CN103728278B - The effect of surfactant, surface plasma resonance detect the method that the signal of low molecular weight substance amplifies - Google Patents
The effect of surfactant, surface plasma resonance detect the method that the signal of low molecular weight substance amplifies Download PDFInfo
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Abstract
The present invention relates to bio information field, the method being specifically related to the signal amplification of the effect of surfactant, surface plasma resonance detection low molecular weight substance。The method is, by surfactant, molecule less for relative molecular mass is gathered into micelle, becomes big molecule and reacts with enzyme, and sensitivity is very high, response speed very fast, is therefore well suited for carrying out quickly, Sensitive Detection；The method that signal used in the present invention amplifies is based on surfactant and is gathered into the principle of micelle, compared with the method that other signals amplify, easy and simple to handle, and experimental repeatability is high。
Description
Technical field
The present invention relates to bio information field, particularly to the method for the effect of surfactant, the signal amplification of surface plasma resonance detection low molecular weight substance。
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 when prism is with metallic film surface generation total reflection phenomenon, can produce evanescent waves in metal film, and when evanescent waves, with surface plasma-wave, resonance occurs, the intensity of the reflection light detected can greatly weaken。Energy transfers to surface plasma from photon, and most of energy of incident illumination is absorbed by surface plasma-wave, so that the energy of reflection light sharply reduces。Now corresponding angle of incidence 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 biosensor of First is born in the world, in practice it has proved that, with traditional detection comparatively, spr sensor has the outstanding advantages such as prominent label-free, rapid sensitive and detection in real time。So, at present to be widely used in protein-protein interaction, nucleic acid interaction, between macromole and macromole, the interaction between macromole and little molecule, 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 is traditional SPR detection technique to combine CCD take the photograph a kind of rapid high throughput assays method of spectrum, it is that the intensity to light is monitored in real time, can obtaining multiple spot resonance curve, retractility is very big simultaneously。Therefore, SPRI except possess label-free, quickly and except the advantage of detection in real time, high throughput testing can be carried out again, it will in genomics, protein science, drug screening, epitope, the aspect such as Chinese medicine fraction screening plays huge application。
Although SPR has many advantages as biosensor, but sensitivity is limited。The measurement scope of SPRI is very big with the molecular mass relation of analyte。The material of high molecular is able to detect that when low concentration, but low-molecular-weight material (< 1000) to it is possible to when higher concentration monitor less reaction signal, and owing to the non-specific adsorption effect of background dot can produce certain signal equally, so will not can determine that making existing signal is positive or false positive in such cases。So, try to explore various highly sensitive little Molecular Detection analysis method and become an important content in SPR research。
Improving in this problem of sensitivity, researcheres have done substantial amounts of work。Have been reported that use nanogold particle carries out signal amplification at present, such as by the submicron micelle containing antigen and the antibodies being fixed on vane surface, carried out iodine signal；In the research of DNA hybridization experiment, single stranded DNA is fixed on gold film surface by researcher, then gold colloidal nanoparticle is sticked on single stranded DNA, it is introduced into sample cell to contact with complementary DNA, there is hybridization, by the field coupling amplification of gold 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 that Avidin-Biotin system is used for amplifying surface plasma resonance immunosensor, stick to the Avidin on gold film to combine with biotinylated antibody the network-like complex of formation, immune response signal is amplified, and the corresponding signal detected increases considerably。It will be appreciated, however, that in the process, the ratio being used for preparing the biotinylated antibody of compound antibody and Avidin is wanted suitably。If Avidin is too much, then being difficult to form big network-like molecular complex, in compound antibody solution, the Avidin in complex can be played competitive inhibition by free Avidin simultaneously。Otherwise, if Avidin is very few, then biotin-binding site thereon be entirely biotinylated antibody close, make compound antibody can not with detection antibody on biotin reaction, so although Avidin-Biotin system improves the sensitivity of detection to a certain extent, but simultaneously complicated experimentation。And micromolecular detection is being analyzed application seldom by these methods, therefore, find a kind of simple to operation and can just seem particularly necessary with the method improving low molecular weight substance detection sensitivity by greatly enlarged reaction signal。
Summary of the invention
In view of this, the method that the present invention provides the effect of surfactant, the signal of surface plasma resonance detection low molecular weight substance amplifies。The method is, by surfactant, molecule less for relative molecular mass is gathered into micelle, becomes big molecule and reacts with enzyme, and sensitivity is very high, response speed very fast, is therefore well suited for carrying out quickly, Sensitive Detection；The method that signal used in the present invention amplifies is based on surfactant and is gathered into the principle of micelle, compared with the method that other signals amplify, easy and simple to handle, and experimental repeatability is high。
The method that signal used in the present invention amplifies is based on surfactant and is gathered into the principle of micelle, compared with the method that other signals amplify, easy and simple to handle, and experimental repeatability is high。
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides the surfactant application as surface plasma resonance sensitizer。
In some embodiments of the invention, surfactant is nonionic surfactant。
Surfactant of the present invention, including ionic surfactant and nonionic surfactant。As preferably, surfactant is Tween-80。
In other case study on implementation of the present invention, amphoteric ionic surfactant provided by the invention: Tween-80(Tween 80), relative molecular mass is 428.6, and molecular formula is C24H44O6。
Present invention also offers the method for amplifying signal of a kind of surface plasma resonance detection low molecular weight substance, it is thus achieved that the mixed liquor of surfactant and buffer；Take and detect through surface plasma resonance after mixed liquor mixes with described low molecular weight substance, to obtain final product。
In some embodiments of the invention, in the method for amplifying signal of a kind of surface plasma resonance provided by the invention detection low molecular weight substance, surfactant is nonionic surfactant。
As preferably, surfactant is Tween-80。
In other case study on implementation of the present invention, amphoteric ionic surfactant provided by the invention: Tween-80(Tween 80), relative molecular mass is 428.6, and molecular formula is C24H44O6。
In some embodiments of the invention, in the method for amplifying signal of a kind of surface plasma resonance provided by the invention detection low molecular weight substance, 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, in the method for amplifying signal of a kind of surface plasma resonance provided by the invention detection low molecular weight substance, low molecular weight substance is 4-Nitrophenyl butyrate, 4-nitrophenyl caprylate, 4-nitrobenzophenone caprylate, 4-Nitrobenzol last of the ten Heavenly stems, 4-nitrobenzophenone laurate, 4-Nitrobenzol myristinate, 4-Nitrobenzol cetylate or stearic acid p-nitrophenyl ester。
In some case study on implementation of the present invention, the little molecule of substrate provided by the invention is: 4-Nitrophenyl butyrate (4-Nitrophenylbutyrate), and molecular formula is C10H11NO4, 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-nitrophenyl caprylate (4-Nitrophenylhexanoate), and molecular formula is C12H15NO4, 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-Nitrophenyloctanoate), and molecular formula is C14H19NO4, 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-Nitrobenzol last of the ten Heavenly stems (4-Nitrophenyldecanoate), and molecular formula is C16H23NO4, 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-Nitrophenyldodecanoate), and molecular formula is C18H27NO4, 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-Nitrobenzol myristinate (4-Nitrophenylmyristate), and molecular formula is C20H31NO4, 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-Nitrobenzol cetylate (4-Nitrophenylmyristate), and molecular formula is C22H35NO4, 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-Nitrophenylstearate), and molecular formula is C24H39NO4, relative molecular mass is 405.57。Structural formula is VIII:
In some embodiments of the invention, in the method for amplifying signal of a kind of surface plasma resonance provided by the invention detection low molecular weight substance, the concentration of low molecular weight substance is 0.3～1mmol/L。
The effect that signal amplifies is had certain impact by the micromolecular concentration of substrate。The concentration keeping surfactant is constant, and when small molecule substrates concentration is higher, the micelle quantity of generation is many, and signal amplification effect is 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, little molecule still exists with single status in the solution, being formed without micelle, now no signal amplification effect, its result is identical with the result being added without surfactant: signal is only small or is barely perceivable the SPRI signal that enzyme-to-substrate interacts。
In other case study on implementation of the present invention, the little molecular concentration of substrate provided by the invention is 1mmol/L, and now signal amplification effect is clearly；
In other case study on implementation of the present invention, the little molecular concentration of substrate provided by the invention is 0.75mmol/L, and now signal amplification effect is clearly；
In other case study on implementation of the present invention, the little molecular concentration of substrate provided by the invention is 0.5mmol/L, and now signal amplification effect is still substantially；
In other case study on implementation of the present invention, the little molecular concentration of substrate provided by the invention is 0.3mmol/L, and now signal amplification effect is still substantially；
In other case study on implementation of the present invention, the little molecular concentration 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 concentration of substrate provided by the invention is 0.1mmol/L, and now signal amplification effect weakens significantly；
In other case study on implementation of the present invention, the little molecular concentration of substrate provided by the invention is 0.05mmol/L, now no signal amplification effect, signal results is identical with the result being added without surfactant: signal is only small or is barely perceivable the SPRI signal that enzyme-to-substrate interacts。
In some embodiments of the invention, in the method for amplifying signal of a kind of surface plasma resonance provided by the invention detection low molecular weight substance, it is 0.05% that surfactant accounts for the percent by volume in described mixed liquor。
The size of the SPRI signal that enzyme-to-substrate is interacted by the concentration of surfactant has very big impact。When the concentration of surfactant is lower than critical micelle concentration (CMC), substrate molecule can not assemble the micelle forming macromole, and now SPR or can not be only able to detect the actuating signal of faint enzyme-to-substrate molecule；When surfactant concentration equal to or during a little higher than critical micelle concentration (CMC), the little molecule of single substrate is gathered into micelle, enzyme catalysis macromole micelle, 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 Tween-80(Tween 80) at buffer PBS(phosphate buffer) in concentration be 0.05%(v/v), process small molecule substrates (10mmol/L) with this solution dilution。
In other case study on implementation of the present invention, surfactant Tween-80(Tween 80) at buffer PBS(phosphate buffer) in concentration be 0.01%(v/v), process small molecule substrates (10mmol/L) with this solution dilution。Concentration now can't see amplification effect。
Buffer of the present invention, including the most buffer commonly used in experiment, such as PBS(phosphate) buffer, glycine-HCI buffer, phthalic acid-hydrochloride buffer, disodium hydrogen phosphate-sodium citrate buffer solution, citric acid-sodium hydroxide-hydrochloride buffer, citric acid-sodium citrate buffer, Acetic acid-sodium acetate buffer, sodium dihydrogen phosphate-sodium hydrate buffer solution, boric acid-borate buffer solution, Glycine-NaOH buffer, sodium carbonate-bicarbonate buffer etc., but Tris buffer (TRIS buffer) is not included。
The SPR device that the present invention relates to, including 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 include based on micro-fluidic simultaneously, the SPR device that automatic sample handling system combines, also includes carrying out, based on SPR character, all the other SPR apparatus of detecting simultaneously, also includes local SPR device simultaneously, and long-range SPR device, SPR device of waveguide mode etc.。
The surface fixing means that the present invention uses, adsorbs including physically based deformation, chemical covalent effect, noncovalent interaction, and if the fixing means that produces of the active force such as hydrogen bond。
The surface substrate that chip of the present invention adopts, including the surface of various metals and alloy, the surface of the inorganics such as various glass and quartz, also includes the surface that macromolecule such as polydimethylsiloxane and polystyrene etc. can be used for the protein of fixed nucleic acid。Also include coarse surface, including the Electrospun nonwoven surface of molecular surface such as different materials processing through micro-nano and modifying, and through adhesive surface prepared by physics and chemistry and biological method simultaneously。
In above-mentioned steps of the present invention, surfactant processes the method for small molecule substrates and includes: directly dilute substrate with the buffer adding surfactant；First dilute substrate to experimental concentration with buffer, add surfactant
The step of the method directly diluting substrate with the buffer adding surfactant in above-mentioned steps includes: prepare buffer；Concentration according to the surfactant set adds surfactant by volume in buffer；With the above-mentioned dilution little molecule mother solution of substrate of the buffer containing surfactant prepared to experimental concentration。
First diluting substrate to experimental concentration with buffer in above-mentioned steps, the step of the method adding surfactant includes: prepare buffer；With the buffer dilution little molecule mother solution of substrate prepared to experimental concentration；Concentration according to the surfactant set adds surfactant by volume in the small molecule solution diluted。
The method step that the probe that the little molecule handled well is fixing with surface is combined is by above-mentioned steps: PBS processes 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, in the method for amplifying signal of a kind of surface plasma resonance provided by the invention detection low molecular weight substance, probe is enzyme。
As preferably, in the method for amplifying signal of a kind of surface plasma resonance provided by the invention detection low molecular weight substance, enzyme is lipase or protease。
The method that present invention also offers a kind of surface plasma resonance detection low molecular weight substance, comprises the steps:
Step 1: obtain the mixed liquor of surfactant and buffer；Take mixed liquor to mix with low molecular weight substance, it is thus achieved that test substance；
Step 2: fix probe and negative control substances at chip surface respectively, passes into mixed liquor and is scanned, and reads light intensity signal value, obtains determinand initial value, negative control initial value respectively；Probe can be specific binding with low molecular weight substance；
Step 3: passing into test substance, the negative control substances that test substance is fixed with chip surface is not combined, and reads light intensity signal value, it is thus achieved that negative control measured value；Pass into mixed liquor again to rinse until baseline is steady；
Passing into test substance, the probe that test substance is fixed with chip surface is combined, and reads light intensity signal value, it is thus achieved that determinand measured value；Pass into mixed liquor again to rinse until baseline is steady；
Step 4: take negative control measured value and deduct negative control initial value acquisition negative control changing value；
Take 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, in the method for a kind of surface plasma resonance provided by the invention detection low molecular weight substance, surfactant is nonionic surfactant。
Surfactant of the present invention, including ionic surfactant and nonionic surfactant。In some embodiments of the invention, in the method for a kind of surface plasma resonance provided by the invention detection low molecular weight substance, surfactant is Tween-80。
In other case study on implementation of the present invention, amphoteric ionic surfactant provided by the invention: Tween-80(Tween 80), relative molecular mass is 428.6, and molecular formula is C24H44O6。
In some embodiments of the invention, in the method for a kind of surface plasma resonance provided by the invention detection low molecular weight substance, 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, in the method for a kind of surface plasma resonance provided by the invention detection low molecular weight substance, low molecular weight substance is 4-Nitrophenyl butyrate, 4-nitrophenyl caprylate, 4-nitrobenzophenone caprylate, 4-Nitrobenzol last of the ten Heavenly stems, 4-nitrobenzophenone laurate, 4-Nitrobenzol myristinate, 4-Nitrobenzol cetylate or stearic acid p-nitrophenyl ester。
In some case study on implementation of the present invention, the little molecule of substrate provided by the invention is: 4-Nitrophenyl butyrate (4-Nitrophenylbutyrate), and molecular formula is C10H11NO4, 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-nitrophenyl caprylate (4-Nitrophenylhexanoate), and molecular formula is C12H15NO4, 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-Nitrophenyloctanoate), and molecular formula is C14H19NO4, 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-Nitrobenzol last of the ten Heavenly stems (4-Nitrophenyldecanoate), and molecular formula is C16H23NO4, 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-Nitrophenyldodecanoate), and molecular formula is C18H27NO4, 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-Nitrobenzol myristinate (4-Nitrophenylmyristate), and molecular formula is C20H31NO4, 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-Nitrobenzol cetylate (4-Nitrophenylmyristate), and molecular formula is C22H35NO4, 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-Nitrophenylstearate), and molecular formula is C24H39NO4, relative molecular mass is 405.57。Structural formula is VIII:
In some embodiments of the invention, in the method for a kind of surface plasma resonance provided by the invention detection low molecular weight substance, the concentration of low molecular weight substance is 0.3～1mmol/L。
The effect that signal amplifies is had certain impact by the micromolecular concentration of substrate。The concentration keeping surfactant is constant, and when small molecule substrates concentration is higher, the micelle quantity of generation is many, and signal amplification effect is 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, little molecule still exists with single status in the solution, being formed without micelle, now no signal amplification effect, its result is identical with the result being added without surfactant: signal is only small or is barely perceivable the SPRI signal that enzyme-to-substrate interacts。
In other case study on implementation of the present invention, the little molecular concentration of substrate provided by the invention is 1mM, and now signal amplification effect is clearly；
In other case study on implementation of the present invention, the little molecular concentration of substrate provided by the invention is 0.75mM, and now signal amplification effect is clearly；
In other case study on implementation of the present invention, the little molecular concentration of substrate provided by the invention is 0.5mM, and now signal amplification effect is still substantially；
In other case study on implementation of the present invention, the little molecular concentration of substrate provided by the invention is 0.3mM, and now signal amplification effect is still substantially；
In other case study on implementation of the present invention, the little molecular concentration 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 concentration of substrate provided by the invention is 0.1mM, and now signal amplification effect weakens significantly；
In other case study on implementation of the present invention, the little molecular concentration of substrate provided by the invention is 0.05mM, now no signal amplification effect, signal results is identical with the result being added without surfactant: signal is only small or is barely perceivable the SPRI signal that enzyme-to-substrate interacts。
In some embodiments of the invention, in the method for a kind of surface plasma resonance provided by the invention detection low molecular weight substance, it is 0.05% that surfactant accounts for the percent by volume in described mixed liquor。
The size of the SPRI signal that enzyme-to-substrate is interacted by the concentration of surfactant has very big impact。When the concentration of surfactant is lower than critical micelle concentration (CMC), substrate molecule can not assemble the micelle forming macromole, and now SPR or can not be only able to detect the actuating signal of faint enzyme-to-substrate molecule；When surfactant concentration equal to or during a little higher than critical micelle concentration (CMC), the little molecule of single substrate is gathered into micelle, enzyme catalysis macromole micelle, 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 Tween-80(Tween 80) at buffer PBS(phosphate buffer) in concentration be 0.05%(v/v), process small molecule substrates (10mM) with this solution dilution。
In other case study on implementation of the present invention, surfactant Tween-80(Tween 80) at buffer PBS(phosphate buffer) in concentration be 0.01%(v/v), process small molecule substrates (10mM) with this solution dilution。Concentration now can't see amplification effect。
Buffer of the present invention, including the most buffer commonly used in experiment, such as PBS(phosphate) buffer, glycine-HCI buffer, phthalic acid-hydrochloride buffer, disodium hydrogen phosphate-sodium citrate buffer solution, citric acid-sodium hydroxide-hydrochloride buffer, citric acid-sodium citrate buffer, Acetic acid-sodium acetate buffer, sodium dihydrogen phosphate-sodium hydrate buffer solution, boric acid-borate buffer solution, Glycine-NaOH buffer, sodium carbonate-bicarbonate buffer etc., but Tris buffer (TRIS buffer) is not included。
SPR device of the present invention, including 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 include based on micro-fluidic simultaneously, the SPR device that automatic sample handling system combines, also includes carrying out, based on SPR character, all the other SPR apparatus of detecting simultaneously, also includes local SPR device simultaneously, and long-range SPR device, SPR device of waveguide mode etc.。
Surface of the present invention fixing means, adsorbs including physically based deformation, chemical covalent effect, noncovalent interaction, and if the fixing means that produces of the active force such as hydrogen bond。
Surface substrate of the present invention, including the surface of various metals and alloy, the surface of the inorganics such as various glass and quartz, also includes the surface that macromolecule such as polydimethylsiloxane and polystyrene etc. can be used for the protein of fixed nucleic acid。Also include coarse surface, including the Electrospun nonwoven surface of molecular surface such as different materials processing through micro-nano and modifying, and through adhesive surface prepared by physics and chemistry and biological method simultaneously。
In above-mentioned steps 1 of the present invention, surfactant processes the method for small molecule substrates and includes: directly dilute substrate with the buffer adding surfactant；First dilute substrate to experimental concentration with buffer, add surfactant
The step of the method directly diluting substrate with the buffer adding surfactant in above-mentioned steps includes: prepare buffer；Concentration according to the surfactant set adds surfactant by volume in buffer；With the above-mentioned dilution little molecule mother solution of substrate of the buffer containing surfactant prepared to experimental concentration。
First diluting substrate to experimental concentration with buffer in above-mentioned steps, the step of the method adding surfactant includes: prepare buffer；With the buffer dilution little molecule mother solution of substrate prepared to experimental concentration；Concentration according to the surfactant set adds surfactant by volume in the small molecule solution diluted。
The method step that the probe that the little molecule handled well is fixing with surface is combined is by above-mentioned steps: PBS processes 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, in the method for a kind of surface plasma resonance provided by the invention detection low molecular weight substance, the concentration of low molecular weight substance is 0.3～1mmol/L。
In some embodiments of the invention, in the method for a kind of surface plasma resonance provided by the invention detection low molecular weight substance, it is 0.05% that surfactant accounts for the percent by volume in described mixed liquor。
In some embodiments of the invention, in the method for a kind of surface plasma resonance provided by the invention detection low molecular weight substance, probe is enzyme。
In some embodiments of the invention, in the method for a kind of surface plasma resonance provided by the invention detection low molecular weight substance, enzyme is lipase or protease。
The method that the present invention provides the effect of surfactant, the signal of surface plasma resonance detection low molecular weight substance amplifies。The method is, by surfactant, molecule less for relative molecular mass is gathered into micelle, becomes big molecule and reacts with enzyme, and sensitivity is very high, response speed very fast, is therefore well suited for carrying out quickly, Sensitive Detection；The method that signal used in the present invention amplifies is based on surfactant and is gathered into the principle of micelle, compared with the method that other signals amplify, easy and simple to handle, and experimental repeatability is high。
By the method using the signal of the surfactant micromolecular SPR of Treatment Analysis thing to amplify, make SPR method not only have the feature of detection label-free, real-time, be also provided with high sensitivity, quickly detection, simple to operation, the repeated advantages of higher of result。Can apply to the qualification of enzyme-to-substrate, the screening of high flux lipase and qualification, it may also be used for the fields such as the quick and accurate of infectious disease detects, the interaction of medicine and macromole。The condition that realizes of the method is simple, and its high sensitivity and high-repetition-rate, so what be well suited for common lab and field quickly tests use。
Accompanying drawing explanation
Fig. 1 shows the surface plasma resonance figure after adding 0.05%Tween-80 buffer in embodiment 1；Wherein, curve 1 shows determinand, and curve 2 shows negative control；
Fig. 2 shows the surface plasma resonance figure after adding 0.01%Tween-80 buffer in embodiment 1；Wherein, curve 1 shows determinand, and curve 2 shows negative control；
Fig. 3 shows the surface plasma resonance figure after adding 0.05%Tween-80 buffer in embodiment 2；Wherein, curve 1 shows determinand, and curve 2 shows negative control；
Fig. 4 shows the surface plasma resonance figure after adding 0.01%Tween-80 buffer in embodiment 2；Wherein, curve 1 shows determinand, and curve 2 shows negative control。
Detailed description of the invention
The method that the invention discloses the signal amplification of the effect of surfactant, surface plasma resonance detection low molecular weight substance, those skilled in the art can use for reference present disclosure, is suitably modified technological parameter and realizes。Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are considered as including in the present invention。Method and the application of the present invention are described already by preferred embodiment, method described herein and application substantially can be modified or suitably change and combination by related personnel in without departing from present invention, spirit and scope, realize and apply the technology of the present invention。
The effect of surfactant provided by the invention, surface plasma resonance detection low molecular weight substance signal amplify method in agents useful for same all can be buied by market。
Below in conjunction with embodiment, the present invention is expanded on further:
Embodiment 1 processes substrate little molecule 4-nitrobenzophenone caprylate (4-Nitrophenyloctanoate) by surfactant Tween-80 and carries out the amplification of SPRI signal
1. at the lipase CalB of the fixing 100ug/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-80 buffer；
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 PBS+0.05%Tween-80 buffer and carries out the scanning of baseline, as shown in Fig. 1 baseline；
4. with the PBS+0.05%Tween-80 buffer prepared by little for substrate molecule 4-nitrobenzophenone caprylate
It is diluted to 0.25mM from mother solution 10mM, obtains sample 1；
5. pass into sample 1 with the association rate of 2ul/s, as shown in Fig. 1 binding curve；
6. passing into PBS+0.05%Tween-80 buffer and be rinsed until baseline is steady, as shown in Fig. 1 dissociation curve, the amplification effect of 0.05%Tween-80 is compared with 0.05%Tween-20 and 0.05%TritonX100, and signal wants more weak。
7. keep above-mentioned experimental procedure constant, change the concentration of Tween-80, namely PBS+0.05%Tween-80 is changed to PBS+0.01%Tween-80。The dilution carrying out substrate with PBS+0.01%Tween-80 processes, and obtains sample 2, and same operation carries out loading, and carries out the flushing of baseline, and signal is as shown in Figure 2。When Tween-80 concentration is down to 0.01%, do not observe the SPRI signal (being equivalent to be not added with the effect of surfactant) that enzyme-to-substrate interacts。
Embodiment 2 processes substrate little molecule 4-nitrobenzophenone caprylate (4-Nitrophenyloctanoate) by surfactant Tween-80 and carries out the amplification of SPRI signal
1. at the lipase CalB of the fixing 100ug/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-80 buffer；
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 PBS and carries out the scanning of baseline, as shown in Fig. 3 baseline；
2. with the PBS+0.05%Tween-80 buffer prepared, little for substrate molecule 4-nitrobenzophenone caprylate is diluted to 0.25mM from mother solution 10mM, obtains sample 1；
3. passing into sample 1 with the association rate of 2ul/s, binding time is 400s, as shown in Fig. 3 binding curve；
4. passing into PBS and be rinsed until baseline is steady, as shown in Fig. 3 dissociation curve, the amplification effect of 0.05%Tween-80 is compared with 0.05%Tween-20 and 0.05%TritonX100 in above two cases, and signal wants more weak。
Keep above-mentioned experimental procedure constant, change the concentration of Tween-80, namely PBS+0.05%Tween-80 is changed to PBS+0.01%Tween-80。The dilution carrying out substrate with PBS+0.01%Tween-80 processes, and obtains sample 2, and same operation carries out loading, and carries out the flushing of baseline, and signal is as shown in Figure 4。When Tween-80 concentration is down to 0.01%, do not observe the SPRI signal (being equivalent to be not added with the effect of surfactant) that enzyme-to-substrate interacts。
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 making some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention。
Claims (12)
1. the method for amplifying signal of a surface plasma resonance detection low molecular weight substance, it is characterised in that
Obtain the mixed liquor of surfactant and buffer；
Take and detect through surface plasma resonance after described mixed liquor mixes with described low molecular weight substance, to obtain final product；
Surfactant is surfactant described in nonionic surfactant is Tween-80。
2. method for amplifying signal according to claim 1, it is characterised 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-nitrophenyl caprylate, 4-nitrobenzophenone caprylate, 4-Nitrobenzol last of the ten Heavenly stems, 4-nitrobenzophenone laurate, 4-Nitrobenzol myristinate, 4-Nitrobenzol cetylate or stearic acid p-nitrophenyl ester。
4. method for amplifying signal according to claim 1, it is characterised in that the concentration of described low molecular weight substance is 0.3～1mmol/L。
5. method for amplifying signal according to claim 1, it is characterised in that it is 0.05% that described surfactant accounts for the percent by volume in described mixed liquor。
6. the method for a surface plasma resonance detection low molecular weight substance, it is characterised in that comprise the steps:
Step 1: obtain the mixed liquor of surfactant and buffer；Take described mixed liquor to mix with low molecular weight substance, it is thus achieved that test substance；
Step 2: fix probe and negative control substances at chip surface respectively, passes into described mixed liquor and is scanned, and reads light intensity signal value, obtains determinand initial value, negative control initial value respectively；Described probe can be specific binding with described low molecular weight substance；
Step 3: passing 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, it is thus achieved that negative control measured value；Pass into described mixed liquor again to rinse until baseline is steady；
Passing into described test substance, the described probe that described test substance is fixed with chip surface is combined, and reads light intensity signal value, it is thus achieved that determinand measured value；Pass into described mixed liquor again to rinse until baseline is steady；
Step 4: take described negative control measured value and deduct described negative control initial value acquisition negative control changing value；
Take described determinand measured value and deduct described determinand initial value acquisition determinand changing value；
Relatively described determinand changing value and described negative control changing value, significant difference, to obtain final product；
Surfactant is nonionic surfactant；
Described surfactant is Tween-80。
7. method according to claim 6, it is characterised 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-nitrophenyl caprylate, 4-nitrobenzophenone caprylate, 4-Nitrobenzol last of the ten Heavenly stems, 4-nitrobenzophenone laurate, 4-Nitrobenzol myristinate, 4-Nitrobenzol cetylate or stearic acid p-nitrophenyl ester。
9. method according to claim 6, it is characterised in that the concentration of described low molecular weight substance is 0.3～1mmol/L。
10. method according to claim 6, it is characterised in that it is 0.05% that described surfactant accounts for the percent by volume in described mixed liquor。
11. method according to claim 6, it is characterised in that described probe is enzyme。
12. method according to claim 11, it is characterised in that described enzyme is lipase or protease。
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| CN103728279B (en) * | 2013-12-11 | 2016-06-22 | 王丽红 | The effect of surfactant, surface plasma resonance detect the method that the signal of low molecular weight substance amplifies |
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