value between 0 DEG C to 60 DEG C, flow velocity between
0.1 mul/min is defined between 1 ml/min, the currency is no longer changed by spr signal, but at least 1 minute.
7. analysis method as claimed in claim 1, it is characterised in that step 5) first Quercetin is dissolved in pure pyridine, obtain
To pyridine solution of the concentration for 1~50mmol/L Quercetin, then buffered with the PBS for the pyridine that with the addition of 3%~15%v/v
One of solution, Tris cushioning liquid or HEPES cushioning liquid are as retarder thinner, and pH is 6.6~8.5 at 25 DEG C, dilute with its
The pyridine solution of Quercetin is released, quercetin concentration is obtained for c1、c2、…、ciSerial solution；Meanwhile, with the retarder thinner with phase
Pure pyridine is diluted in proportion, obtains the pyridine solution of various concentrations as contrast solution.
8. analysis method as claimed in claim 7, it is characterised in that be no more than 5wt% containing concentration in the retarder thinner
Additive.
9. analysis method as claimed in claim 7, it is characterised in that the retarder thinner is to contain 10mmol/L
HEPES, 0.15mol/L sodium chloride, 3mmol/L EDTA, 0.005%v/v surfactant P20, pH is 7.4 at 25 DEG C
8%v/v pyridines are with the addition of in buffer solution.
10. analysis method as claimed in claim 7, it is characterised in that step 6) by step 5) solution prepared is according to first right
Order according to Quercetin solution after solution is sequentially sent to surface plasma body resonant vibration instrument and analyzed, and flow velocity is between 0.1 micro- liter/min
Clock is between 1 ml/min, and between 0 DEG C to 60 DEG C of chip area temperature control, equal tracer signal after each sample introduction is until no longer become
Change, and at least maintain 1 minute.
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| CN1603828A (en) * | 2004-10-20 | 2005-04-06 | 国家海洋环境监测中心 | Surface plasma resonance rapid detection method for paralytic shellfish poisoning |
| CN104962633A (en) * | 2015-07-03 | 2015-10-07 | 南京理工大学 | Nucleic acid detection method based on surface plasmon resonance technology |
| US20160011216A1 (en) * | 2014-07-10 | 2016-01-14 | International Business Machines Corporation | Biosensors including surface resonance spectroscopy and semiconductor devices |
| US20170176450A1 (en) * | 2012-02-27 | 2017-06-22 | The University Of Kansas | Systems and methods for identifying protein stabilizers |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1603828A (en) * | 2004-10-20 | 2005-04-06 | 国家海洋环境监测中心 | Surface plasma resonance rapid detection method for paralytic shellfish poisoning |
| US20170176450A1 (en) * | 2012-02-27 | 2017-06-22 | The University Of Kansas | Systems and methods for identifying protein stabilizers |
| US20160011216A1 (en) * | 2014-07-10 | 2016-01-14 | International Business Machines Corporation | Biosensors including surface resonance spectroscopy and semiconductor devices |
| CN104962633A (en) * | 2015-07-03 | 2015-10-07 | 南京理工大学 | Nucleic acid detection method based on surface plasmon resonance technology |
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