CN101710118A - Optical immunity detecting method based on porous silicon three-element structure microcavity - Google Patents
Optical immunity detecting method based on porous silicon three-element structure microcavity Download PDFInfo
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Abstract
The invention relates to an optical immunity detecting method based on a porous silicon three-element structure microcavity, which belongs to the technical field of biological medicines, food security and environmental monitoring. An upper Bragg structure and a lower Bragg structure in a porous silicon microcavity adopted by the method are respectively formed by alternately carrying out electrochemical corrosion through three current densities; a probe molecule is fixed in a porous silicon hole at first; then the concentration of a target molecule is detected by the light spectrum peak position change before and after biological reaction; and meanwhile, a carrier is coded by a reflecting light spectrum or a photoluminescence light spectrum of the porous silicon microcavity prepared under different corrosion conditions to realize the identification to an antigen or antibody variety. The optical immunity detecting method has a plurality of excellent properties of a porous silicon and photonic bandgap structure sensor and good structural stability and can realize multielement detection by a coding detection technology. In addition, because the adopted preparation method is simpler and has relatively low cost, the invention has certain commercial application prospects.
Description
Technical field
The present invention relates to a kind of with the porous silicon three-element structure microcavity be the solid phase sensor carrier exempt from the mark optical immunity detecting method, belong to the technical field of biological medicine, food security and environmental monitoring.
Background technology
At present, the immunoassay detection technique is very active in the research in fields such as biological medicine, food security, environmental monitoring, hormone determination.This technology mainly is based on the specificity combination principle between antigen and the antibody and combines with modern means of testing, at first stationary probe biomolecule (antigen or antibody) on the test substrate is carried out the high-sensitivity analysis detection of target molecule by the change that target molecule (antigen or antibody) and probe molecule specific reaction meron signal take place.Wherein, the technology of utilizing optical signalling to carry out immune detection is exactly the optics immunoassay technology.
Immunoassay technology mainly comprises mark and exempts from mark two big classes, by tracer (enzyme, fluorescein, ferritin, collaurum, chemiluminescence agent etc.) target molecule is carried out fluorescence labeling during immune labeled detection, antigen-antibody reaction takes place then, utilize exact instrument such as optical microscope that reaction result is analyzed (referring to chief editor'ss " practical immunological experiment technology " such as Wu Xiongwen at last, Hubei science tech publishing house, 2002,57-84).Because immunolabelling technique is to change by the signal that labeled molecule produces to detect, so it is a kind of indirect analysis method.Over the past two decades, a kind of labelled immune detection technique of exempting from of direct detection reaction molecular signal has developed rapidly.As surface plasma resonance technology (the surface plasmon resonance of sensing detection is carried out in the change of refractive index of superficial layer that utilizes the substrate sensor material of fixing biological molecules, SPR) and interfere measurement technique (interferometry), and the optical immunity detecting method that utilizes liquid crystal aligning to change, elliptic polarization optics detects, based on the optical immunity detecting method of inverse opal photonic crystal or the like technology and method.In addition, on the basis of the detection technique of the sensor material outstanding based on these, the purpose that just can realize multivariate detection by the detecting unit that need not mark is encoded is (referring to Gu Zhongze etc., non-marked multi-component immune detection method based on inverse opal photonic crystal, patent publication No.: CN101251538A), so just can measure simultaneously the kind and the concentration information of biomolecule.
In the last few years, along with the fast development of photonic crystal theory, based on the hot spot technology that the labelled immune detection method has become people's research of exempting from of photon crystal material and photonic bandgap material.Photonic crystal is a kind of artificial photonic bandgap material that is made of the periodic arrangement of dielectric material, according to specific inductive capacity in spatial distributions, it is divided into one dimension, two dimension, three-dimensional three kinds of structures, wherein no matter the 1-D photon crystal structure is Design Theory, or actual fabrication is all easy, and study most often one dimension diadactic structure photonic crystal, the just various photonic bandgap materials that constitute by the two media material in the 1-D photon crystal.People such as Wang Xudong find the photon crystal structure that is made of three kinds of dielectric materials in the photonic crystal Study on Theory, one dimension ternary structural photonic crystal just, it changes not obvious by the degree of disorder that stochastic error causes, be more to be tending towards a kind of ideal and stable photon crystal structure (referring to Wang Xudong etc., photoelectron laser, 2004,15 (1), 104-107).
The photonic band gap structure sensor is because its variations in refractive index occurs in the strongest characteristics such as zone of electric field (referring to Huimin Ouyang, et al, Analytical Chemistry, 2007,79,1502-1506) make it have highly sensitive, characteristics such as response is fast, real-time is good, exempt from mark, remote-controlled, compact conformation, no electromagnetic interference (EMI) and security height.The porous silicon of galvanic corrosion preparation is exactly a kind of outstanding base material for preparing photonic band gap structure.Porous silicon, have unique porous structure and very large internal surface area, and having can be at room temperature luminous, cheap, characteristics such as energy and the complete compatibility of existing integrated circuit, itself be exactly that a kind of desirable immunosensor base material is (referring to Victor S.-Y.Linet al, Science, 1997,278,840-843), people such as the Andrew Jane of Australia Flinders University behind system summary porous silicon biosensor technology, point out the cost problem be influence the porous silicon biology sensor move towards from the laboratory one of commercial subject matter (referring to Andrew Jane, et al, Trends inBiotechnology, 2009,27 (4), 230-239), therefore selecting the less expensive electrochemical method of preparation method is more favourable a kind of method.In addition, the porous silicon porous silicon Bragg catoptron that the at present main galvanic corrosion of using that hockets of passing through high and low two kinds of electric currents forms, porous silica microsphere cavity configuration or based on the various porous silicon sandwich constructions of porous silicon photoluminescence property, owing to be subjected to many extraneous enchancement factor effect meetings to cause the departing from of physical size of porous silicon photonic band gap structure, can produce certain randomness, be difficult to big batch and repeat to prepare desirable porous silicon photonic crystal.Therefore, not only have the excellent properties of porous silicon and photonic band gap structure sensor simultaneously concurrently based on the one dimension ternary photonic band gap structure biology sensor of porous silicon, and can play good maintenance effect to the stability of porous silicon photonic band gap structure, encode at the porous silicon one dimension ternary photonic band gap structure biology sensor of different condition preparation simultaneously, can also realize the purpose of multivariate detection.
Summary of the invention
Technical matters: the purpose of this invention is to provide and a kind ofly exempt from the mark optical immunity detecting method based on porous silicon three-element structure microcavity, this detection method not only has more stable underlying structure, and the detection time that has porous silicon and photonic band gap structure sensor concurrently is short, many excellent properties such as sensitivity height, can realize multivariate detection by the Porous Silicon Microcavity of obstructed condition preparation is encoded simultaneously, determine the kind and the concentration of testing molecule.
Technical scheme: purpose of the present invention can realize by following scheme:
Adopt accurate Control current of computing machine and the electrochemical etching method of time to prepare Porous Silicon Microcavity, upper and lower Bragg structure is formed by the alternating corrosion of three kinds of big or small electric currents of difference in the Porous Silicon Microcavity.It is this that to carry out the porous silicon three-element structure microcavity that galvanic corrosion forms by hydrofluorite and alcohol mixed solution be the photonic bandgap material that a kind of one dimension direction is arranged, similar with photon crystal material, it is a kind of special optical microcavity, and the defective in the microcavity can cause occurring in the forbidden photon band extremely narrow defective peak.A lot of theoretical calculation methods are arranged in the photonic crystal Study on Theory at present, as the transition matrix method etc., therefore can be according to the refractive index and the physical thickness of every layer material, i.e. the frequency range position of optical thickness accurate Calculation forbidden photon band.The refractive index of every layer of porous silicon is relevant with the porosity of this porous layer in this multi-layer porous silicon, therefore just can obtain the porous silicon layer of interior refractive index in a big way by changing the current density size; And the accurate control of etching time can obtain the porous layer of different-thickness, so different electric currents, defective peak-to-peak position or luminescent center peak position are different in the reflectance spectrum of the Porous Silicon Microcavity that different time is prepared, and defective peak-to-peak position or luminescent center peak position just can be used as the coding of Porous Silicon Microcavity in the different like this reflectance spectrums.If multivariate detection can be encoded to the Porous Silicon Microcavity of different condition preparation, monobasic detects does not then need coding, make the combination in the hole of porous silicon three-element structure microcavity of antibody or antigen then, the kind of antigen or antibody utilizes the coding of Porous Silicon Microcavity to identify in the multivariate detection, and carry out corresponding antigen or antibody concentration in the test sample by the variation of the spectrum peak position before and after the biological respinse, described detection method may further comprise the steps:
1. antigen or antibody are fixed in the hole of Porous Silicon Microcavity, if need the multivariate detection of coding, the specific antigen or the antibody of the corresponding fixing a kind of biomolecule to be measured of so a kind of Porous Silicon Microcavity of coding, wash unconjugated molecule and seal the blank key position of the not binding biomolecules in the Porous Silicon Microcavity, the Porous Silicon Microcavity spectrum that is fixed with antigen or antibody measured in record;
2. different Porous Silicon Microcavity and variable concentrations solution to be measured react, and make Ag-Ab specificity combination in the hole of Porous Silicon Microcavity, wash after the reaction, and record Porous Silicon Microcavity spectrum and coding are determined kind and concentration.
In the optical immunity detecting method based on porous silicon three-element structure microcavity, the coding of Porous Silicon Microcavity can pass through reflection spectrum measuring, also can be by the fluorescence spectrometry of porous silicon.Porous Silicon Microcavity by reflection spectrum measuring be encoded to the pairing resonant transmission peak of its defective position, Porous Silicon Microcavity by fluorescence spectrometry be encoded to the pairing peak position of its luminescent center, the coding of Porous Silicon Microcavity Porous Silicon Microcavity characterizes and can design in the UV, visible light zone, also can be at region of ultra-red.
The pore size of porous silicon three-element structure microcavity can be designed to nano-pore microcavity, the mesoporous microcavity of 10～500 nanometers or the macropore micro-cavity structure of micron number magnitude about several nanometers according to different biological molecules.Antigen or antibody can be combined in the hole of porous silicon three-element microcavity by physics or chemical method.Testing sample can be the soluble recepter molecule of serum, tissue fluid, drugs, excitant, various cells.
Beneficial effect: the present invention has the following advantages:
1) kept the original cheapness of Porous Silicon Microcavity immunosensor, surface area of electrochemical preparation big, can and advantage such as the complete compatibility of existing integrated circuit the time, have the excellent properties of photonic band gap structure sensor concurrently, in addition, the employing of ternary structural makes Porous Silicon Microcavity more stable, is reduced significantly with the influence of advancing factor in the preparation process.
2) only need to adjust the electric current and the time of corroding, just can obtain having the Porous Silicon Microcavity of different spectrum peak positions, thereby can utilize the Porous Silicon Microcavity of different spectrum peak positions to encode, realized the purpose of multivariate detection.
3) coding of Porous Silicon Microcavity can be encoded according to the defective peak position in the Porous Silicon Microcavity reflectance spectrum, also can utilize the exclusive photoluminescent spectrum of porous silicon structure, and promptly the pairing peak position in Luminescence in Porous Silicon center is encoded.
3) according to the needs of actual detected, use the Theory of Electromagnetic Field in the photonic crystal, the peak position at reflectance spectrum defective peak can be designed into and be positioned at the vast zone of ultraviolet band to infrared band, encoding amount is very huge, can satisfy the requirement of high throughput testing.
Description of drawings
Accompanying drawing is the structural representation of porous silicon three-element structure microcavity.
Embodiment
Mode by physisorption or covalent bond coupling is fixed on antigen of probe or antibody molecule in the hole of the Porous Silicon Microcavity for preparing, if chemical bond coupled mode, then utilize buffer solution to wash unconjugated molecule and seal blank key position, and carry out spectroscopic assay, can be reflectance spectrum, also can be luminescent spectrum.Concentrate some Porous Silicon Microcavity of different coding and add target molecule, make probe, target molecule in the hole of Porous Silicon Microcavity, antigen-antibody reaction take place; Reaction unreacted antigen of post-flush or antibody molecule carry out spectroscopic assay once more, and the spectrum peak bit position changes relevant with the concentration of testing molecule, and the mensuration of coding can identify the kind of testing molecule.
Wherein, the pore size of porous silicon three-element structure microcavity can be designed to the nano-pore microcavity according to different biological molecules, mesoporous microcavity or macropore micro-cavity structure.Testing sample then can be the soluble recepter molecule of serum, tissue fluid, drugs, excitant, various cells.In the mensuration of Porous Silicon Microcavity coding, Porous Silicon Microcavity by reflection spectrum measuring be encoded to the pairing resonant transmission peak of its defective position, Porous Silicon Microcavity by fluorescence spectrometry be encoded to the pairing peak position of its luminescent center, the coding of Porous Silicon Microcavity Porous Silicon Microcavity characterizes can be in the UV, visible light zone, or at region of ultra-red.
Optical immunity detecting method based on porous silicon three-element structure microcavity may further comprise the steps:
1. antigen or antibody are fixed in the hole of Porous Silicon Microcavity, if need the multivariate detection of coding, the specific antigen or the antibody of the corresponding fixing a kind of biomolecule to be measured of so a kind of Porous Silicon Microcavity of coding, wash unconjugated molecule and seal the blank key position of the not binding biomolecules in the Porous Silicon Microcavity, the Porous Silicon Microcavity spectrum that is fixed with antigen or antibody measured in record;
2. different Porous Silicon Microcavity and variable concentrations solution to be measured react, and make Ag-Ab specificity combination in the hole of Porous Silicon Microcavity, wash after the reaction, and record Porous Silicon Microcavity spectrum and coding are determined kind and concentration.
Embodiment one.The concentration of using porous silicon three-element structure microcavity to carry out target antigen-bovine serum albumin(BSA) (BSA) detects:
1. fixedly BSA antibody is in the hole of Porous Silicon Microcavity for the way of employing chemical bond coupling, and pre-treatment step is as follows:
1) porous silicon is placed on oxidation 15min in 900 ℃ the environment;
2) Silanization reaction was carried out in immersion in one hour among the 4%APTES of toluene solution dilution;
3) use toluene respectively, methyl alcohol-toluene, methyl alcohol, deionized water washes repeatedly, places the N2 environment dry, then sample is toasted 10min in 100 ℃ of environment;
4) 30min in 2% glutaraldehyde water solution, and wash repeatedly with PBS (phosphate buffer PH=7.4) and to remove remaining glutaraldehyde;
The Porous Silicon Microcavity of handling is divided into 2 groups, and one group is carried out the immersion of BSA positive serum, and another group is carried out the BSA negative serum and soaked as contrast.Sample was placed 2 hours down for 37 ℃ afterwards, used PBST (mixed solution of 0.1%Tween-20 and phosphoric acid) to wash repeatedly then, and sealed with 3% oralbumin (OVA).Defective peak-to-peak position in the reflectance spectrum of record Porous Silicon Microcavity.
2. fixedly the multi-disc Porous Silicon Microcavity of serum is fixed different BSA respectively, and all samples was placed 2 hours down for 37 ℃, made it that antigen-antibody reaction fully take place, and again with the PBST flushing, removes unreacted BSA after the reaction, and sample is at N then
2Dry in the environment.Note the reflectance spectrum of every Porous Silicon Microcavity and do contrast, judge the concentration of BSA according to the long red shift distance of defective spike with the reflectance spectrum before the antigen-antibody reaction.
Embodiment two.Use the Porous Silicon Microcavity of coding to carry out human papilloma virus in the solution (HPV) L1 antigen and grassland rabbit tail mouse egg vitellary membrane 3 genes (LZP3) detection of antigens:
1. adopt the preprocess method of Porous Silicon Microcavity among the embodiment one that the Porous Silicon Microcavity of two kinds of structures is carried out the functionalization pre-service.The Porous Silicon Microcavity of two kinds of structures is fixedly HPV L1 antibody and LZP3 antibody respectively, another Porous Silicon Microcavity fixedly BSA as contrast.Wash repeatedly with PBST (mixed solution of 0.1%Tween-20 and phosphoric acid), and seal with 3%BSA.The defective transmission peaks wavelength of measuring 3 kinds of Porous Silicon Microcavity is respectively at 1500nm, 3500nm, and 5500nm encodes respectively according to defective transmission peaks wavelength.
2. 3 kinds of Porous Silicon Microcavity of putting together are immersed in and mix in the solution to be measured, all samples was placed 2 hours down for 37 ℃, made it that antigen-antibody reaction fully take place, and again with the PBST flushing, removed unreacted BSA after the reaction, and sample is at N then
2Dry in the environment.Measurement result at first with coding contrast, defective transmission peaks wavelength is a HPV L1 antigen near 1500nm's, near 3500nm be LZP3 antigen, near 3500nm is contrast antigen.Judge the concentration of testing molecule then according to the distance of defective transmission peaks red shift of wavelength.
Claims (7)
1. optical immunity detecting method based on porous silicon three-element structure microcavity, this method adopts the electrochemical etching method of accurately controlling based on computing machine to prepare Porous Silicon Microcavity, on wherein in the Porous Silicon Microcavity, under the Bragg structure hocket galvanic corrosion by three kinds of current densities and form, it is characterized in that encoding and to realize multivariate detection for the Porous Silicon Microcavity of different condition preparation, if detecting, monobasic then do not need coding, make the combination in the hole of porous silicon three-element structure microcavity of antibody or antigen, the kind of antigen or antibody utilizes the coding of Porous Silicon Microcavity to identify in the multivariate detection, and carry out corresponding antigen or antibody concentration in the test sample by the variation of the spectrum peak position before and after the biological respinse, described detection method may further comprise the steps:
1) antigen or antibody are fixed in the hole of Porous Silicon Microcavity, if need the multivariate detection of coding, the specific antigen or the antibody of the corresponding fixing a kind of biomolecule to be measured of so a kind of Porous Silicon Microcavity of coding, wash unconjugated molecule and seal the blank key position of the not binding biomolecules in the Porous Silicon Microcavity, the Porous Silicon Microcavity spectrum that is fixed with antigen or antibody measured in record;
2) different Porous Silicon Microcavity and variable concentrations solution to be measured reacts, and makes Ag-Ab specificity combination in the hole of Porous Silicon Microcavity, washes after the reaction, and record Porous Silicon Microcavity spectrum and coding are determined kind and concentration.
2. the optical immunity detecting method based on porous silicon three-element structure microcavity according to claim 1 is characterized in that the coding of Porous Silicon Microcavity can pass through reflection spectrum measuring, also can be by the fluorescence spectrometry of porous silicon.
3. the optical immunity detecting method based on porous silicon three-element structure microcavity according to claim 1 and 2, it is characterized in that by reflection spectrum measuring Porous Silicon Microcavity be encoded to the pairing resonant transmission peak of its defective position, the Porous Silicon Microcavity by fluorescence spectrometry be encoded to the pairing peak position of its luminescent center.
4. the optical immunity detecting method based on porous silicon three-element structure microcavity according to claim 3 is characterized in that the coding sign of Porous Silicon Microcavity Porous Silicon Microcavity can design in the UV, visible light zone, also can design at region of ultra-red.
5. the optical immunity detecting method based on porous silicon three-element structure microcavity according to claim 1, the pore size that it is characterized in that porous silicon three-element structure microcavity can be designed to the nano-pore structure microcavity according to different biological molecules, meso-hole structure microcavity or macroporous structure micro-cavity structure.
6. the optical immunity detecting method based on porous silicon three-element structure microcavity according to claim 1 is characterized in that antigen or antibody can be combined in the hole of Porous Silicon Microcavity by physics or chemical method.
7. the optical immunity detecting method based on porous silicon three-element structure microcavity according to claim 1 is characterized in that testing sample can be serum, tissue fluid, drugs, excitant and various soluble recepter molecule.
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Application publication date: 20100519 |