CN103728279A - Function of surfactant, and signal amplification method for SPR (Surface Plasma Resonance) detection on low molecular weight substances - Google Patents
Function of surfactant, and signal amplification method for SPR (Surface Plasma Resonance) detection on low molecular weight substances Download PDFInfo
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Abstract
The invention relates to the field of biological information, in particular to the function of a surfactant, and a signal amplification method for SPR (Surface Plasma Resonance) detection on low molecular weight substances. According to the method, molecules with smaller relative molecular mass are aggregated into micelles through the surfactant to form macromolecules, and the macromolecules react with enzymes, the sensitivity is very high, and the reaction speed is very fast, so that the method is suitable for quick and sensitive detection; compared with other signal amplification methods, the signal amplification method provided by the invention is based on the principle that the micelles can be formed by aggregating through the surfactant, simple and convenient to operate, and high in experimental repeatability.
Description
Technical field
The present invention relates to biological information field, the particularly effect of surfactant, surface plasma resonance detect the method for the signal amplification of low molecular weight substance.
Background technology
Surface plasma resonance (Surface Plasmon Resonances, SPR) sensing technology is a kind of biomolecule detection technology growing up the nineties in 20th century.Its principle is: when light is during at prism and metallic film surface generation total reflection phenomenon, can in metal film, produce disappearance ripple, when disappearance ripple occurs to resonate with surface plasma-wave, the catoptrical intensity detecting can weaken greatly.Energy is transferred to surface plasma from photon, and most of energy of incident light is absorbed by surface plasma-wave, thereby catoptrical energy is sharply reduced.Now corresponding incident angle is resonance angle.SPR angle changes with the variation of gold surface refractive index, and the variation of refractive index is because the molecular mass of gold surface combination is directly proportional.Nineteen ninety, the business-like biology sensor of First is born in the world, facts have proved, with traditional detection comparatively speaking, spr sensor has the outstanding outstanding advantages such as mark, rapid sensitive and detection in real time of exempting from.So, at present to be widely used in protein-protein interaction, nucleic acid interaction, between large molecule and large molecule, the interaction between large molecule and little molecule, in medical diagnosis, biotechnology, biological monitoring, the fields such as food safety detection and drug screening have broad application prospects.Surface plasma resonance imaging (Surface Plasmon Resonances Imaging, SPRI) technology is a kind of fast high-flux analytical approach that traditional SPR detection technique is combined to CCD and takes the photograph spectrum, that light intensity is carried out to Real-Time Monitoring, can obtain multiple spot resonance curve, retractility is very large simultaneously.Therefore, SPRI exempts from, mark, advantage quick and that detect in real time, can carry out again high flux detection except possessing, will be in genomics, and protein science, drug screening, epitope, huge application is brought into play in the aspects such as Chinese medicine cut screening.
Although SPR has many advantages as biology sensor, sensitivity is limited.The molecular mass relation of the measurement range of SPRI and analyte is very large.The material of high molecular can detect when low concentration, but low-molecular-weight material (<1000) will just likely monitor less reaction signal when higher concentration, and because the non-specific adsorption effect of background dot can produce certain signal equally, so will can not determine that making existing signal is positive or false positive in such cases.So trying to explore various highly sensitive little Molecular Detection analytical approachs has become an important content in SPR research.
Improving in this problem of sensitivity, researchers have done a large amount of work.There is at present report to use nanogold particle to carry out signal amplification, such as the sub-micron micelle that contains antigen is combined with the antibody that is fixed on vane surface, carried out iodine signal; In the research of DNA hybrid experiment, researcher is fixed on golden film surface by single stranded DNA, then collaurum nano particle is sticked on single stranded DNA, being introduced into sample cell contacts with complementary DNA, there is hybridization reaction, by the field coupled amplification of golden film and golden nanometer particle, improved the sensitivity of measuring DNA, but the efficiency that the method is amplified remains limited.Separately have Avidin-Biotin system for amplifying the signal of surface plasma resonance immunosensor, sticking to Avidin on golden film combines with biotinylated antibody and forms network-like compound, immune response signal is after amplifying, and the corresponding signal detecting increases considerably.But it should be noted that in this process, be used for preparing the biotinylated antibody of compound antibody and the ratio of Avidin and want suitably.If Avidin is too much, be difficult to form large network-like molecular complex, in compound antibody solution, free Avidin can play competitive inhibition to the Avidin in compound simultaneously.Otherwise, if Avidin is very few, the biotin binding site on it is sealed by biotinylated antibody entirely, make compound antibody can not with the biotin reaction detecting on antibody, so although Avidin-Biotin system has improved the sensitivity detecting to a certain extent, simultaneously complicated experimentation.And these methods are micromolecular detection to be analyzed to application few, therefore, find a kind of simple to operation and significantly iodine signal to improve the method for low molecular weight substance detection sensitivity, just seem particularly necessary.
Summary of the invention
In view of this, the effect, surface plasma resonance that the invention provides surfactant detects the method that the signal of low molecular weight substance amplifies.The method is, by surfactant, molecular aggregates less relative molecular mass is become to micella, becomes large molecule and reacts with enzyme, and sensitivity is very high, very fast of reaction velocity, is therefore well suited for carrying out fast, Sensitive Detection; The method that signal used in the present invention amplifies is the principle that is gathered into micella based on surfactant, and compared with the method for amplifying with other signals, easy and simple to handle, experimental repeatability is high.
The method that signal used in the present invention amplifies is the principle that is gathered into micella based on surfactant, and compared with the method for amplifying with other signals, easy and simple to handle, experimental repeatability is high.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides the application of surfactant as surface plasma resonance sensitizer.
In some embodiments of the invention, surfactant is non-ionics.
Surfactant of the present invention, comprises ionic surfactant and non-ionics.As preferably, surfactant is Tween-20.
In other case study on implementation of the present invention, non-ionics provided by the invention: Tween-20(polysorbas20), relative molecular mass is 346.5, molecular formula is C
18h
34o
6.
The method for amplifying signal that the present invention also provides a kind of surface plasma resonance to detect low molecular weight substance, the mixed liquor of acquisition surfactant and damping fluid; After getting mixed liquor and mixing with described low molecular weight substance, through surface plasma resonance, detect, obtain.
In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for amplifying signal of low molecular weight substance, and surfactant is non-ionics.
As preferably, surfactant is Tween-20.
In other case study on implementation of the present invention, non-ionics provided by the invention: Tween-20(polysorbas20), relative molecular mass is 346.5, molecular formula is C
18h
34o
6.
In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for amplifying signal of low molecular weight substance, and the relative molecular weight of low molecular weight substance is less than 500.In the present invention, as the artificial substrates molecule of lipase, by the catalytic action fast decoupled of enzyme.
As preferably, a kind of surface plasma resonance provided by the invention detects in the method for amplifying signal of low molecular weight substance, and low molecular weight substance is 4-nitrobenzophenone butyric ester, 4-nitrobenzophenone capronate, 4-nitrobenzophenone caprylate, 4-nitrobenzene last of the ten Heavenly stems, 4-nitrobenzophenone laurate, 4-nitrobenzene myristinate, 4-nitrobenzene palmitate or stearic acid p-nitrophenyl ester.
In case study on implementation more of the present invention, the little molecule of substrate provided by the invention is: 4-nitrobenzophenone butyric ester (4-Nitrophenyl butyrate), molecular formula is C
10h
11nO
4, relative molecular mass is 209.20.Structural formula is I:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: 4-nitrobenzophenone capronate (4-Nitrophenyl hexanoate), molecular formula is C
12h
15nO
4, relative molecular mass is 237.26.Structural formula is II:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: 4-nitrobenzophenone caprylate (4-Nitrophenyl octanoate), molecular formula is C
14h
19nO
4, relative molecular mass is 265.30.Structural formula is III:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: the 4-nitrobenzene last of the ten Heavenly stems (4-Nitrophenyl decanoate), molecular formula is C
16h
23nO
4, relative molecular mass is 293.36.Structural formula is IV:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: 4-nitrobenzophenone laurate (4-Nitrophenyl dodecanoate), molecular formula is C
18h
27nO
4, relative molecular mass is 321.41.Structural formula is V:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: 4-nitrobenzene myristinate (4-Nitrophenyl myristate), molecular formula is C
20h
31nO
4, relative molecular mass is 349.46.Structural formula is VI:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: 4-nitrobenzene palmitate (4-Nitrophenyl myristate), molecular formula is C
22h
35nO
4, relative molecular mass is 377.52.Structural formula is VII:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: stearic acid p-nitrophenyl ester (4-Nitrophenyl stearate), molecular formula is C
24h
39nO4, relative molecular mass is 405.57.Structural formula is VIII:
In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for amplifying signal of low molecular weight substance, and the concentration of low molecular weight substance is 0.3～1mmol/L.
The effect that the micromolecular concentration of substrate is amplified signal has certain influence.Keep the concentration of surfactant constant, when little molecule substrate concentration is higher, the micella quantity of generation is many, and signal amplification effect is very obvious; Along with the reduction of little molecule substrate concentration, signal amplification effect reduces; When the micromolecular concentration of substrate is worth lower than certain, little molecule still exists with single status in solution, do not form micella, now no signal amplification effect, its result with do not add coming to the same thing of surfactant: signal is very little or almost do not observe the interactional SPRI signal of enzyme-to-substrate.
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 1mmol/L, and now signal amplification effect is very obvious;
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 0.75mM, and now signal amplification effect is very obvious;
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 0.5mmol/L, and now signal amplification effect still obviously;
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 0.3mmol/L, and now signal amplification effect still obviously;
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 0.2mmol/L, and now signal amplification effect weakens to some extent;
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 0.1mmol/L, and now signal amplification effect weakens greatly;
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 0.05mmol/L, now no signal amplification effect, signal results with do not add coming to the same thing of surfactant: signal is very little or almost do not observe the interactional SPRI signal of enzyme-to-substrate.
In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for amplifying signal of low molecular weight substance, and the percent by volume that surfactant accounts in described mixed liquor is 0.0038～0.05%.
The concentration of surfactant has very large impact to the size of the interactional SPRI signal of enzyme-to-substrate.When the concentration of surfactant is during lower than critical micelle concentration (CMC), substrate molecule can not be assembled and forms macromolecular micella, and now SPR can not maybe can only detect the actuating signal of faint enzyme-to-substrate molecule; When the concentration of surfactant equals or during a little higher than critical micelle concentration (CMC), the little molecular aggregates of single substrate becomes micella, the large molecule micella of enzymatic, produces stronger SPRI signal; When the concentration of surfactant reaches certain value, SPRI signal enhancing trend slows down, and approaches to saturation.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.05%(v/v), process little molecule substrate (10mmol/L) with this solution dilution.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.01%(v/v), process little molecule substrate (10mmol/L) with this solution dilution.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.00625%(v/v), process little molecule substrate (10mmol/L) with this solution dilution.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.005%(v/v), process little molecule substrate (10mmol/L) with this solution dilution.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.0038%(v/v), process little molecule substrate (10mmol/L) with this solution dilution.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.0025%(v/v), process little molecule substrate (10mmol/L) with this solution dilution.Concentration is now not enough to carry out signal amplification.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.0005%(v/v), process little molecule substrate (10mmol/L) with this solution dilution.Concentration is now not enough to carry out signal amplification.
Damping fluid of the present invention, comprise most damping fluids of commonly using in experiment, as PBS(phosphate) damping fluid, glycocoll-hydrochloride buffer, phthalic acid-hydrochloride buffer, sodium hydrogen phosphate-sodium citrate buffer solution, citric acid-NaOH-hydrochloride buffer, citric acid-sodium citrate damping fluid, acetic acid-sodium-acetate buffer, sodium dihydrogen phosphate-sodium hydrate buffer solution, boric acid-borate buffer solution, glycocoll-sodium hydrate buffer solution, sodium carbonate-sodium bicarbonate buffer liquid etc., but Tris damping fluid (TRIS buffer) is not included.
The SPR device the present invention relates to, comprise the SPR device detecting based on angle, the SPR device detecting based on wavelength, the SPR device based on intensity detection, the SPR device based on phase-detection, also comprise based on micro-fluidic simultaneously, the SPR device of automatic sample handling system combination also comprises all the other SPR apparatuss that detect based on SPR character simultaneously, also comprises local SPR device simultaneously, and long-range SPR device, SPR device of waveguide mode etc.
The surperficial fixing means that the present invention uses, comprises based on physisorption, chemical covalent effect, and noncovalent interaction, and if the fixing means that produces of the acting force such as hydrogen bond.
The surperficial substrate that chip of the present invention adopts, comprises the surface of various metals and alloy, and the surface of the inorganics such as various glass and quartz, also comprises that macromolecule can be used for the surface of the protein of fixed nucleic acid such as dimethyl silicone polymer and polystyrene etc.Also comprise coarse surface simultaneously, comprise that the molecular surface of processing and modifying through micro-nano is as the Electrospun nonwoven surface of different materials, and the adhesive surface of preparing through physics and chemistry and biological method.
In above-mentioned steps of the present invention, the method that surfactant is processed little molecule substrate comprises: directly with adding the damping fluid of surfactant to dilute substrate; First with damping fluid, dilute substrate to experimental concentration, then add surfactant
The step that in above-mentioned steps, directly use adds the damping fluid of surfactant to dilute the method for substrate comprises: prepare damping fluid; According to the concentration of the surfactant of setting, in damping fluid, add by volume surfactant; The little molecule mother liquor of the damping fluid that contains surfactant dilution substrate having prepared with above-mentioned is to experimental concentration.
In above-mentioned steps, first with damping fluid, dilute substrate to experimental concentration, then add the step of the method for surfactant to comprise: prepare damping fluid; With the little molecule mother liquor of damping fluid dilution substrate preparing to experimental concentration; According to the concentration of the surfactant of setting, in the little molecular solution having diluted, add by volume surfactant.
The method step of in above-mentioned steps, the little molecule of handling well the probe fixing with surface being combined is: PBS processes biochip; The little molecule substrate of handling well is passed into the surface of SPR chip, carry out in conjunction with catalytic reaction.
In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for amplifying signal of low molecular weight substance, and probe is enzyme.
As preferably, a kind of surface plasma resonance provided by the invention detects in the method for amplifying signal of low molecular weight substance, and enzyme is lipase or proteinase.
The present invention also provides a kind of surface plasma resonance to detect the method for low molecular weight substance, comprises the steps:
Step 1: the mixed liquor that obtains surfactant and damping fluid; Get mixed liquor and mix with low molecular weight substance, obtain test substance;
Step 2: at chip surface stationary probe and negative control material respectively, pass into mixed liquor and scan, read light intensity signal value, obtain respectively determinand initial value, negative control initial value; Probe can with low molecular weight substance specific binding;
Step 3: pass into test substance, the test substance negative control material fixing with chip surface is not combined, and reads light intensity signal value, obtains negative control measured value; Passing into mixed liquor rinses until baseline is steady again;
Pass into test substance, the test substance probe fixing with chip surface is combined, and reads light intensity signal value, obtains determinand measured value; Passing into mixed liquor rinses until baseline is steady again;
Step 4: get negative control measured value and deduct negative control initial value acquisition negative control changing value;
Get determinand measured value and deduct determinand initial value acquisition determinand changing value;
Relatively determinand changing value and negative control changing value, significant difference, obtains.
In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for low molecular weight substance, and surfactant is non-ionics.
Surfactant of the present invention, comprises ionic surfactant and non-ionics.In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for low molecular weight substance, and surfactant is Tween-20.
In other case study on implementation of the present invention, non-ionics provided by the invention: Tween-20(polysorbas20), relative molecular mass is 346.5, molecular formula is C
18h
34o
6.
In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for low molecular weight substance, and the relative molecular weight of low molecular weight substance is less than 500.In the present invention, as the artificial substrates molecule of lipase, by the catalytic action fast decoupled of enzyme.
In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for low molecular weight substance, and low molecular weight substance is 4-nitrobenzophenone butyric ester, 4-nitrobenzophenone capronate, 4-nitrobenzophenone caprylate, 4-nitrobenzene last of the ten Heavenly stems, 4-nitrobenzophenone laurate, 4-nitrobenzene myristinate, 4-nitrobenzene palmitate or stearic acid p-nitrophenyl ester.
In case study on implementation more of the present invention, the little molecule of substrate provided by the invention is: 4-nitrobenzophenone butyric ester (4-Nitrophenyl butyrate), molecular formula is C
10h
11nO
4, relative molecular mass is 209.20.Structural formula is I:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: 4-nitrobenzophenone capronate (4-Nitrophenyl hexanoate), molecular formula is C
12h
15nO
4, relative molecular mass is 237.26.Structural formula is II:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: 4-nitrobenzophenone caprylate (4-Nitrophenyl octanoate), molecular formula is C
14h
19nO
4, relative molecular mass is 265.30.Structural formula is III:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: the 4-nitrobenzene last of the ten Heavenly stems (4-Nitrophenyl decanoate), molecular formula is C
16h
23nO
4, relative molecular mass is 293.36.Structural formula is IV:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: 4-nitrobenzophenone laurate (4-Nitrophenyl dodecanoate), molecular formula is C
18h
27nO
4, relative molecular mass is 321.41.Structural formula is V:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: 4-nitrobenzene myristinate (4-Nitrophenyl myristate), molecular formula is C
20h
31nO
4, relative molecular mass is 349.46.Structural formula is VI:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: 4-nitrobenzene palmitate (4-Nitrophenyl myristate), molecular formula is C
22h
35nO
4, relative molecular mass is 377.52.Structural formula is VII:
In other case study on implementation of the present invention, the little molecule of substrate provided by the invention is: stearic acid p-nitrophenyl ester (4-Nitrophenyl stearate), molecular formula is C
24h
39nO4, relative molecular mass is 405.57.Structural formula is VIII:
In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for low molecular weight substance, and the concentration of low molecular weight substance is 0.3～1mmol/L.
The effect that the micromolecular concentration of substrate is amplified signal has certain influence.Keep the concentration of surfactant constant, when little molecule substrate concentration is higher, the micella quantity of generation is many, and signal amplification effect is very obvious; Along with the reduction of little molecule substrate concentration, signal amplification effect reduces; When the micromolecular concentration of substrate is worth lower than certain, little molecule still exists with single status in solution, do not form micella, now no signal amplification effect, its result with do not add coming to the same thing of surfactant: signal is very little or almost do not observe the interactional SPRI signal of enzyme-to-substrate.
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 1mM, and now signal amplification effect is very obvious;
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 0.75mM, and now signal amplification effect is very obvious;
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 0.5mM, and now signal amplification effect still obviously;
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 0.3mM, and now signal amplification effect still obviously;
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 0.2mM, and now signal amplification effect weakens to some extent;
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 0.1mM, and now signal amplification effect weakens greatly;
In other case study on implementation of the present invention, the little molecular conecentration of substrate provided by the invention is 0.05mM, now no signal amplification effect, signal results with do not add coming to the same thing of surfactant: signal is very little or almost do not observe the interactional SPRI signal of enzyme-to-substrate.
In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for low molecular weight substance, and the percent by volume that surfactant accounts in described mixed liquor is 0.0038～0.05%.
The concentration of surfactant has very large impact to the size of the interactional SPRI signal of enzyme-to-substrate.When the concentration of surfactant is during lower than critical micelle concentration (CMC), substrate molecule can not be assembled and forms macromolecular micella, and now SPR can not maybe can only detect the actuating signal of faint enzyme-to-substrate molecule; When the concentration of surfactant equals or during a little higher than critical micelle concentration (CMC), the little molecular aggregates of single substrate becomes micella, the large molecule micella of enzymatic, produces stronger SPRI signal; When the concentration of surfactant reaches certain value, SPRI signal enhancing trend slows down, and approaches to saturation.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.05%(v/v), process little molecule substrate (10mM) with this solution dilution.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.01%(v/v), process little molecule substrate (10mM) with this solution dilution.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.00625%(v/v), process little molecule substrate (10mM) with this solution dilution.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.005%(v/v), process little molecule substrate (10mM) with this solution dilution.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.0038%(v/v), process little molecule substrate (10mM) with this solution dilution.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.0025%(v/v), process little molecule substrate (10mM) with this solution dilution.Concentration is now not enough to carry out signal amplification.
In other case study on implementation of the present invention, surfactant Tween-20(polysorbas20) at damping fluid PBS(phosphate buffer) in concentration be 0.0005%(v/v), process little molecule substrate (10mM) with this solution dilution.Concentration is now not enough to carry out signal amplification.
Damping fluid of the present invention, comprise most damping fluids of commonly using in experiment, as PBS(phosphate) damping fluid, glycocoll-hydrochloride buffer, phthalic acid-hydrochloride buffer, sodium hydrogen phosphate-sodium citrate buffer solution, citric acid-NaOH-hydrochloride buffer, citric acid-sodium citrate damping fluid, acetic acid-sodium-acetate buffer, sodium dihydrogen phosphate-sodium hydrate buffer solution, boric acid-borate buffer solution, glycocoll-sodium hydrate buffer solution, sodium carbonate-sodium bicarbonate buffer liquid etc., but Tris damping fluid (TRIS buffer) is not included.
SPR device of the present invention, comprise the SPR device detecting based on angle, the SPR device detecting based on wavelength, the SPR device based on intensity detection, the SPR device based on phase-detection, also comprise based on micro-fluidic simultaneously, the SPR device of automatic sample handling system combination also comprises all the other SPR apparatuss that detect based on SPR character simultaneously, also comprises local SPR device simultaneously, and long-range SPR device, SPR device of waveguide mode etc.
Surperficial fixing means of the present invention, comprises based on physisorption, chemical covalent effect, and noncovalent interaction, and if the fixing means that produces of the acting force such as hydrogen bond.
Surperficial substrate of the present invention, comprises the surface of various metals and alloy, and the surface of the inorganics such as various glass and quartz, also comprises that macromolecule can be used for the surface of the protein of fixed nucleic acid such as dimethyl silicone polymer and polystyrene etc.Also comprise coarse surface simultaneously, comprise that the molecular surface of processing and modifying through micro-nano is as the Electrospun nonwoven surface of different materials, and the adhesive surface of preparing through physics and chemistry and biological method.
In above-mentioned steps 1 of the present invention, the method that surfactant is processed little molecule substrate comprises: directly with adding the damping fluid of surfactant to dilute substrate; First with damping fluid, dilute substrate to experimental concentration, then add surfactant
The step that in above-mentioned steps, directly use adds the damping fluid of surfactant to dilute the method for substrate comprises: prepare damping fluid; According to the concentration of the surfactant of setting, in damping fluid, add by volume surfactant; The little molecule mother liquor of the damping fluid that contains surfactant dilution substrate having prepared with above-mentioned is to experimental concentration.
In above-mentioned steps, first with damping fluid, dilute substrate to experimental concentration, then add the step of the method for surfactant to comprise: prepare damping fluid; With the little molecule mother liquor of damping fluid dilution substrate preparing to experimental concentration; According to the concentration of the surfactant of setting, in the little molecular solution having diluted, add by volume surfactant.
The method step of in above-mentioned steps, the little molecule of handling well the probe fixing with surface being combined is: PBS processes biochip; The little molecule substrate of handling well is passed into the surface of SPR chip, carry out in conjunction with catalytic reaction.
In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for low molecular weight substance, and probe is enzyme.
In some embodiments of the invention, a kind of surface plasma resonance provided by the invention detects in the method for low molecular weight substance, and enzyme is lipase or proteinase.
The effect, surface plasma resonance that the invention provides surfactant detects the method that the signal of low molecular weight substance amplifies.The method is, by surfactant, molecular aggregates less relative molecular mass is become to micella, becomes large molecule and reacts with enzyme, and sensitivity is very high, very fast of reaction velocity, is therefore well suited for carrying out fast, Sensitive Detection; The method that signal used in the present invention amplifies is the principle that is gathered into micella based on surfactant, and compared with the method for amplifying with other signals, easy and simple to handle, experimental repeatability is high.
By the method for using the signal of the micromolecular SPR of surfactant Treatment Analysis thing to amplify, SPR method has not only been had and exempt from mark, the real-time feature detecting, also there is high sensitivity, fast detecting, simple to operation, the repeated advantages of higher of result.Can be applied to the evaluation of enzyme-to-substrate, the screening of high flux lipase and evaluation, also can be used for detecting fast and accurately of infectious disease, the fields such as medicine and macromolecular interaction.The realization condition of the method is simple, and its high sensitivity and high-repetition-rate, so be well suited for the quick test in common lab and field, uses.
Accompanying drawing explanation
Fig. 1 shows and in embodiment 1, adds the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 2 shows and in embodiment 1, does not add the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 3 shows and in embodiment 2, adds the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 4 shows and in embodiment 2, does not add the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 5 shows and in embodiment 3, adds the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 6 shows and in embodiment 3, does not add the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 7 shows and in embodiment 4, adds the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 8 shows and in embodiment 4, does not add the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 9 shows and in embodiment 5, adds the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Figure 10 shows and in embodiment 5, does not add the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Figure 11 shows and in embodiment 6, adds the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Figure 12 shows and in embodiment 6, does not add the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Figure 13 shows and in embodiment 7, adds the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Figure 14 shows and in embodiment 7, does not add the surface plasma resonance figure after surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control.
Embodiment
The effect, surface plasma resonance that the invention discloses surfactant detects the method that the signal of low molecular weight substance amplifies, and those skilled in the art can use for reference content herein, suitably improve technological parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications as herein described in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply the technology of the present invention.
In the method that the effect of surfactant provided by the invention, the signal that surface plasma resonance detects low molecular weight substance amplify, agents useful for same all can be buied by market.
Below in conjunction with embodiment, further set forth the present invention:
1. at the lipase CalB of the fixing 200ug/ml of the chip surface of SPRI, using the bovine serum albumin BSA of 200ug/ml as negative control;
2. preparation PBS+0.05%Tween-20 damping fluid;
3. chip is fixed on SPRI device, through procedure Selection lipase CalB and bovine serum albumin BSA institute fixed position, carries out the scanning of intensity, surface is passed into PBS+0.05%Tween-20 damping fluid and carry out the scanning of baseline, as shown in Fig. 1 baseline;
4. with the PBS+0.05%Tween-20 damping fluid preparing, little substrate molecule 4-nitrobenzophenone caprylate is diluted to 0.25mM from mother liquor 10mM;
5. with the association rate of 2ul/s, pass into the little molecule of substrate after treatment, as shown in Fig. 1 binding curve;
6. pass into PBS+0.05%Tween-20 damping fluid and rinse until baseline is steady, as shown in Fig. 1 dissociation curve.
7. keep above-mentioned experimental procedure constant, change damping fluid, PBS+0.05%Tween-20 is changed to PBS (phosphate buffer), do not add Tween-20.With PBS, carry out the dilution process of substrate, and carry out the flushing of baseline, signal as shown in Figure 2, does not observe the interactional SPRI signal of enzyme-to-substrate.
1. at the lipase CalB of the fixing 200ug/ml of the chip surface of SPRI, using the bovine serum albumin BSA of 200ug/ml as negative control;
2. preparation PBS+0.01%Tween-20 damping fluid;
3. chip is fixed on SPRI device, through procedure Selection lipase CalB and bovine serum albumin BSA institute fixed position, carries out the scanning of intensity, surface is passed into PBS+0.01%Tween-20 damping fluid and carry out the scanning of baseline, as shown in Fig. 3 baseline;
4. use the PBS+0.01%Tween-20 damping fluid preparing by little substrate molecule 4-nitrobenzophenone caprylate
From mother liquor, 10mM is diluted to 0.25mM;
5. with the association rate of 2ul/s, pass into the little molecule of substrate after treatment, as shown in Fig. 3 binding curve;
6. pass into PBS+0.01%Tween-20 damping fluid and rinse until baseline is steady, as shown in Fig. 3 dissociation curve.
7. keep above-mentioned experimental procedure constant, change damping fluid, PBS+0.01%Tween-20 is changed to PBS (phosphate buffer), do not add Tween-20.With PBS, carry out the dilution process of substrate, and carry out the flushing of baseline, signal as shown in Figure 4, does not observe the interactional SPRI signal of enzyme-to-substrate.
Embodiment 3 processes substrate little molecule 4-nitrobenzophenone caprylate (4-Nitrophenyl octanoate) by surfactant Tween-20 and carries out the amplification of SPRI signal
1. at the lipase CalB of the fixing 200ug/ml of the chip surface of SPRI, using the bovine serum albumin BSA of 200ug/ml as negative control;
2. preparation PBS+0.00625%Tween-20 damping fluid;
3. chip is fixed on SPRI device, through procedure Selection lipase CalB and bovine serum albumin BSA institute fixed position, carries out the scanning of intensity, surface is passed into PBS+0.00625%Tween-20 damping fluid and carry out the scanning of baseline, as shown in Fig. 5 baseline;
4. with the PBS+0.00625%Tween-20 damping fluid preparing, little substrate molecule 4-nitrobenzophenone caprylate is diluted to 0.25mM from mother liquor 10mM;
5. with the association rate of 2ul/s, pass into the little molecule of substrate after treatment, as shown in Fig. 5 binding curve;
6. pass into PBS+0.00625%Tween-20 damping fluid and rinse until baseline is steady, as shown in Fig. 5 dissociation curve.
7. keep above-mentioned experimental procedure constant, change damping fluid, PBS+0.00625%Tween-20 is changed to PBS(phosphate buffer), do not add Tween-20.With PBS, carry out the dilution process of substrate, and carry out the flushing of baseline, signal as shown in Figure 6, does not observe the interactional SPRI signal of enzyme-to-substrate.
Embodiment 4 processes substrate little molecule 4-nitrobenzophenone caprylate (4-Nitrophenyl octanoate) by surfactant Tween-20 and carries out the amplification of SPRI signal
1. at the lipase CalB of the fixing 200ug/ml of the chip surface of SPRI, using the bovine serum albumin BSA of 200ug/ml as negative control;
2. preparation PBS+0.005%Tween-20 damping fluid;
3. chip is fixed on SPRI device, through procedure Selection lipase CalB and bovine serum albumin BSA institute fixed position, carries out the scanning of intensity, surface is passed into PBS+0.005%Tween-20 damping fluid and carry out the scanning of baseline, as shown in Fig. 7 baseline;
4. with the PBS+0.005%Tween-20 damping fluid preparing, little substrate molecule 4-nitrobenzophenone caprylate is diluted to 0.25mM from mother liquor 10mM;
5. with the association rate of 2ul/s, pass into the little molecule of substrate after treatment, as shown in Fig. 7 binding curve;
6. pass into PBS+0.005%Tween-20 damping fluid and rinse until baseline is steady, as shown in Fig. 7 dissociation curve.
7. keep above-mentioned experimental procedure constant, change damping fluid, PBS+0.005%Tween-20 is changed to PBS(phosphate buffer), do not add Tween-20.With PBS, carry out the dilution process of substrate, and carry out the flushing of baseline, signal as shown in Figure 8, does not observe the interactional SPRI signal of enzyme-to-substrate.
Embodiment 5 processes substrate little molecule 4-nitrobenzophenone caprylate (4-Nitrophenyl octanoate) by surfactant Tween-20 and carries out the amplification of SPRI signal
1. at the lipase CalB of the fixing 200ug/ml of the chip surface of SPRI, using the bovine serum albumin BSA of 200ug/ml as negative control;
2. preparation PBS+0.0038%Tween-20 damping fluid;
3. chip is fixed on SPRI device, through procedure Selection lipase CalB and bovine serum albumin BSA institute fixed position, carries out the scanning of intensity, surface is passed into PBS+0.0038%Tween-20 damping fluid and carry out the scanning of baseline, as shown in Fig. 9 baseline;
4. with the PBS+0.0038%Tween-20 damping fluid preparing, little substrate molecule 4-nitrobenzophenone caprylate is diluted to 0.25mM from mother liquor 10mM;
5. with the association rate of 2ul/s, pass into the little molecule of substrate after treatment, as shown in Fig. 9 binding curve;
6. pass into PBS+0.0038%Tween-20 damping fluid and rinse until baseline is steady, as shown in Fig. 9 dissociation curve.
7. keep above-mentioned experimental procedure constant, change damping fluid, PBS+0.0038%Tween-20 is changed to PBS(phosphate buffer), do not add Tween-20.With PBS, carry out the dilution process of substrate, and carry out the flushing of baseline, signal as shown in figure 10, does not observe the interactional SPRI signal of enzyme-to-substrate.
Embodiment 6 processes substrate little molecule 4-nitrobenzophenone caprylate (4-Nitrophenyl octanoate) by surfactant Tween-20 and carries out the amplification of SPRI signal
Step is as follows:
1. at the lipase CalB of the fixing 200ug/ml of the chip surface of SPRI, using the bovine serum albumin BSA of 200ug/ml as negative control;
2. preparation PBS+0.0025%Tween-20 damping fluid;
3. chip is fixed on SPRI device, through procedure Selection lipase CalB and bovine serum albumin BSA institute fixed position, carries out the scanning of intensity, surface is passed into PBS+0.0025%Tween-20 damping fluid and carry out the scanning of baseline, as shown in Figure 11 baseline;
4. with the PBS+0.0025%Tween-20 damping fluid preparing, little substrate molecule 4-nitrobenzophenone caprylate is diluted to 0.25mM from mother liquor 10mM;
5. with the association rate of 2ul/s, pass into the little molecule of substrate after treatment;
6. passing into PBS+0.0025%Tween-20 damping fluid rinses until baseline is steady; No signal amplification, does not observe the interactional SPRI signal of enzyme-to-substrate.
7. keep above-mentioned experimental procedure constant, change damping fluid, PBS+0.0025%Tween-20 is changed to PBS(phosphate buffer), do not add Tween-20.With PBS, carry out the dilution process of substrate, and carry out the flushing of baseline, signal as shown in figure 12, does not observe the interactional SPRI signal of enzyme-to-substrate.
Embodiment 7 processes substrate little molecule 4-nitrobenzophenone caprylate (4-Nitrophenyl octanoate) by surfactant Tween-20 and carries out the amplification of SPRI signal
1. at the lipase CalB of the fixing 200ug/ml of the chip surface of SPRI, using the bovine serum albumin BSA of 200ug/ml as negative control;
2. preparation PBS+0.0005%Tween-20 damping fluid;
3. chip is fixed on SPRI device, through procedure Selection lipase CalB and bovine serum albumin BSA institute fixed position, carries out the scanning of intensity, surface is passed into PBS+0.0005%Tween-20 damping fluid and carry out the scanning of baseline, as shown in Figure 13 baseline;
4. with the PBS+0.0005%Tween-20 damping fluid preparing, little substrate molecule 4-nitrobenzophenone caprylate is diluted to 0.25mM from mother liquor 10mM;
5. with the association rate of 2ul/s, pass into the little molecule of substrate after treatment;
6. passing into PBS+0.0005%Tween-20 damping fluid rinses until baseline is steady; No signal amplification, does not observe the interactional SPRI signal of enzyme-to-substrate.
7. keep above-mentioned experimental procedure constant, change damping fluid, PBS+0.0005%Tween-20 is changed to PBS(phosphate buffer), do not add Tween-20.With PBS, carry out the dilution process of substrate, and carry out the flushing of baseline, signal as shown in figure 14, does not observe the interactional SPRI signal of enzyme-to-substrate.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (21)
1. surfactant is as the application of surface plasma resonance sensitizer.
2. application according to claim 1, is characterized in that, described surfactant is non-ionics.
3. application according to claim 2, is characterized in that, described surfactant is Tween-20.
4. surface plasma resonance detects a method for amplifying signal for low molecular weight substance, it is characterized in that,
Obtain the mixed liquor of surfactant and damping fluid;
After getting described mixed liquor and mixing with described low molecular weight substance, through surface plasma resonance, detect, obtain.
5. method for amplifying signal according to claim 4, is characterized in that, surfactant is non-ionics.
6. method for amplifying signal according to claim 4, is characterized in that, described surfactant is Tween-20.
7. method for amplifying signal according to claim 4, is characterized in that, the relative molecular weight of described low molecular weight substance is less than 500.
8. method for amplifying signal according to claim 4, it is characterized in that, described low molecular weight substance is 4-nitrobenzophenone butyric ester, 4-nitrobenzophenone capronate, 4-nitrobenzophenone caprylate, 4-nitrobenzene last of the ten Heavenly stems, 4-nitrobenzophenone laurate, 4-nitrobenzene myristinate, 4-nitrobenzene palmitate or stearic acid p-nitrophenyl ester.
9. method for amplifying signal according to claim 4, is characterized in that, the concentration of described low molecular weight substance is 0.3～1mmol/L.
10. method for amplifying signal according to claim 4, is characterized in that, the percent by volume that described surfactant accounts in described mixed liquor is 0.0038～0.05%.
11. method for amplifying signal according to claim 4, is characterized in that, described probe is enzyme.
12. method for amplifying signal according to claim 11, is characterized in that, described enzyme is lipase or proteinase.
13. 1 kinds of surface plasma resonances detect the method for low molecular weight substance, it is characterized in that, comprise the steps:
Step 1: the mixed liquor that obtains surfactant and damping fluid; Get described mixed liquor and mix with low molecular weight substance, obtain test substance;
Step 2: at chip surface stationary probe and negative control material respectively, pass into described mixed liquor and scan, read light intensity signal value, obtain respectively determinand initial value, negative control initial value; Described probe can with described low molecular weight substance specific binding;
Step 3: pass into described test substance, the described test substance described negative control material fixing with chip surface is not combined, and reads light intensity signal value, obtains negative control measured value; Passing into described mixed liquor rinses until baseline is steady again;
Pass into described test substance, the described test substance described probe fixing with chip surface is combined, and reads light intensity signal value, obtains determinand measured value; Passing into described mixed liquor rinses until baseline is steady again;
Step 4: get described negative control measured value and deduct described negative control initial value acquisition negative control changing value;
Get described determinand measured value and deduct described determinand initial value acquisition determinand changing value;
More described determinand changing value and described negative control changing value, significant difference, obtains.
14. methods according to claim 13, is characterized in that, surfactant is non-ionics.
15. methods according to claim 13, is characterized in that, described surfactant is Tween-20.
16. methods according to claim 13, is characterized in that, the relative molecular weight of described low molecular weight substance is less than 500.
17. methods according to claim 13, it is characterized in that, described low molecular weight substance is 4-nitrobenzophenone butyric ester, 4-nitrobenzophenone capronate, 4-nitrobenzophenone caprylate, 4-nitrobenzene last of the ten Heavenly stems, 4-nitrobenzophenone laurate, 4-nitrobenzene myristinate, 4-nitrobenzene palmitate or stearic acid p-nitrophenyl ester.
18. methods according to claim 13, is characterized in that, the concentration of described low molecular weight substance is 0.3～1mmol/L.
19. methods according to claim 13, is characterized in that, the percent by volume that described surfactant accounts in described mixed liquor is 0.0038～0.05%.
20. methods according to claim 13, is characterized in that, described probe is enzyme.
21. methods according to claim 20, is characterized in that, described enzyme is lipase or proteinase.
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