CN103728273B - The method that the signal that the effect of surfactant, surface plasma resonance detect low molecular weight substance amplifies - Google Patents
The method that the signal that the effect of surfactant, surface plasma resonance detect low molecular weight substance amplifies Download PDFInfo
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Abstract
The present invention relates to biological information field, be specifically related to the effect of surfactant, surface plasma resonance detect low molecular weight substance signal amplify method.The method is, by surfactant, molecule less for relative molecular mass is gathered into micella, becomes large molecule and enzyme reacts, and sensitivity is very high, reaction velocity very fast, is therefore well suited for carrying out fast, Sensitive Detection; The method that signal used in the present invention amplifies is the principle being 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.
Description
Technical field
The present invention relates to biological information field, the method that the particularly effect of surfactant, the signal of surface plasma resonance detection low molecular weight substance are amplified.
Background technology
Surface plasma resonance (SurfacePlasmonResonances, SPR) sensing technology is a kind of biomolecule detection technology grown up the nineties in 20th century.Its principle is: when light is at prism and metallic film surface generation total reflection phenomenon, and can produce evanescent waves in metal film, when evanescent waves and surface plasma-wave occur to resonate, the intensity of the reflected light detected can weaken greatly.Energy transfers to surface plasma from photon, and most of energy of incident light is absorbed by surface plasma-wave, thus the energy of reflected light is sharply reduced.Now corresponding incident angle is resonance angle.SPR angle changes with the change of gold surface refractive index, and the molecular mass that the change of refractive index combines due to gold surface 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 outstanding exempts from the outstanding advantages such as mark, rapid sensitive and detection in real time.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 Small molecular, in medical diagnosis, biotechnology, biological monitoring, the field such as food safety detection and drug screening has broad application prospects.Surface plasma resonance imaging (SurfacePlasmonResonancesImaging, SPRI) technology traditional SPR detection technique is combined a kind of rapid high throughput assays method that CCD takes the photograph spectrum, that Real-Time Monitoring is carried out to the intensity of light, can obtain multiple spot resonance curve, retractility is very large simultaneously.Therefore, SPRI exempts from, except mark, advantage that is quick and that detect in real time, can carry out high flux detection again except possessing, will in genomics, and protein science, drug screening, epitope, the aspects such as Chinese medicine cut screening play huge application.
Although SPR has many advantages as biology sensor, sensitivity is limited.The measurement range of SPRI is very large with the molecular mass relation analyzing thing.The material of high molecular can detect when low concentration, but low-molecular-weight material (<1000) just will likely monitor less reaction signal when higher concentration, and certain signal can be produced equally due to the non-specific adsorption effect of background dot, so will can not determine that making existing signal is positive or false positive in such cases.So, try to explore various highly sensitive Small molecular determination method become SPR research in an important content.
In this problem of raising sensitivity, researchers have done a large amount of work.There is report to use nanogold particle to carry out signal amplification at present, such as the sub-micron micelle containing antigen has been combined with the antibody being fixed on vane surface, carrys out iodine signal; In the research of DNA hybridization experiment, single stranded DNA is fixed on golden film surface by researcher, then collaurum nano particle is sticked on single stranded DNA, be introduced into sample cell to contact with complementary DNA, there is hybridization reaction, by the field coupled amplification of golden film and golden nanometer particle, improve the sensitivity measuring DNA, but the efficiency that the method is amplified remains limited.Separately there is the signal being used for Avidin-Biotin system to amplify surface plasma resonance immunosensor, the Avidin sticked on golden film combines with biotinylated antibody and forms network-like compound, immune response signal is after amplifying, and the corresponding signal detected increases considerably.But it should be noted that in the process, the ratio of the biotinylated antibody and Avidin that are used for preparing compound antibody is wanted suitably.If Avidin is too much, be then 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, then the biotin-binding site on it is closed by biotinylated antibody entirely, makes compound antibody can not with the biotin reaction detected on antibody, so although Avidin-Biotin system improves the sensitivity of detection 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 can significantly just to seem particularly necessity with the method improving low molecular weight substance detection sensitivity by iodine signal.
Summary of the invention
In view of this, the invention provides the effect of surfactant, surface plasma resonance detect low molecular weight substance signal amplify method.The method is, by surfactant, molecule less for relative molecular mass is gathered into micella, becomes large molecule and enzyme reacts, and sensitivity is very high, reaction velocity very fast, is therefore well suited for carrying out fast, Sensitive Detection; The method that signal used in the present invention amplifies is the principle being 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 being 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 TritonX100.
In other case study on implementation of the present invention, non-ionics provided by the invention: TritonX100(Value 3608), relative molecular mass is 324.45, and molecular formula is C
18h
28o
5.
Present invention also offers the method for amplifying signal that a kind of surface plasma resonance detects low molecular weight substance, obtain the mixed liquor of surfactant and damping fluid; Get after mixed liquor mixes with described low molecular weight substance and detect through surface plasma resonance, to obtain final product.
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 TritonX100.
In other case study on implementation of the present invention, non-ionics provided by the invention: TritonX100(Value 3608), relative molecular mass is 324.45, and molecular formula is C
18h
28o
5.
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-Nitrophenyl butyrate, 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, substrate Small molecular provided by the invention is: 4-Nitrophenyl butyrate (4-Nitrophenylbutyrate), and 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, substrate Small molecular provided by the invention is: 4-nitrobenzophenone capronate (4-Nitrophenylhexanoate), and 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, substrate Small molecular provided by the invention is: 4-nitrobenzophenone caprylate (4-Nitrophenyloctanoate), and 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, substrate Small molecular provided by the invention is: the 4-nitrobenzene last of the ten Heavenly stems (4-Nitrophenyldecanoate), and 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, substrate Small molecular provided by the invention is: 4-nitrobenzophenone laurate (4-Nitrophenyldodecanoate), and 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, substrate Small molecular provided by the invention is: 4-nitrobenzene myristinate (4-Nitrophenylmyristate), and 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, substrate Small molecular provided by the invention is: 4-nitrobenzene palmitate (4-Nitrophenylmyristate), and 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, substrate Small molecular provided by the invention is: stearic acid p-nitrophenyl ester (4-Nitrophenylstearate), and 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 micromolecular concentration of substrate has certain influence to the effect that signal amplifies.Keep the concentration of surfactant constant, when small molecule substrates concentration is higher, the micella quantity of generation is many, and signal amplification effect clearly; Along with the reduction of small molecule substrates concentration, signal amplification effect reduces; When the micromolecular concentration of substrate is lower than certain value, Small molecular still exists with single status in the 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, substrate Small molecular concentration provided by the invention is 1mmol/L, and now signal amplification effect clearly;
In other case study on implementation of the present invention, substrate Small molecular concentration provided by the invention is 0.75mmol/L, and now signal amplification effect clearly;
In other case study on implementation of the present invention, substrate Small molecular concentration 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, substrate Small molecular concentration 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, substrate Small molecular concentration 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, substrate Small molecular concentration 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, substrate Small molecular concentration 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 surfactant percent by volume accounted in described mixed liquor is 0.005 ~ 0.05%.
The size of concentration on enzyme-to-substrate interactional SPRI signal of surfactant has very large impact.When the concentration of surfactant is lower than critical micelle concentration (CMC), substrate molecule can not assemble the macromolecular micella of formation, and now SPR maybe can not can only detect the actuating signal of faint enzyme-to-substrate molecule; When the concentration of surfactant equal or a little higher than critical micelle concentration (CMC) time, single substrate Small molecular is gathered into micella, and the large molecule micella of enzymatic, produces stronger SPRI signal; When the concentration of surfactant reaches certain value, SPRI signal strengthens trend and slows down, and approaches to saturation.
In other case study on implementation of the present invention, surfactant TritonX100(Value 3608) at damping fluid PBS(phosphate buffer) in concentration be 0.05%(v/v), with this solution dilution process small molecule substrates (10mmol/L).
In other case study on implementation of the present invention, surfactant TritonX100(Value 3608) at damping fluid PBS(phosphate buffer) in concentration be 0.01%(v/v), with this solution dilution process small molecule substrates (10mmol/L).
In other case study on implementation of the present invention, surfactant TritonX100(Value 3608) at damping fluid PBS(phosphate buffer) in concentration be 0.005%(v/v), with this solution dilution process small molecule substrates (10mmol/L).
Damping fluid of the present invention, comprise the most damping fluids commonly used in experiment, as PBS(phosphate) damping fluid, glycine-HCI damping fluid, phthalic acid-hydrochloride buffer, sodium hydrogen phosphate-sodium citrate buffer solution, citric acid-sodium hydroxide-hydrochloride buffer, citric acid-sodium citrate damping fluid, Acetic acid-sodium acetate damping fluid, sodium dihydrogen phosphate-sodium hydrate buffer solution, boric acid-borate buffer solution, Glycine-NaOH damping fluid, sodium carbonate-bicarbonate damping fluid etc., but Tris damping fluid (TRIS buffer) is not included.
The SPR device that the present invention relates to, comprise the SPR device detected based on angle, based on the SPR device of wavelength detecting, based on the SPR device of intensity detection, based on the SPR device of phase-detection, also comprise based on micro-fluidic simultaneously, the SPR device that automatic sample handling system combines, also comprises simultaneously and carries out based on SPR character all the other SPR apparatuss of detecting, also comprise 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 physically based deformation absorption, chemical covalent effect, noncovalent interaction, and if the fixing means that produces of the acting force such as hydrogen bond.
The surface substrate that chip of the present invention adopts, comprises the surface of various metal and alloy, and the surface of the inorganics such as various glass and quartz, also comprises the surface that macromolecule such as dimethyl silicone polymer and polystyrene etc. can be used for the protein of fixed nucleic acid.Also comprise coarse surface simultaneously, comprise molecular surface through micro-nano process and modification as the Electrospun nonwoven surface of different materials, and through adhesive surface prepared by physics and chemistry and biological method.
In above-mentioned steps of the present invention, the method for surfactant process small molecule substrates comprises: directly dilute substrate with the damping fluid adding surfactant; First dilute substrate to experimental concentration with damping fluid, then add surfactant
Directly comprise by the step that the damping fluid adding surfactant dilutes the method for substrate in above-mentioned steps: prepare damping fluid; Concentration according to the surfactant of setting adds surfactant by volume in damping fluid; With the above-mentioned dilution of the damping fluid containing the surfactant substrate Small molecular mother liquor prepared to experimental concentration.
First dilute substrate to experimental concentration with damping fluid in above-mentioned steps, then the step of the method adding surfactant comprises: prepare damping fluid; With the damping fluid dilution substrate Small molecular mother liquor prepared to experimental concentration; Concentration according to the surfactant of setting adds surfactant by volume in the small molecule solution diluted.
In above-mentioned steps by the method step that the probe that the Small molecular handled well and surface are fixed is combined be: PBS process biochip; The small molecule substrates handled well is passed into the surface to SPR chip, carries 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.
Present invention also offers a kind of method that surface plasma resonance detects low molecular weight substance, comprise the steps:
Step 1: the mixed liquor obtaining surfactant and damping fluid; Get mixed liquor to mix with low molecular weight substance, obtain test substance;
Step 2: at chip surface stationary probe and negative control substances respectively, pass into mixed liquor and scan, read light intensity signal value, obtain determinand initial value, negative control initial value respectively; Probe can with low molecular weight substance specific binding;
Step 3: pass into test substance, the negative control substances that test substance is fixed with chip surface is not combined, and reads light intensity signal value, obtains negative control measured value; Pass into mixed liquor again to rinse until baseline is steady;
Pass into test substance, the probe that test substance is fixed with chip surface is combined, and reads light intensity signal value, obtains determinand measured value; Pass into mixed liquor again to rinse until baseline is steady;
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, to obtain final product.
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 TritonX100.
In other case study on implementation of the present invention, non-ionics provided by the invention: TritonX100(Value 3608), relative molecular mass is 324.45, and molecular formula is C
18h
28o
5.
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-Nitrophenyl butyrate, 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, substrate Small molecular provided by the invention is: 4-Nitrophenyl butyrate (4-Nitrophenylbutyrate), and 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, substrate Small molecular provided by the invention is: 4-nitrobenzophenone capronate (4-Nitrophenylhexanoate), and molecular formula is C
12h
15nO
4, relative molecular mass is 237.26.Structural formula is II:
Formula II
In other case study on implementation of the present invention, substrate Small molecular provided by the invention is: 4-nitrobenzophenone caprylate (4-Nitrophenyloctanoate), and 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, substrate Small molecular provided by the invention is: the 4-nitrobenzene last of the ten Heavenly stems (4-Nitrophenyldecanoate), and 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, substrate Small molecular provided by the invention is: 4-nitrobenzophenone laurate (4-Nitrophenyldodecanoate), and 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, substrate Small molecular provided by the invention is: 4-nitrobenzene myristinate (4-Nitrophenylmyristate), and 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, substrate Small molecular provided by the invention is: 4-nitrobenzene palmitate (4-Nitrophenylmyristate), and 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, substrate Small molecular provided by the invention is: stearic acid p-nitrophenyl ester (4-Nitrophenylstearate), and molecular formula is C
24h
39nO
4, 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 micromolecular concentration of substrate has certain influence to the effect that signal amplifies.Keep the concentration of surfactant constant, when small molecule substrates concentration is higher, the micella quantity of generation is many, and signal amplification effect clearly; Along with the reduction of small molecule substrates concentration, signal amplification effect reduces; When the micromolecular concentration of substrate is lower than certain value, Small molecular still exists with single status in the 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, substrate Small molecular concentration provided by the invention is 1mM, and now signal amplification effect clearly;
In other case study on implementation of the present invention, substrate Small molecular concentration provided by the invention is 0.75mM, and now signal amplification effect clearly;
In other case study on implementation of the present invention, substrate Small molecular concentration provided by the invention is 0.5mM, and now signal amplification effect still obviously;
In other case study on implementation of the present invention, substrate Small molecular concentration provided by the invention is 0.3mM, and now signal amplification effect still obviously;
In other case study on implementation of the present invention, substrate Small molecular concentration 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, substrate Small molecular concentration provided by the invention is 0.1mM, and now signal amplification effect weakens greatly;
In other case study on implementation of the present invention, substrate Small molecular concentration 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 surfactant percent by volume accounted in described mixed liquor is 0.005 ~ 0.05%.
The size of concentration on enzyme-to-substrate interactional SPRI signal of surfactant has very large impact.When the concentration of surfactant is lower than critical micelle concentration (CMC), substrate molecule can not assemble the macromolecular micella of formation, and now SPR maybe can not can only detect the actuating signal of faint enzyme-to-substrate molecule; When the concentration of surfactant equal or a little higher than critical micelle concentration (CMC) time, single substrate Small molecular is gathered into micella, and the large molecule micella of enzymatic, produces stronger SPRI signal; When the concentration of surfactant reaches certain value, SPRI signal strengthens trend and slows down, and approaches to saturation.
In other case study on implementation of the present invention, surfactant TritonX100(Value 3608) at damping fluid PBS(phosphate buffer) in concentration be 0.05%(v/v), with this solution dilution process small molecule substrates (10mM).
In other case study on implementation of the present invention, surfactant TritonX100(Value 3608) at damping fluid PBS(phosphate buffer) in concentration be 0.01%(v/v), with this solution dilution process small molecule substrates (10mM).
In other case study on implementation of the present invention, surfactant TritonX100(Value 3608) at damping fluid PBS(phosphate buffer) in concentration be 0.005%(v/v), with this solution dilution process small molecule substrates (10mM).
Damping fluid of the present invention, comprise the most damping fluids commonly used in experiment, as PBS(phosphate) damping fluid, glycine-HCI damping fluid, phthalic acid-hydrochloride buffer, sodium hydrogen phosphate-sodium citrate buffer solution, citric acid-sodium hydroxide-hydrochloride buffer, citric acid-sodium citrate damping fluid, Acetic acid-sodium acetate damping fluid, sodium dihydrogen phosphate-sodium hydrate buffer solution, boric acid-borate buffer solution, Glycine-NaOH damping fluid, sodium carbonate-bicarbonate damping fluid etc., but Tris damping fluid (TRIS buffer) is not included.
SPR device of the present invention, comprise the SPR device detected based on angle, based on the SPR device of wavelength detecting, based on the SPR device of intensity detection, based on the SPR device of phase-detection, also comprise based on micro-fluidic simultaneously, the SPR device that automatic sample handling system combines, also comprises simultaneously and carries out based on SPR character all the other SPR apparatuss of detecting, also comprise local SPR device simultaneously, and long-range SPR device, SPR device of waveguide mode etc.
Surperficial fixing means of the present invention, comprises physically based deformation absorption, chemical covalent effect, noncovalent interaction, and if the fixing means that produces of the acting force such as hydrogen bond.
Surface substrate of the present invention, comprises the surface of various metal and alloy, and the surface of the inorganics such as various glass and quartz, also comprises the surface that macromolecule such as dimethyl silicone polymer and polystyrene etc. can be used for the protein of fixed nucleic acid.Also comprise coarse surface simultaneously, comprise molecular surface through micro-nano process and modification as the Electrospun nonwoven surface of different materials, and through adhesive surface prepared by physics and chemistry and biological method.
In above-mentioned steps 1 of the present invention, the method for surfactant process small molecule substrates comprises: directly dilute substrate with the damping fluid adding surfactant; First dilute substrate to experimental concentration with damping fluid, then add surfactant
Directly comprise by the step that the damping fluid adding surfactant dilutes the method for substrate in above-mentioned steps: prepare damping fluid; Concentration according to the surfactant of setting adds surfactant by volume in damping fluid; With the above-mentioned dilution of the damping fluid containing the surfactant substrate Small molecular mother liquor prepared to experimental concentration.
First dilute substrate to experimental concentration with damping fluid in above-mentioned steps, then the step of the method adding surfactant comprises: prepare damping fluid; With the damping fluid dilution substrate Small molecular mother liquor prepared to experimental concentration; Concentration according to the surfactant of setting adds surfactant by volume in the small molecule solution diluted.
In above-mentioned steps by the method step that the probe that the Small molecular handled well and surface are fixed is combined be: PBS process biochip; The small molecule substrates handled well is passed into the surface to SPR chip, carries 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 the concentration of low molecular weight substance is 0.3 ~ 1mmol/L.
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 surfactant percent by volume accounted in described mixed liquor is 0.005 ~ 0.05%.
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 invention provides the effect of surfactant, surface plasma resonance detect low molecular weight substance signal amplify method.The method is, by surfactant, molecule less for relative molecular mass is gathered into micella, becomes large molecule and enzyme reacts, and sensitivity is very high, reaction velocity very fast, is therefore well suited for carrying out fast, Sensitive Detection; The method that signal used in the present invention amplifies is the principle being 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 using the method for the signal amplification of the micromolecular SPR of surfactant Treatment Analysis thing, SPR method not only being had and exempts from mark, the real-time feature detected, being also provided with high sensitivity, fast detection, simple to operation, the repeated advantages of higher of result.The qualification of enzyme-to-substrate can be applied to, the selection systems of high flux lipase, also can be used for detecting fast and accurately of infectious disease, the field such as medicine and macromolecular interaction.The realization condition of the method is simple, and its high sensitivity and high-repetition-rate, so the quick test being well suited for common lab and field uses.
Accompanying drawing explanation
Fig. 1 shows in embodiment 1 the surface plasma resonance figure after adding 0.05%TritonX100 damping fluid; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 2 shows in embodiment 1 the surface plasma resonance figure after adding 0.01%TritonX100 damping fluid; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 3 shows in embodiment 1 the surface plasma resonance figure after adding 0.005%TritonX100 damping fluid; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 4 shows in embodiment 1 the surface plasma resonance figure after not adding surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 5 shows in embodiment 2 the surface plasma resonance figure after adding surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control;
Fig. 6 shows in embodiment 2 the surface plasma resonance figure after not adding surfactant; Wherein, curve 1 shows determinand, and curve 2 shows negative control.
Embodiment
The invention discloses the effect of surfactant, method that signal that surface plasma resonance detects low molecular weight substance amplifies, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, 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 not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In the method that the signal that the effect of surfactant provided by the invention, surface plasma resonance detect low molecular weight substance amplifies, agents useful for same all can be buied by market.
Below in conjunction with embodiment, set forth the present invention further:
Embodiment 1 carries out the amplification of SPRI signal by surfactant TritonX-100 process substrate Small molecular 4-nitrobenzophenone caprylate (4-Nitrophenyloctanoate)
1. fix the lipase CalB of 100ug/ml at the chip surface of SPRI, using the bovine serum albumin BSA of 200ug/ml as negative control;
2. prepare PBS+0.05%TritonX100 damping fluid;
3. chip is fixed on SPRI device, through the scanning that procedure Selection lipase CalB and bovine serum albumin BSA institute fixed position carry out intensity, surface is passed into the scanning that PBS+0.05%TritonX100 damping fluid carries out baseline, as shown in Fig. 1 baseline;
4. with the PBS+0.05%TritonX100 damping fluid prepared, substrate Small molecular 4-nitrobenzophenone caprylate is diluted to 0.25mM from mother liquor 10mM, obtains sample 1;
5. pass into sample 1 with the association rate of 2ul/s, as shown in Fig. 1 binding curve;
6. pass into PBS+0.05%TritonX100 damping fluid to carry out rinsing until baseline is steady, as shown in Fig. 1 dissociation curve.
7. keep above-mentioned experimental procedure constant, change the concentration of TritonX100, namely PBS+0.05%TritonX100 is changed to PBS+0.01%TritonX100.Carry out the dilution process of substrate with PBS+0.01%TritonX100, obtain sample 2, loading is carried out in same operation, and carries out the flushing of baseline, and signal as shown in Figure 2; PBS+0.05%TritonX100 is changed to PBS+0.005%TritonX100, carries out the dilution process of substrate with it, obtain sample 3, loading is carried out in same operation, and carries out the flushing of baseline, and signal as shown in Figure 3; After TritonX100 concentration reduces, the effect that signal amplifies reduces.
8. keep above-mentioned experimental procedure constant, change damping fluid, namely PBS+0.05%TritonX100 is changed to PBS(phosphate buffer), do not add TritonX100.Carry out the dilution process of substrate with PBS, obtain sample 4, 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 2 carries out the amplification of SPRI signal by surfactant TritonX100 process substrate Small molecular 4-nitrobenzophenone caprylate (4-Nitrophenyloctanoate)
1. fix the lipase CalB of 100ug/ml at the chip surface of SPRI, using the bovine serum albumin BSA of 200ug/ml as negative control;
2. prepare PBS+0.05%TritonX100 damping fluid;
1. chip is fixed on SPRI device, through the scanning that procedure Selection lipase CalB and bovine serum albumin BSA institute fixed position carry out intensity, surface is passed into the scanning that PBS damping fluid carries out baseline, as shown in Fig. 5 baseline;
2. with the PBS+0.05%TritonX100 damping fluid prepared, substrate Small molecular 4-nitrobenzophenone caprylate is diluted to 0.25mM from mother liquor 10mM, obtains sample 1;
3. pass into sample 1 with the association rate of 2ul/s, binding time is 400s, as shown in Fig. 5 binding curve;
4. pass into PBS damping fluid to carry out rinsing until baseline is steady, as shown in Fig. 5 dissociation curve.
Keep above-mentioned experimental procedure constant, change the concentration of TritonX100, namely
PBS+0.05%Tween-20 is changed to PBS+0.01%Tween-20.Carry out the dilution process of substrate with PBS+0.01%Tween-20, obtain sample 2, loading is carried out in same operation, and carries out the flushing of baseline, and signal as shown in Figure 6.After TritonX100 concentration reduces, the effect that signal amplifies reduces.
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 (12)
1. 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;
Get after described mixed liquor mixes with described low molecular weight substance and detect through surface plasma resonance, to obtain final product;
Surfactant is non-ionics;
Described surfactant is TritonX100.
2. method for amplifying signal according to claim 1, is characterized in that, the relative molecular weight of described low molecular weight substance is less than 500.
3. method for amplifying signal according to claim 1, it is characterized in that, described low molecular weight substance is 4-Nitrophenyl butyrate, 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.
4. method for amplifying signal according to claim 1, is characterized in that, the concentration of described low molecular weight substance is 0.3 ~ 1mmol/L.
5. method for amplifying signal according to claim 1, is characterized in that, the described surfactant percent by volume accounted in described mixed liquor is 0.005 ~ 0.05%.
6. surface plasma resonance detects a method for low molecular weight substance, it is characterized in that, comprises the steps:
Step 1: the mixed liquor obtaining surfactant and damping fluid; Get described mixed liquor to mix with low molecular weight substance, obtain test substance;
Step 2: at chip surface stationary probe and negative control substances respectively, pass into described mixed liquor and scan, read light intensity signal value, obtain determinand initial value, negative control initial value respectively; Described probe can with described low molecular weight substance specific binding;
Step 3: pass into described test substance, the described negative control substances that described test substance is fixed with chip surface is not combined, and reads light intensity signal value, obtains negative control measured value; Pass into described mixed liquor again to rinse until baseline is steady;
Pass into described test substance, the described probe that described test substance is fixed with chip surface is combined, and reads light intensity signal value, obtains determinand measured value; Pass into described mixed liquor again to rinse until baseline is steady;
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, to obtain final product;
Surfactant is non-ionics;
Described surfactant is TritonX100.
7. method according to claim 6, is characterized in that, the relative molecular weight of described low molecular weight substance is less than 500.
8. method according to claim 6, it is characterized in that, described low molecular weight substance is 4-Nitrophenyl butyrate, 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 according to claim 6, is characterized in that, the concentration of described low molecular weight substance is 0.3 ~ 1mmol/L.
10. method according to claim 6, is characterized in that, the described surfactant percent by volume accounted in described mixed liquor is 0.005 ~ 0.05%.
11. methods according to claim 6, is characterized in that, described probe is enzyme.
12. methods according to claim 11, is characterized in that, described enzyme is lipase or proteinase.
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