CN102262069A - Prism coupled type surface plasma resonance biosensor - Google Patents
Prism coupled type surface plasma resonance biosensor Download PDFInfo
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- CN102262069A CN102262069A CN2010101899933A CN201010189993A CN102262069A CN 102262069 A CN102262069 A CN 102262069A CN 2010101899933 A CN2010101899933 A CN 2010101899933A CN 201010189993 A CN201010189993 A CN 201010189993A CN 102262069 A CN102262069 A CN 102262069A
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Abstract
The invention relates to a prism coupled type surface plasma resonance (SPR) biosensor. The biosensor comprises an optical plastic prism, an internal metal film layer, an external metal film layer and a biotic component layer, which are orderly arranged as follows: the internal metal film layer is deposited on an upper surface of the optical plastic prism, the external metal film layer is deposited on the internal metal film layer, the internal metal film layer and the external metal film layer can be same or different metal film layers, and sum of the thickness of the internal metal film layer and the thickness of the external metal film layer is 20-80 nm, which satisfies the optical requirements for exciting the surface plasma resonance, and the biotic component layer is fixed on the surface of the external metal film layer. The invention further relates to the use of the prism coupled type SPR biosensor in manufacturing of biochips, and the use of the biosensor in detection of interaction between biological samples.
Description
Technical field
The present invention relates to a kind of biology sensor, particularly a kind of prism-coupled formula surface plasma resonance biosensor.
Background technology
Sensor is a kind of device that obtains with process information.Device to the material composition sensing is exactly a chemical sensor, it be a kind of miniaturization, can be single-minded and reversibly certain chemical constitution is carried out the device of responsing reaction, and can produce and the proportional signal of surveying of this constituent concentration.And biology sensor is the special chemical sensor of a class, and it is as the bio-sensing primitive, the measured target thing to be had the detecting device of high selectivity with biologically active unit (as antibody, nucleic acid, albumen etc.).It is expressed the degree of reaction then by the reaction between various physics, chemical type signal converter captured target thing and the responsive primitive with discrete or continuous electric signal, thereby draws the information such as concentration of measured object.
(Surface Plasmon Resonance, SPR) biology sensor mainly is chemistry and the physisorption situation that reflects material by the variation of measuring the metal surface material refractive index as a kind of emerging biology sensor to surface plasma body resonant vibration.The principle that the SPR phenomenon produces is: when light incided on the interface with the angle of total reflection, a part of incident light was coupled in the surface plasma, promptly existed the evanescent field of light in surface plasma.If the wave vector of incident light equates with the wave vector of surface plasma-wave along the component that is parallel to the interface direction, surface plasma resonates in the evanescent field of light, energy loss takes place in light wave in communication process, on macroscopic view, show as some spectrum by strong absorption, can on the response curve of reflective light intensity, see a minimum spike, this spike is called as resonance absorbing peak, and this moment, corresponding lambda1-wavelength was called resonant wavelength, and corresponding incident angle is a resonance angle.The SPR phenomenon is very responsive to the dielectric refractive index attached to the metal surface.Theoretical Calculation shows, when imperceptible variation takes place the optically thinner medium refractive index, will change the position of resonance absorbing peak accordingly.
Surface plasmon resonance biosensor utilizes above-mentioned principle that the analyte of metal surface is detected.When sample contacted with the interface, owing to have absorption or chemical reaction, medium refraction index will change at the interface.We can understand the physics and the chemisorption process at interface in high sensitivity by the state of surveying the metal surface plasma body based on this principle.Surface plasmon resonance biosensor is applied to the detection of biochip, not only can give full play to its detection sensitivity height, testing process is quick, can obtain real time data, easy to operate, the biologically active that can keep molecule, sample need not outstanding features such as mark and purifying, the false positive of effectively having avoided the traditional biological chip detecting method to need mark and having brought, sensitivity is low, poor repeatability, the easy inactivation of sample, problems such as radioactive contamination, and again can be, thereby fast further combined with the high flux characteristics of biochip technology, simply, exactly biological sample is carried out qualitative and quantitative detection.
Existing commercialization surface plasmon resonance biosensor mainly is based on Kretschmann type prism-coupled structural model design, and used prism mainly is to process by satisfying the optical glass that certain refractive index requires, as shown in Figure 8.Though the prism bottom is the support metal film directly, but directly gold-plated meeting brings inconvenience to operation in the prism bottom surface, because all ingredients that adds in testing process is influential to sensitive membrane, reliable for guaranteeing experimental data, need use sensitive membrane once generally speaking.So just caused the prism utilization ratio lower, use cost increases.Since optical glass device have manufacturing cost higher, be difficult to Design and Machining and become complicated shape and be difficult to adapt to shortcoming such as productions in enormous quantities, so seriously limited its applying in clinical detection and experimental study.
For overcoming above-mentioned shortcoming, metal film can be plated in the one side of optical glass sheet, be attached on the prism bottom surface with the optical glass sheet of optical match medium again metal-coated membrane, rib has reduced cost to use repeatedly like this.The surface plasmon resonance biosensor that Sweden Biacore company commonly used at present produces directly is plated in golden film on the optical glass sheet exactly, glass sheet is embedded in a plastic board underedge again and constitutes sensing chip.The optical glass face of sensing chip contacts with optical glass prism bottom surface.Utilize the silicones optical interface between prism and the sensing chip surface to guarantee prism and sensing chip good optical coupled relation.Yet this has caused again, and the sample detection process operation is loaded down with trivial details, surface plasmon resonance biosensor complex structure and sensing chip price problem of higher, still can not satisfy the actual demand of extensive detection.
The another kind of method of dealing with problems is to utilize the optical plastic prism to replace the optical glass prism.That optical plastic has is cheap for manufacturing cost, be easy to Design and Machining becomes very complicated shape, the plurality of advantages that can produce in enormous quantities.The SPReeta that Texas ,Usa instrument company (TexasInstruments Co.) produces
TMThe type surface plasmon resonance biosensor (is commonly called as organic glass with the optical plastic polymethylmethacrylate exactly, be called for short PMMA) as the light wave coupling element, golden film is plated on the optical glass sheet constitutes sensing chip, again the optical glass face of this sensing chip and a side of organic glass are combined by the optical match medium.As shown in Figure 1.The structure of this sensor is very simple, and optics and electricity system are integrated on the disposable trapezoidal plexiglass block, and the size of plexiglass block approximately is 3cm * 2cm * 1cm.But this surface plasmon resonance biosensor has following deficiency:
(1) it is good to adopt the detection method of multiple spot list angle reflection to can not show a candle to the surface plasmon resonance biosensor of conventional single-point multi-angle.Since biomacromolecules such as albumen evenly distribution in the large tracts of land scope that the synthetic SPR spectrum of multiple spot reflection requires golden film and is adsorbed on golden film surface, and in fact this even absorption is difficult to realize.
(2) adopt optical glass sheet and the organic glass prism method by optical match media implementation optical coupled to have complex operation, problem that signal to noise ratio (S/N ratio) is lower.Have a catadioptric interface between glass sheet and prism, incident intensity can lose a part inevitably, and increases along with the increase of incident angle.And, in detecting device, produce indelible noise because the difference of applying skill causes disorderly film interference striped easily.
(3) adopt organic glass as the light wave coupling element, because its refractive index only has 1.491, relatively low, the result causes SPReeta
TMThe substance classes that the type surface plasmon resonance biosensor can detect than conventional surface plasmon resonance biosensor still less is difficult to satisfy the actual demand of clinical detection.
Metal film is one of condition that produces surface plasma body resonant vibration, its optical constant, directly determined the degree of depth, width and the resonance angle position of SPR spectrum as refractive index, absorption coefficient and thickness, so the selection of metallic film and preparation detect effect to SPR and play direct effect.Existing commercialization SPR instrument mainly with the Au film as the metal film that excites SPR because its stability is better, be difficult for reacting with other materials, be fit to long-term the use, and preparation easily.But, with regard to 3 characteristic parameters (resonant wavelength or resonance angle, resonance width and the resonance degree of depth) of SPR spectrum, no matter be with incident angle as variable, still with lambda1-wavelength as variable, the Ag film all is better than the Au film, as shown in Figure 2.Promptly under similarity condition, the resonance degree of depth of silverskin is slightly larger than golden film, and the resonance half-peak breadth is significantly less than golden film, and the variation of resonant wavelength or resonance angle is obviously than golden film sensitivity.In addition, the reflectivity of Al film also is higher than the Au film, and the SPR phenomenon of its generation is better than the Au film equally.The price of Ag film and Al film is all cheaply a lot of than Au film, is convenient to realize producing in enormous quantities.But, if directly with pure Ag film and pure Al film as the metal film that excites SPR, they are all oxidized easily in air, thereby can influence the detection effect of surface plasmon resonance biosensor, this has just limited their application in surface plasmon resonance biosensor.A solution utilizes part silverskin or aluminium film to replace golden film exactly; excite SPR phenomenon (Stepan A.Zynio et al by the thermometal rete; Sensors 2002; 2; 62-70 and AnujK.Sharma et al; JOURANL OF APPLIED PHYSICS 2007; 101; 093111); so golden film can prevent the oxidation of silverskin or aluminium film as diaphragm; can either reduce the manufacturing cost of surface plasmon resonance biosensor, can utilize silverskin and aluminium film effectively to improve the detection sensitivity and the signal to noise ratio (S/N ratio) of surface plasmon resonance biosensor again.
Yet because optical plastic has some characteristics different with optical glass, people also are not implemented in the optical plastic prism surface that satisfies the surface plasmon resonance biosensor requirement and directly plate many metallic diaphragms at present.For example, SPReeta
TMSelected organic glass in the type surface plasmon resonance biosensor, it is the highest a kind of optical plastic of water-intake rate, if at the direct plated film in its surface, when vacuum coating is bled so, steam can constantly discharge, thereby vacuum tightness is sharply reduced, the metallic diaphragm quality is descended, influence the testing result in later stage.In addition, the not anti-alcohol of organic glass, ketone are easily corroded by highly basic and warm NaOH, are difficult to satisfy the needs of the manufacturing process of follow-up biochip.
In sum, making use cost height, method for making complexity that existing surface plasmon resonance biosensor has are difficult to extensive use; Low, the narrow application range of the signal to noise ratio (S/N ratio) that has limits its application equally.Need a kind of low cost, high-quality surface plasmon resonance biosensor at present badly.To achieve these goals, carried out this research.
Summary of the invention
The invention provides a kind of new surface plasmon resonance biosensor, it is the prism-coupled formula surface plasmon resonance biosensor that directly deposits many metallic diaphragms at the optical plastic prism surface, has solved the problem that existing surface plasmon resonance biosensor exists.Particularly,
One aspect of the present invention relates to a kind of prism-coupled formula surface plasmon resonance biosensor, comprises optical plastic prism, interior metallic diaphragm, outer metallic diaphragm and biological components layer; It puts in order and is metallic diaphragm in: the top deposition of optical plastic prism, deposit outer metallic diaphragm on the interior metallic diaphragm, interior metallic diaphragm can be two kinds of metallic diaphragms identical or inequality with outer metallic diaphragm, outer metallic diaphragm surface is fixed with the biological components layer, and this biological components layer can carry out association reaction with the acceptor in the test substance.
Optical plastic material wherein is selected from a kind of in the following material: the multipolymer of the multipolymer of polycarbonate, allyl diglycol carbonates, styrene and acrylate, styrene and vinyl cyanide, cyclic olefine copolymer, cyclic olefin polymer or poly-three ring methacrylates in the last of the ten Heavenly stems, be preferably polycarbonate or cyclic olefine copolymer, the method processing and fabricating by injection mo(u)lding forms;
The cross sectional shape of optical plastic prism wherein is selected from a kind of in the following shape: triangle, trapezoidal, semicircle or irregular polygon are preferably triangle or semicircle;
Interior metallic diaphragm wherein is a kind of in golden film, silverskin or the aluminium film, and the method by vacuum coating is deposited on above the optical plastic prism, and its thickness is 1nm-50nm, is preferably 20nm-45nm; In one embodiment of the invention, the thickness of interior metallic diaphragm is 23nm; In another embodiment of the invention, the thickness of interior metallic diaphragm is 42nm.
Also have one deck binding metal rete between optical plastic prism wherein and the interior metallic diaphragm, the method by vacuum coating is deposited on above the optical plastic prism, and this metallic diaphragm is selected from a kind of in chromium film, tungsten film or the titanium film, and thickness is 1-5nm; In embodiments of the invention, sticking metallic diaphragm is the chromium film, and its thickness is 3nm.
Outer metallic diaphragm wherein is golden film or silverskin, and above the metallic diaphragm, its thickness was 1nm-50nm, is preferably 1nm-25nm in the method by vacuum coating was deposited on; In one embodiment of the invention, the thickness of outer metallic diaphragm is 24nm; In another embodiment of the invention, the thickness of outer metallic diaphragm is 5nm.
The thickness sum of its China and foreign countries' metallic diaphragm, interior metallic diaphragm is 20-80nm, is preferably 40-60nm.
Biological components wherein is selected from one or more in following: part, nucleic acid, polypeptide, protein, antigen, antibody, carbohydrate, lipid, bacteriophage, virus, cell, tissue and micromolecular compound etc.In one embodiment of the invention, biological components layer molecule is HPV16 type and two kinds of dna probe molecules of HPV 18 types.The biological components layer both can have been put a kind of biomolecule, can contain different biomolecule at diverse location again.
Also comprise a self assembled monolayer between outer metallic diaphragm wherein and the biological components layer, described self assembled monolayer is the unimolecular layer of a sulfocompound, sulfocompound is selected from one or more in the following material: mercaptan, thioether, disulfide and their derivant, and molecule one end of this material contains a kind of in the following group: sulfydryl (HS-), amido (NH
2-), aldehyde radical (CHO-), carboxylic acid group (COOH), epoxy radicals (CH (O) CH-), ester group or biotin (biotin); In one embodiment of the invention, sulfocompound is dithio dipropyl acid.
Fig. 3 is the diagrammatic cross-section of a kind of prism-coupled formula of the present invention surface plasmon resonance biosensor.This prism-coupled formula surface plasmon resonance biosensor 16 comprises optical plastic prism 10, interior metallic diaphragm 12, outer metallic diaphragm 13 and biological components layer 15.Metallic diaphragm 12 in the deposition on the optical plastic prism 10, deposit outer metallic diaphragm 13 on the interior metallic diaphragm 12, interior metallic diaphragm 12 is two kinds of different metallic diaphragms with outer metallic diaphragm 13, and the thickness sum of interior metallic diaphragm 12 and outer metallic diaphragm 13 satisfies the optics requirement of excitating surface plasma resonance between 20-80nm; Outer metallic diaphragm 13 surfaces are fixed with biological components layer 15, and this biological components layer can carry out association reaction with the acceptor in the test substance; A self assembled monolayer 14 is arranged between outer metallic diaphragm and the biological components layer.
Fig. 4 is the sensing synoptic diagram of a kind of prism-coupled formula of the present invention surface plasmon resonance biosensor.Constructional device among Fig. 3 is positioned in the medium 20 of testing sample 19 the biological detection program of being scheduled to.Incident light 17 incides the bottom of optical plastic prism 10 with proper angle, cause the resonance of the free electron at the interface of outer metallic diaphragm 13 and medium 20, thereby cause reflection ray 18 to weaken greatly in certain angle, the mode of accepting reflection ray 18 at reflection direction detects.Light signal is delivered to the COMPUTER CALCULATION response situation.Because the ligand 15 on the sensor 16 only can combine with specific acceptor, if therefore testing sample 19 is an object, association reaction can take place with biological components layer 15 in testing sample 19.Please refer to Fig. 5, if association reaction takes place with biological components layer 15 in the testing sample in the medium 20 19, to cause outer metallic diaphragm 13 surface refractive indexs to change so, and change of refractive is directly proportional with the biological molecular mass that is combined in the metal surface, thereby can be by the dynamic change at SPR angle in the bioprocesses being obtained the special signal of biological components layer 15 and testing sample 19 bio-molecular interactions, and form result as Fig. 6.
The invention still further relates to the purposes of prism-coupled formula surface plasmon resonance biosensor in the preparation biochip.
The invention still further relates to prism-coupled formula surface plasmon resonance biosensor and be used for the purposes of detection of biological component interphase interaction.
The beneficial effect of the invention
The invention provides a kind of prism-coupled formula surface plasmon resonance biosensor, by in optical plastic prism bottom surface directly the many metallic diaphragms of deposition realize detection to biological sample, optical plastic and many metallic diaphragms advantage had separately both been made full use of, the structure and the operating process of surface plasmon resonance biosensor have been simplified in the operation of having avoided existing surface plasmon resonance biosensor in use to exist sensing chip to fit with prism again.Compare with existing surface plasmon resonance biosensor, the present invention has following outstanding advantage:
(1) with low cost, make simply, be convenient to realize producing in enormous quantities, be beneficial in clinical detection and apply;
(2) highly sensitive, satisfy the requirement of clinical diagnosis to the low concentration sample context of detection;
(3) easy to operate, signal to noise ratio (S/N ratio) is higher, has eliminated unnecessary reflection and the veiling glare that the catadioptric interface produces between the prism and optical glass sheet in original surface plasmon resonance biosensor and has interfered;
(4) sensing range is extensive, can be applicable to the detection of various biomolecule;
Description of drawings
Fig. 1 is SPReeta
TMType surface plasmon resonance biosensor structural representation;
Fig. 2 is the comparison of the SPR spectrum spectrogram of silverskin and golden film under the same conditions;
Fig. 3 is a kind of prism-coupled formula of the present invention surface plasmon resonance biosensor diagrammatic cross-section;
Fig. 4 is the sensing synoptic diagram of a kind of prism-coupled formula of the present invention surface plasmon resonance biosensor;
Fig. 5 is the explanation synoptic diagram of the detection mode of detection one specific molecular;
Fig. 6 is for detecting the association reaction kinetic curve synoptic diagram of a specific molecular gained;
Horizontal ordinate is time (unit is min), and ordinate is that (unit is the milli degree to the SPR angle, RU).
Fig. 7 is for detecting the association reaction kinetic curve figure of HPV 18 types virus gained;
Horizontal ordinate is time (unit is min), and ordinate is that (unit is the milli degree to the SPR angle, RU).
Fig. 8 is a Kretschmann type prism-coupled structural representation;
Wherein, Reference numeral:
The 1-LED light source;
The 2-polaroid
3-gold film
The 4-optical glass sheet
5-organic glass prism
The 6-photodiode array
The 7-storer
The SPR spectrum spectrogram of silverskin under 8-the same terms
The SPR spectrum spectrogram of golden film under 9-the same terms
10-optical plastic prism
11-binding metal rete
Metallic diaphragm in the 12-
The outer metallic diaphragm of 13-
The 14-self assembled monolayer
15-biological components layer
16-prism-coupled formula surface plasmon resonance biosensor
The 17-incident light
The 18-reflected light
The 19-testing sample
The 20-medium
21-optical glass prism
Embodiment
Below in conjunction with embodiment embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used to illustrate the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person among the embodiment carries out according to the condition of normal condition or manufacturer's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
The explanation of relational language and explanation among the present invention
According to the present invention, term " surface plasmon resonance biosensor " comprises following components: coupling prism, metal film and the biological components that is fixed on metallic film surface.
According to the present invention, term " sensitive membrane " comprises metal film and biological components.
According to the present invention, term " biochip " is meant the miniature organism chemical analysis system that makes up on the solid-phase matrix surface by micro-processing technology and microelectric technique, with the detection of realization to accurate, the quick and large information capacity of tissue, cell, protein, nucleic acid, carbohydrate and the other biological component of viable organism.
According to the present invention, term " part (ligand) " is meant itself to have its special biologically active, and energy and acceptor (receptor) combination, presents specific bioactive molecule.
1 one kinds of prism-coupled formulas of embodiment surface plasmon resonance biosensor
In the present embodiment, the material of optical plastic prism 10 is selected from polycarbonate, and its refractive index is 1.586, and manufacturing cross sectional shape by the method for injection mo(u)lding is semicircular prism.Because the refractive index of polycarbonate is higher, can measure material with big refractive index.The water-intake rate of polycarbonate optical plastic is lower, soaks 24h in water, only increases weight 0.13%.In addition, it not only has chemical-resistant and thermotolerance preferably, and has very high impact strength, and ductility is good, has uniform molding shrinkage, is convenient to injection moulding.
In the present embodiment, interior metallic diaphragm 12 is a silverskin, and the method by vacuum coating is deposited on above the optical plastic prism 10, and its thickness is 23nm; Because the surface energy of plastics is generally lower, surface polarity is poor, for metallic diaphragm 12 in preventing comes off from optical plastic prism 10 surfaces, also has one deck binding metal rete 11 between optical plastic prism 10 and interior metallic diaphragm 12, this metallic diaphragm is the chromium film, and thickness is 3nm;
In the present embodiment, outer metallic diaphragm 13 is golden film, and above the metallic diaphragm, its thickness was 24nm in the method by vacuum coating was deposited on; The thickness of interior metallic diaphragm 12 is 23nm, and interior metallic diaphragm 12 is 47nm with the thickness sum of outer metallic diaphragm 13, satisfies the optics requirement of excitating surface plasma resonance.
The self assembled monolayer that this surface plasmon resonance biosensor connects, biological components layer and uses thereof see also embodiment 3.
2 one kinds of prism-coupled formulas of embodiment surface plasmon resonance biosensor
In the present embodiment, the material of optical plastic prism 10 is selected from cyclic olefine copolymer, and its refractive index is 1.535, and manufacturing cross sectional shape by the method for injection mo(u)lding is semicircular prism.The thermal stability of cyclic olefine copolymer and humidity-proof ability are good, and water-intake rate (24h) is less than 0.01%.Its chemical stability is very good, and the meticulous surface profile of reproducible.
In the present embodiment, interior metallic diaphragm 12 is a silverskin, and the method by vacuum coating is deposited on above the optical plastic prism 10, and its thickness is 42nm; Because the surface energy of plastics is generally lower, surface polarity is poor, for metallic diaphragm 12 in preventing comes off from optical plastic prism 10 surfaces, also has one deck binding metal rete 11 between optical plastic prism 10 and interior metallic diaphragm 12, this metallic diaphragm is the chromium film, and thickness is 3nm;
In the present embodiment, outer metallic diaphragm 13 is golden film, and above the metallic diaphragm, its thickness was 5nm in the method by vacuum coating was deposited on; The thickness of interior metallic diaphragm 12 is 42nm, and interior metallic diaphragm 12 is 47nm with the thickness sum of outer metallic diaphragm 13, satisfies the optics requirement of excitating surface plasma resonance.
On this basis, can connect different self assembled monolayers and biological components according to actual needs.
The application of 3 one kinds of prism-coupled formulas of embodiment surface plasmon resonance biosensor in detecting human papilloma virus
(Human papillomavirus HPV) can cause the optimum and malignant neoplastic disease of the multiple mankind to Human infectious warts virus, and its type and pathogenic closely related.HPV especially high-risk HPV is detected the important step that has become early diagnosis of tumor and prevention with somatotype.Traditional HPV detection method mainly is by morphological method and immunological method it to be detected, specificity and sensitivity are all not ideal enough, mainly be that the applied molecular biology method is carried out DNA detection to HPV at present, comprise based on the method for nucleic acid hybridization with based on the method for PCR (PCR).The present invention is applied to the detection of human papilloma virus with prism-coupled formula surface plasmon resonance biosensor, has the mark of need not, simple to operate, detection efficiency advantage of higher.
The selection of material:
(1) surface plasmon resonance biosensor: the used surface plasmon resonance biosensor of present embodiment is pressed embodiment 1 described method preparation;
(2) biological components: HPV 16 types and HPV 18 type dna probes are bought from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Probe 1 sequence: 5 '-CATTATGTGCTGCCATATCTACTTCAGAAMCTAC-3 ' (SEQ ID NO:1), wherein m=A/C
Probe 2 sequences: 5 '-GCTGCCATATCTACTTCAGAAmct-3 ' (SEQ ID NO:2), wherein m=A/C
Probe 3 sequences: 5 '-TTCTACACAGTCTCCTGTACCTGGGCAA-3 ' (SEQ IDNO:3)
Probe 4 sequences: 5 '-TCTCCTGTACCTGGGCAATA-3 ' (SEQ ID NO:4)
For dna probe being fixed to the self assembled monolayer surface of sensor, 5 ' end of probe (NH2) carries out mark with amino; Amino (NH2) and connect polyT between the probe sequence, to improve hybridization signal.
(3) self assembled monolayer: dithio dipropyl acid, molecule one end of this sulfocompound contains the carboxylic acid group (COOH), after the mixed solution activation of nitrogen-N-Hydroxysuccinimide (NHS) and nitrogen-ethyl-nitrogen-(dimethyl aminopropyl) carbodiimide (EDC), with the amino of probe 3 ' end (NH2) react, thus with the golden film surface of probe stationary at sensor;
(4) medium: phosphate buffered solution (100mM PBS, PH=7.5)
(5) testing sample: gather the swab sample from cervix, after treatment, extract DNA and carry out PCR as template.
The primer sequence of PCR reaction is respectively:
F1:5 '-TTCTCGGATGgtcactagcttttkttachgtkgtdgatacyac-3 ' (SEQ ID NO:5) and
R1：5’-GAAAHATAAAYTGYAADTCATAYTC-3’(SEQ?ID?NO：6)，
K=G/T in the above primer; H=A/C/T; D=A/G/T; Y=C/T.
Reaction system:
10 * PCR damping fluid (does not contain MgCl
2) 5 μ l,
25mM?MgCl
2 25μl，
2.5mM?dNTP 4μl，
5U/ μ l archaeal dna polymerase 0.5 μ l,
20 μ M primers F, 15 μ l,
20 μ M primer R1,5 μ l,
Dna profiling 1 μ l,
Add water system is supplied 50 μ l.
Use ABI 9700 amplification instrument amplifications.Response procedures is as follows:
94℃，3min；
94 ℃ of 1min, 48 ℃ of 1min, 72 ℃ of 1min, totally 30 circulations;
94 ℃ of 1min, 65 ℃ of 1min, 72 ℃ of 1min, totally 20 circulations;
72℃7min。
The about 200bp of the size of PCR product verifies by agarose electrophoresis.
The modification of surface plasmon resonance biosensor
(1) with the surface of hydrogen peroxide and the concentrated sulphuric acid (1: 3) treatment S PR biology sensor gold film, remove impurity such as golden film surface organic matter, then with pure water rinsing several times, high pure nitrogen dries up;
(2) the golden film surface of surface plasmon resonance biosensor is dipped in the ethanol solution of dithio dipropyl acid that concentration is 5mM, room temperature assembling 18 hours is cleaned 5min successively with absolute ethyl alcohol and ultrapure water then, and high pure nitrogen dries up;
(3) handle golden film 20min with 0.05mol/L nitrogen-N-Hydroxysuccinimide (NHS) and the mixed solution of 0.2mol/L nitrogen-ethyl-nitrogen-(dimethyl aminopropyl) carbodiimide (EDC), clean repeatedly with ultrapure water, high pure nitrogen dries up;
(3) with the diverse location of micropipettor on golden film surface, point is made HPV 16 types and HPV 18 type dna probe solution side by side, and the point sample concentration of probe solution is 5 μ M, and the point sample amount is 0.5 μ l, and distance between points is 2mm.
(4) sensor after the system is hatched 4h at 25 ℃ of wet boxes;
(5) hatch and clean repeatedly with ultrapure water after finishing, high pure nitrogen dries up;
(5) be 1M with concentration, the ethanolamine solutions deactivation of pH=8.4 is cleaned with ultrapure water subsequently again, and high pure nitrogen dries up;
Testing result
Detect the pcr amplification product of HPV 18 types of above-mentioned preparation with the surface plasmon resonance biosensor after the above-mentioned modification, testing result as shown in Figure 7.Article two, curve is respectively surface plasmon resonance biosensor has the golden film surface of HPV 16 types and HPV 18 type dna probe solution to point sensing synoptic diagram.The bottom line of initial part all represents to have only buffer solution to flow through and is fixed on the dna probe molecule surface on the outer metallic diaphragm.When continuously injecting testing sample, if itself and the dna probe generation association reaction on outer metallic diaphragm surface, just the molecule process of mutually combining of dna probe and testing sample could show from the resonance signal change so.Increase with minute, the increase of the testing sample amount of injection, thereby resonance signal change amplitude increases.When metallic diaphragm is surperficial outside buffer solution replacement testing sample flows through, can observe the dissociation process of dna probe and the formed compound of testing sample.We measure testing sample according to surface plasmon resonance biosensor and flow through before the outer metallic film surface with the signal difference in respective streams pond afterwards to judge whether the existence of HPV 18 types virus is arranged.Wherein, the golden film surface that is fixed with HPV 16 type dna probes is surperficial in contrast, and the golden film surface that is fixed with HPV 18 type dna probes is as surface to be measured.The change of resonance signal is represented with milli degree (RU).
In the present embodiment, determine whether HPV 18 type viruses exist with according to being: if the signal difference of contrast surface＞0, positive findings then can be regarded as in ratio＞2 of the signal difference on surface to be measured and the signal difference of contrast surface, and the signal difference absolute value＞200RU on this surface to be measured; If the signal difference of contrast surface＜0, the signal difference absolute value＞200RU on surface to be measured then also can regard as positive findings.As shown in Figure 7, dotted line is a surface plasmon resonance biosensor of the present invention has the sensing figure on the golden film surface (contrast surface) of HPV 16 type dna probes to point, and the signal that feeds behind the testing sample is 28.3 milli degree with feeding the front signal difference; Solid line is a surface plasmon resonance biosensor of the present invention has the sensing figure on the golden film surface (surface to be measured) of HPV 18 type dna probes to point, and the signal that feeds behind the testing sample is 216 milli degree with feeding the front signal difference.Because signal difference＞0 of contrast surface, ratio＞2 of the signal difference on surface to be measured and the signal difference of contrast surface, and the signal difference absolute value＞200RU on this surface to be measured, so can regard as positive findings.
Above-mentioned experimental result shows: testing sample flows through before and after the golden film surface that is fixed with HPV 16 type dna probes, does not almost have the sample combination to get on, and the front and back signal difference is very little.Yet when flowing through the golden film surface that is fixed with HPV 18 type dna probes, the testing sample specificity is attached to golden film surface and gets on, thereby cause the variation of golden film surface refractive index, signal difference is apparent in view before and after causing, and shows to have HPV 18 type viruses in the testing sample.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Sequence table
＜110〉Beijing JinPuJia Medical Treatment Science Co., Ltd
＜120〉a kind of prism-coupled formula surface plasma resonance biosensor
<130>IDC100016
<160>6
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＜213〉artificial sequence
<400>5
ttctcggatg?gtcactagct?tttkttachg?tkgtdgatac?yac 43
<210>6
<211>25
<212>DNA
＜213〉artificial sequence
<400>6
gaaahataaa?ytgyaadtca?taytc 25
Claims (12)
1. a prism-coupled formula surface plasmon resonance biosensor (16) comprises optical plastic prism (10), interior metallic diaphragm (12), outer metallic diaphragm (13) and biological components layer (15); Its put in order into, on the optical plastic prism deposition in metallic diaphragm, deposit outer metallic diaphragm on the interior metallic diaphragm, interior metallic diaphragm can be two kinds of metallic diaphragms identical or inequality with outer metallic diaphragm, outer metallic diaphragm surface is fixed with the biological components layer.
2. surface plasmon resonance biosensor according to claim 1, wherein said optical plastic is selected from a kind of in the following material: the multipolymer of the multipolymer of polycarbonate, allyl diglycol carbonates, styrene and acrylate, styrene and vinyl cyanide, cyclic olefine copolymer, cyclic olefin polymer or poly-three ring methacrylates in the last of the ten Heavenly stems are preferably polycarbonate or cyclic olefine copolymer.
3. surface plasmon resonance biosensor according to claim 1, wherein said optical plastic prism, its shape of cross section are selected from a kind of in the following shape: triangle, trapezoidal, semicircle or irregular polygon are preferably triangle or semicircle.
4. surface plasmon resonance biosensor according to claim 1, wherein said in metallic diaphragm be a kind of in golden film, silverskin or the aluminium film.
5. surface plasmon resonance biosensor according to claim 1, the thickness of wherein said interior metallic diaphragm is 1nm-50nm, is preferably 20nm-45nm.
6. surface plasmon resonance biosensor according to claim 1 also has one deck binding metal rete between wherein said optical plastic prism and the interior metallic diaphragm, this metallic diaphragm is selected from a kind of in chromium film, tungsten film or the titanium film, and thickness is 1-5nm.
7. surface plasmon resonance biosensor according to claim 1, wherein said outer metallic diaphragm is golden film or silverskin, the thickness of outer metallic diaphragm is 1nm-50nm, is preferably 1nm-25nm.
8. surface plasmon resonance biosensor according to claim 1, the thickness sum of its China and foreign countries' metallic diaphragm, interior metallic diaphragm is 20-80nm, is preferably 40-60nm.
9. surface plasmon resonance biosensor according to claim 1, wherein said biological components layer is selected from one or more in the following component: part, nucleic acid, polypeptide, protein, antigen, antibody, carbohydrate, lipid, bacteriophage, virus, cell, tissue and micromolecular compound; The biological components layer both can have been put a kind of biomolecule, can contain different biomolecule at diverse location again.
10. surface plasmon resonance biosensor according to claim 1, also comprise a self assembled monolayer between wherein said outer metallic diaphragm and the biological components layer, described self assembled monolayer is the unimolecular layer of a sulfocompound, sulfocompound is selected from one or more in the following material: mercaptan, thioether, disulfide and their derivant, and molecule one end of this material contains a kind of in the following group: sulfydryl, amido, aldehyde radical, carboxylic acid group, epoxy radicals, ester group or biotin; Particularly, described self assembled monolayer compound is dithio dipropyl acid.
11. the purposes of the described surface plasmon resonance biosensor of claim 1-10 in the preparation biochip.
12. the described surface plasmon resonance biosensor of claim 1-10 is used for the purposes of detection of biological component interphase interaction.
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| CN103323429A (en) * | 2013-05-30 | 2013-09-25 | 中南大学 | Surface plasmon resonance (SPR) sensor chip for detection of melamine content of milk and its preparation method and detection method |
| WO2014075222A1 (en) * | 2012-11-13 | 2014-05-22 | 国家纳米科学中心 | Spr sensor and manufacturing method thereof |
| CN104155453A (en) * | 2014-07-11 | 2014-11-19 | 天津大学 | Hyaluronic acid modified surface plasma resonance spectrometer chip and preparation method thereof |
| CN104359867A (en) * | 2014-10-27 | 2015-02-18 | 李博 | Method for preparing surface plasma resonance chip by taking palladium as target |
| CN104374746A (en) * | 2014-10-27 | 2015-02-25 | 李博 | Method for preparing surface plasma resonance chip by taking silver as target |
| CN104713851A (en) * | 2015-03-24 | 2015-06-17 | 吉林大学 | Miniature real-time on-line offshore oil spill detection system |
| CN104142315B (en) * | 2013-05-07 | 2016-08-17 | 中国科学院理化技术研究所 | A kind of surface plasmon resonance sensing chip for tumor cell detection and preparation method thereof, application |
| CN105954233A (en) * | 2016-06-17 | 2016-09-21 | 北京理工大学 | Preparation method of surface plasma resonance sensor chip for detecting D-glucose in aqueous phase |
| CN106124457A (en) * | 2016-06-17 | 2016-11-16 | 北京理工大学 | A kind of preparation method of surface plasma resonance sensor chip based on nano material |
| CN106940302A (en) * | 2017-03-13 | 2017-07-11 | 中国科学院青岛生物能源与过程研究所 | A kind of SPR sensorgram chip, preparation method and applications |
| CN108693144A (en) * | 2018-04-28 | 2018-10-23 | 天津大学 | Durability single cell protein group Hygienic monitoring on hands of childhood method is carried out based on SPRM technologies |
| CN109709047A (en) * | 2018-03-07 | 2019-05-03 | 长沙学院 | A kind of bimetallic long-range surface plasmon resonance sensor and preparation method thereof |
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| CN104142315B (en) * | 2013-05-07 | 2016-08-17 | 中国科学院理化技术研究所 | A kind of surface plasmon resonance sensing chip for tumor cell detection and preparation method thereof, application |
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| CN104155453A (en) * | 2014-07-11 | 2014-11-19 | 天津大学 | Hyaluronic acid modified surface plasma resonance spectrometer chip and preparation method thereof |
| CN104359867A (en) * | 2014-10-27 | 2015-02-18 | 李博 | Method for preparing surface plasma resonance chip by taking palladium as target |
| CN104374746A (en) * | 2014-10-27 | 2015-02-25 | 李博 | Method for preparing surface plasma resonance chip by taking silver as target |
| CN104713851A (en) * | 2015-03-24 | 2015-06-17 | 吉林大学 | Miniature real-time on-line offshore oil spill detection system |
| CN105954233A (en) * | 2016-06-17 | 2016-09-21 | 北京理工大学 | Preparation method of surface plasma resonance sensor chip for detecting D-glucose in aqueous phase |
| CN106124457A (en) * | 2016-06-17 | 2016-11-16 | 北京理工大学 | A kind of preparation method of surface plasma resonance sensor chip based on nano material |
| CN105954233B (en) * | 2016-06-17 | 2018-08-24 | 北京理工大学 | The preparation method of the surface plasma resonance sensor chip of detection D-Glucose in a kind of water phase |
| CN106124457B (en) * | 2016-06-17 | 2019-03-19 | 北京理工大学 | A kind of preparation method of the surface plasma resonance sensor chip based on nano material |
| CN106940302A (en) * | 2017-03-13 | 2017-07-11 | 中国科学院青岛生物能源与过程研究所 | A kind of SPR sensorgram chip, preparation method and applications |
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Application publication date: 20111130 |