CN105548577B - A kind of Lyme disease detection protein-chip of succinyl beta cyclodextrin modification and its preparation and application - Google Patents

A kind of Lyme disease detection protein-chip of succinyl beta cyclodextrin modification and its preparation and application Download PDF

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CN105548577B
CN105548577B CN201610084174.XA CN201610084174A CN105548577B CN 105548577 B CN105548577 B CN 105548577B CN 201610084174 A CN201610084174 A CN 201610084174A CN 105548577 B CN105548577 B CN 105548577B
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chip
protein
lyme disease
solid phase
succinyl
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CN105548577A (en
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杜卫东
黄娜丽
叶雷
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Anhui Medical University
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Anhui Medical University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/20—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Spirochaetales (O), e.g. Treponema, Leptospira
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention belongs to field of biological medicine, and in particular to a kind of Lyme disease detection protein-chip of succinyl beta cyclodextrin modification and its preparation and application.The first aspect of the present invention contains the sequence table of Borrelia burgdoyferi variable major protein sample up to E protein there is provided a kind of protein-chip detected for Lyme disease, including solid phase carrier and the capture molecule being fixed on the solid phase carrier, the capture molecule（VlsE）.In detection, every chip can detect many cases serum sample simultaneously, and sensitivity, specificity are higher, and the occurrence probability of false positive and false negative is detected between reduction group.When compared with conventional method, carry out extensive examination with its crowd to Lyme disease high-risk areas, it is possible to provide more easy and reliable detection method, improve screening efficiency and positive detection rate and the degree of accuracy.

Description

A kind of Lyme disease detection protein-chip of succinyl-beta-cyclodextrin modification and its Preparation and application
Technical field
The invention belongs to field of biological medicine, and in particular to a kind of Lyme disease inspection of succinyl-beta-cyclodextrin modification Survey protein-chip and its preparation and application.
Background technology
Lyme disease (Lyme Diseas, LD) be it is a kind of propagated by tick worm rotation body-sensing dye caused by involve skin, nerve, The disease of many injuries of tissues and organs such as joint, heart, also known as lime borreliosis (Lymeborreliosis).Lyme disease Main pathogens are Borrelia burgdoyferi, are broadly divided into three hypotypes：Narrow sense Borrelia burgdoyferi (Borrelia Burgdorferi sensue stricto), Ga Shi Borrelia burgdoyferis (Borrelia afzelii) and Jia Shi Bai Shi dredge spiral shell Revolve body (Borrelia garinii).Lyme disease bacterial strain is also classified into three kinds of genotype, wherein B.burgdorferi in China Sensue stricto, B.afzelii, B.garinii gene kind ratios are respectively 5.81%, 23.26%, 66.28%.Lime Disease is its hotspot in the U.S. and Europe, the especially Scandinavian Peninsula and Central European area；In China, Lyme disease Morbidity be mainly distributed on mountain area and forest zone, child morbidity is higher than adult.
Annular migratory erythema (Erythema migrans, EM) is Lyme disease early stage classical symptom, the Lay of untreated Nurse disease patient disease development several months to can occur neuropathy between the several years, peripheral nerve and nervous centralis all will be by It is tired, cause nerve Lyme disease (neuroborreliosis, NB).But be due to Lyme disease initial infection clinical manifestation it is non- Performance and many skin diseases of specificity, i.e. its annular migratory erythema have similar and overlapping performance, and clinical doctor The raw harm to Lyme disease recognizes the reasons such as deficiency, causes the easy clinically mistaken diagnosis of Lyme disease or fails to pinpoint a disease in diagnosis.Experimental study shows place Active thing is different to clinical symptoms caused by three kinds of genotype B. barrgdorferi infections, and narrow sense Borrelia burgdoyferi is main Cause the disease relevant with joint, Ga Shi Borrelia burgdoyferis mainly cause disease of skin, and Jia Shi Borrelia burgdoyferis mainly draw Play disease relevant with nervous system etc..Method currently used for laboratory diagnosis B. barrgdorferi infection includes cell culture Technology, immunohistochemistry technology, polymerase chain reaction and carried out with full cell pyrolysis liquid, recombinant protein and polypeptide it is enzyme-linked The technology such as immune detection or the Serum Antibody Detection of Western blotting.Nevertheless, routine serum immunization method is in Lyme disease Value in diagnosis remains problems.American National Disease Control and Prevention Center recommends the prefered method of Lyme disease detection to be to use The detection for the antibody that ELISA or direct immunofluorescence are produced to Borellia in serum, but result stills need Diagnosis of Sghistosomiasis The confirmation of mark method.
The content of the invention
In order to overcome the problems of in the prior art, it is an object of the invention to provide a kind of succinyl-β-ring paste The Lyme disease detection protein-chip of refine decorations and its preparation and application.
To achieve these goals and other related purposes, the present invention is adopted the following technical scheme that：
The first aspect of the present invention there is provided it is a kind of for Lyme disease detect protein-chip, including solid phase carrier and The capture molecule being fixed on the solid phase carrier, the capture molecule contains Borrelia burgdoyferi variable major protein sample sequence Express E protein (VlsE).
Preferably, the dot matrix that is fixed as is fixed.
Preferably, the solid phase carrier is goldleaf chip.
The substrate of goldleaf chip employed in the preferred embodiments of the present invention is sheet glass, and one layer of 10nm thickness is covered thereon It is array (the row * 12 of 96 hole * 2,8 of 50 μm of TEFLON films of a compartmentalization on the proof gold (purity 99.9%) of degree, goldleaf Row), array aperture is 1.25mm.
It is further preferred that the solid phase carrier is surface is modified with 16- amino -1- hexadecanethiols and amber successively The goldleaf chip of acyl-beta-schardinger dextrin.
Preferably, on succinyl-beta-cyclodextrin by the succinimido of activated carboxylic with being repaiied on goldleaf chip Amino in the 16- amino -1- hexadecanethiols of decorations is combined.
Preferably, the sequence table of Borrelia burgdoyferi variable major protein sample reaches E protein (VlsE) and succinyl-beta-cyclodextrin With reference to.
Preferably, the sequence table of Borrelia burgdoyferi variable major protein sample reaches E protein (VlsE) on the solid phase carrier Density range is：3.9x 10-4ng/mm2~0.41ng/mm2。
It is further preferred that B. burgdorferi surface albumen VlsE density range is on the solid phase carrier：1.3x 10-2ng/mm2~0.41ng/mm2。
There is provided the preparation of solid phase carrier in the above-mentioned protein-chip detected for Lyme disease for the second aspect of the present invention Method, including step：
(1) it is succinimido by the activated carboxylic on succinyl-beta-cyclodextrin, the succinyl-β after being activated- Cyclodextrin；
(2) 16- amino -1- hexadecanethiols are modified on goldleaf chip, the modification of 16- amino -1- hexadecanethiols is obtained Goldleaf chip；
(3) on the goldleaf chip that gained 16- amino -1- hexadecanethiols are modified in step (2) obtained by modification step (1) Succinyl-beta-cyclodextrin after activation, obtains solid phase carrier.
Preferably, in step (1), the carboxyl on succinyl-beta-cyclodextrin is activated using EDC and NHS.
The third aspect of the present invention there is provided it is above-mentioned for Lyme disease detect protein-chip construction method, including Step：
Capture molecule is diluted, capture molecule solution is obtained；Using conventional method by capture molecule solution point respectively Battle array is fixed in the diverse location of surface of solid phase carriers.
The fourth aspect of the present invention additionally provides the protein-chip for being previously used for Lyme disease detection in the thin spiral shells of preparation Bai Shi Revolve the purposes in body detection kit.
The fifth aspect of the present invention provides a kind of Borrelia burgdoyferi detection kit, and the kit includes foregoing use The protein-chip detected in Lyme disease.
Preferably, negative control is also contained in the Borrelia burgdoyferi detection kit.
The negative control is PBST-BSA solution or negative serum.
The PBST-BSA solution is the phosphate buffer PBS (PH 7.2-7.4) by concentration 0.01M；0.1% (v/v) Tween 20；0.1% (w/v) hyclone BSA mixing is constituted.
The negative serum is the serum for the Healthy People that clinical definite is uninfected by Borrelia burgdoyferi.
Preferably, positive control is also contained in the Borrelia burgdoyferi detection kit.
Described positive control selects human immunoglobulin(HIg) IgG, IgM or positive serum.
Described positive serum is the serum of the B. barrgdorferi infection patient of clinical definite.
The selection of special antigen and goldleaf chip is the key innovations of present protein chip.Institute based on the present invention State kit to use fluorescent method to be detected, so some other reagent can also be included in kit.For example：Mark One or more in quasi- product, dilution, cleaning fluid.Specifically need which reagent is fitted into kit, can be according to reality Need configuration.
The standard items include the IgG or IgM that the sequence table of Borrelia burgdoyferi variable major protein sample reaches E protein (VlsE) Antibody.
Preferably, described kit also includes fluorescein-labeled IgG secondary antibodies, fluorescein-labeled IgM secondary antibodies.
Preferably, the fluorescein is marked or Cy5 marks labeled as Cy3.
Preferably, dilution PBST-BSA solution is also contained in the kit.The dilution can be used for dilution antigen, Antibody, serum etc..The PBST-BSA solution is the phosphate buffer PBS (PH 7.2-7.4) by concentration 0.01M；0.1% (v/v)Tween 20；0.1% (w/v) hyclone BSA mixing is constituted.
The sixth aspect of the present invention additionally provides the application method of the kit, comprises the following steps：
By serum sample point on aforementioned proteins chip, be incubated, cleaning, then put plus fluorescein-labeled IgG secondary antibodies and Fluorescein-labeled IgM secondary antibodies mixed solution.
Clean for the serum not reacted completely to be removed from chip, can select this area routine is used for antigen-antibody The washing lotion of reaction.Preferably, it can select PBST (pH7.4) and be used as washing lotion.
Preferably, during cleaning, PBST is cleaned 3 times, 2 minutes every time, nitrogen drying.
Preferably, the fluorescein-labeled IgG secondary antibodies are the IgG antibody that Cy3 is marked.More preferably Cy3 mark donkeys resist Human IgG antibody.
Preferably, the fluorescein-labeled IgM secondary antibodies are the IgM antibody that Cy5 is marked.More preferably Cy5 marks goat Anti-human IgM antibodies.
Compared with prior art, the present invention has the advantages that：
(1) on the one hand the present invention is used as matrix by the use of goldleaf.Itself and traditional glass piece, silicon chip and high polymer material matrix Advantage be that goldleaf sheet is as inert metal, but it is for the combination of chemical substance is compared with traditional glass piece and silicon chip It is more firm.And the biological affinity of goldleaf is relatively low, it is difficult to produce non-specific adsorption with the material such as gene or protein, Greatly reduce non-specificity, it is to avoid the generation of testing result false positive.
(2) for actually detected clinical Lyme disease patients serum moderate resistance Borrelia burgdoyferi variable major protein sample sequence When expressing E protein (VlsE) antigen, every chip can detect many cases serum sample simultaneously, and sensitivity, specificity are higher, reduce The occurrence probability of false positive and false negative is detected between group.When compared with conventional method, with it to Lyme disease high-risk areas Crowd carry out extensive examination, it is possible to provide more easy and reliable detection method, improve screening efficiency and positive examine Survey rate and the degree of accuracy.
Brief description of the drawings
Fig. 1：Structured the formation schematic diagram for goldleaf chip point sample.
Fig. 2：Utilize sign situation before AFM (AFM) observation goldleaf chip modification.
Fig. 3：Situation is characterized after being modified using AFM (AFM) observation goldleaf chip.
Fig. 4：For goldleaf chip plane phenogram after Fourier infrared spectrograph scanning chemical modification.
Fig. 5 A：The goldleaf chip modified using chip scanner succinyl-beta-cyclodextrin in embodiment 2 is to different albumen The Quality Control experiment gained protein-chip of matter compatibility is scanned acquired results figure respectively.
Fig. 5 B：Quality Control of the goldleaf chip that succinyl-beta-cyclodextrin is modified in embodiment 2 to different proteins compatibility is real Test the fluorescence curve figure of gained protein-chip.
Fig. 6：The fluorescence curve figure of antigen incubation temperature-time Quality Control experiment gained protein-chip in embodiment 2.
Fig. 7 A：Quality Control is incubated using chip scanner to rabbit-anti VlsE antigen I gG antibody concentrations gradient in embodiment 2 to test Gained protein-chip is scanned acquired results figure respectively.
Fig. 7 B：Rabbit-anti VlsE antigen Is gG antibody concentrations gradient is incubated Quality Control experiment gained protein-chip in embodiment 2 Fluorescence curve figure.
Fig. 8 A：Three groups of fluorescence results in repeated experiment in 3 groups of embodiment, not by different loading wells and different time Limitation, preferably, stability is higher for repeatability.
Fig. 8 B：Each group fluorescence results in repeatability experiment between 3 groups of embodiment, not by different loading wells and different time Limitation, repeatability preferably, stability is higher.
Fig. 9 A：Gained is scanned using each protein-chip of the serum dilution of chip scanner embodiment 4 experiment gained Result figure.
Fig. 9 B：Using serum dilution as abscissa in embodiment 4, mean value of fluorescence intensity A is ordinate, draws gained glimmering Light intensity figure.
Figure 10 A：Gained protein-chip is scanned using chip scanner in embodiment 5, Lyme disease patients serum Middle IgM detections figure.
Figure 10 B：Gained protein-chip is scanned using chip scanner in embodiment 5, Lyme disease patients serum Middle IgG detections figure.
Embodiment
Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe Embodiment, the protection domain being not intended to be limiting of the invention.The test method of unreceipted actual conditions in the following example, Generally according to normal condition, or according to the condition proposed by each manufacturer.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range Any one numerical value can select between point and two end points.Unless otherwise defined, in the present invention all technologies for using and Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except the specific method used in embodiment, equipment, Outside material, according to those skilled in the art to the grasp of prior art and the record of the present invention, it can also use and this Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method using this technology lead Domain conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The source of various embodiments of the present invention raw material is with preparing as follows：
1. goldleaf chip-derivation：Goldleaf chip used of the invention comes from Interactiva companies (Germany, ULM), its Substrate is sheet glass, and 50 μm for a compartmentalization on the proof gold (purity 99.9%) of one layer of 10nm thickness, goldleaf are covered thereon TEFLON films array (the row * 12 of 96 hole * 2,8 are arranged), array aperture is 1.25mm, as shown in Figure 1.
2. goldleaf chip surface chemical modifying agent：
Decorating liquid 1：Concentration is the ethanol solution of 0.8mM 16- amino -1- hexadecanethiols.16- amino -1- hexadecanes The molecular formula of mercaptan is C16N36ClNS；
Chemical structural formula is：Purchased from Japanese DOJINDO companies.
Decorating liquid 2：Concentration is the PBS of 1mM succinyl-beta-cyclodextrin (Succinyl- β-CD, Mr=1135g/mol) The aqueous solution.Can be with preparation method specifically：Weigh 1.14mg succinyl-beta-cyclodextrins (Succinyl- β-CD, Mr= 1135g/mol) it is dissolved in the solution for being configured to that concentration is 1mM in 10mM PBS (PH=7.4).Succinyl-beta-cyclodextrin is purchased from SIGMA-ALDRICH companies of the U.S..
Decorating liquid 3：Concentration is 430mM N- hydroxysuccinimides (NHS, Mr=115.09g/mol) aqueous solution.Specifically Compound method can be：Weigh 49.49mg N- hydroxysuccinimides (NHS, Mr=115.09g/mol) and be dissolved in 1ml high pressures The deionized water that steam treatment is crossed obtains the solution that concentration is 430mM.N- hydroxysuccinimides are purchased from U.S. SIGMA-ALDRICH Company.
Decorating liquid 4：Concentration is 50mM 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC, Mr= 191.70g/mol) the aqueous solution.Concrete configuration method can be：Weigh 9.59mg1- (3- dimethylamino-propyls) -3- ethyls carbon two Inferior amine salt hydrochlorate (EDC, Mr=191.70g/mol) is dissolved in the deionized water that 1ml high steam processs crosses and obtains concentration for 50mM Solution 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides be purchased from SIGMA-ALDRICH companies of the U.S..
Decorating liquid 5：Isometric EDC and NHS solution is mixed, and mixed solution presses body with Succinyl- β-CD solution Product is than 1/10 mixing, and 4 DEG C are incubated the mixed solution obtained by 2h.
3. antigen, antibody, fluoresceins
Antigen：The sequence table of Borrelia burgdoyferi variable major protein sample reaches E protein (restructuring) (variable major Protein-like sequence E protein, VlsE), Borrelia burgdoyferi flagellin (restructuring) (Flagellin) purchase From MYBIOSOURCE companies；Borrelia burgdoyferi outer membrane protein C (restructuring) (Outer space protein C, OspC) is purchased from Aviva companies；
Antibody：The sequence table of rabbit-anti Borrelia burgdoyferi variable major protein sample is purchased from up to E protein antigen I gG antibody ROCKLAND companies, rabbit-anti Borrelia burgdoyferi flagellin antigen I gG antibody, rabbit-anti Borrelia burgdoyferi outer membrane protein C resist Former IgG antibody；
Fluorescein：Cy3 mark goat anti-rabbit IgG antibodies are purchased from Sangon companies；Cy3 mark donkey anti-human IgG antibodies are purchased from Sangon companies；Cy3 mark goat anti-human IgM antibodies are purchased from KPL companies.
4. PBST solution is to be mixed to constitute with 0.01M Tween 20 by phosphate buffer PBS.
PBST is prepared：The bags of PBS mono- are dissolved in 1000ml deionized waters, then add 1ml Tween-20, are mixed, are kept flat In shaker overnight.PBS：0.01M, PH 7.2-7.4；Tween-20：0.1% (v/v).
5. PBST-BSA solution is the phosphate buffer PBS by 0.01M, PH 7.2-7.4；0.1% (v/v) Tween 20 And 0.1% (w/v) hyclone BSA mixing constitute.
PBST-BSA solution is prepared：50 μ l BSA (200mg/ml) are taken in 10ml PBST solution, are shaken, are stood. PBST-BSA solution (needs BSA mass concentrations to be 0.1% (w/v)).
Phosphate buffer (PBS), Tween 20 and hyclone (BSA) are purchased from SIGMA-ALDRICH companies.
The selection of the carrier of embodiment 1 and chemical surface treatment
The present invention selects goldleaf chip, from Interactiva companies (Germany, ULM), and its substrate is sheet glass, its It is the array of 50 μm of TEFLON films of a compartmentalization on the proof gold (purity 99.9%) of one layer of 10nm thickness of upper covering, goldleaf (the row * 12 of 96 hole * 2,8 are arranged), array aperture is 1.25mm, as shown in Figure 1.
The cleaning of step 1. goldleaf chip
Prepare TL1 solution (H2O:H2O2:NH3·H2O=5:1:1) pour into stainless steel box, chip is put into box, 82 DEG C Water-bath 5min, deionized water rinsing 4~5 times, ethanol 2 times, each 3min；Nitrogen is air-dried, kept dry.
Goldleaf chip carries out surface chemical modification after step 2. pair cleaning, obtains solid phase carrier
After being cleaned to goldleaf chip, immerse in foregoing decorating liquid 1, room temperature sways incubation 12 hours under dark condition, Cleaned after taking-up with ethanol solution, nitrogen is dried up, and obtains the goldleaf chip of 16- amino -1- hexadecanethiols modification；Take out After be dipped in foregoing decorating liquid 5 and be incubated 4h；Then take out the deionized water that chip crossed with high steam processs and clean 3 times, every time 2 Minute, nitrogen is dried, and is modified on succinyl-beta-cyclodextrin by activated carboxylic into succinimido with goldleaf chip Amino in 16- amino -1- hexadecanethiols is combined, and obtains the goldleaf chip of succinyl-beta-cyclodextrin modification, stand-by.
Sign situation before and after being modified using AFM (AFM) observation goldleaf chip, respectively as shown in Figures 2 and 3, As can be seen that the plane surface after modification is more coarse compared with the plane surface before modification, represent after modification, chemical group is covalent It is connected on goldleaf chip surface.
Goldleaf chip plane after modification is scanned using Fourier infrared spectrograph, chemical characterization figure refers to Fig. 4： 1232cm-1Peak value belongs to C-O in hydroxyl and vibrated；1423 and 1560cm-1Peak value is respectively belonging to C-N and C=O in amido link Vibration；1708cm-1Peak value belongs to C=O in anhydride group and vibrated；2935cm-1Peak value belongs to C-C in hexadecane and vibrated, 3278-3357cm-1Peak value may Master Home in hydroxyl O-H vibrations, O-H vibrates in N-H vibrations and carboxylic acid group in amino.
The Quality Control of embodiment 2 is tested
Prepare Incubating Solution 1：VlsE antigens are dissolved in PBST-BSA solution, concentration gradient are each configured to for 0.5 μ g/ml, 0.25 μ g/ml, 0.125 μ g/ml, 0.0625 μ g/ml, 0.0313 μ g/ml, 0.0156 μ g/ml, 0.0078 μ g/ml, 0.0039 μ G/ml, 0.0019 μ g/ml, 0.00095 μ g/ml, 0.000475 μ g/ml antigenic solution.
Prepare Incubating Solution 2：Human IgG is dissolved in PBST-BSA solution, concentration gradient is configured to for 0.5 μ g/ml, 0.25 μ g/ Ml, 0.125 μ g/ml, 0.0625 μ g/ml, 0.0313 μ g/ml, 0.0156 μ g/ml, 0.0078 μ g/ml, 0.0039 μ g/ml, 0.0019 μ g/ml, 0.00095 μ g/ml, 0.000475 μ g/ml solution；
Prepare Incubating Solution 3：Flagellin antigens are dissolved in PBST-BSA solution, concentration gradient are configured to for 0.5 μ g/ml, 0.25 μ g/ml, 0.125 μ g/ml, 0.0625 μ g/ml, 0.0313 μ g/ml, 0.0156 μ g/ml, 0.0078 μ g/ml, 0.0039 μ G/ml, 0.0019 μ g/ml, 0.00095 μ g/ml, 0.000475 μ g/ml solution；
Prepare Incubating Solution 4:OspC antigens are dissolved in PBST-BSA solution, concentration gradient are configured to for 0.5 μ g/ml, 0.25 μ G/ml, 0.125 μ g/ml, 0.0625 μ g/ml, 0.0313 μ g/ml, 0.0156 μ g/ml, 0.0078 μ g/ml, 0.0039 μ g/ml, 0.0019 μ g/ml, 0.00095 μ g/ml, 0.000475 μ g/ml solution；
Prepare Incubating Solution 5：Rabbit-anti VlsE IgG antibodies are dissolved in PBST-BSA solution, antibody concentration gradient is each configured to For 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, 3.13 μ g/ml, 1.56 μ g/ml, 0.78 μ g/ml, 0.39 μ g/ Ml, 0.19 μ g/ml, 0.095 μ g/ml, 0.0475 μ g/ml solution；
Prepare Incubating Solution 6：Cy3 mark donkey anti-human IgG antibodies are dissolved in PBST-BSA solution, fluorescence secondary antibody concentration is configured to For 2.5 μ g/ml solution；
Prepare Incubating Solution 7：Rabbit-anti Flagellin IgG antibodies are dissolved in PBST-BSA solution, are configured to antibody concentration for 50 μ G/mL solution；
Prepare Incubating Solution 8：Rabbit-anti OspC IgG antibodies are dissolved in PBST-BSA solution respectively, it is 50 to be configured to antibody concentration μ g/mL solution；
Prepare Incubating Solution 9：Cy3 mark goat anti-rabbit IgG antibodies are dissolved in PBST-BSA solution, fluorescence secondary antibody are configured to dense Spend for 2.5 μ g/mL solution；
Prepare Incubating Solution 10：Cy3 mark donkey anti-human IgG antibodies are dissolved in PBST-BSA solution, fluorescence secondary antibody are configured to dense Spend for 2.5 μ g/mL solution；
Prepare Incubating Solution 11：Donkey anti-human IgG antibodies and Cy5 mark goat anti-human IgM antibody's equivalent is marked to be dissolved in Cy3 PBST-BSA solution, is configured to Cy3 mark donkey anti-human IgG antibodies and Cy5 mark goats that fluorescence secondary antibody concentration is 2.5 μ g/ml Anti-human IgM antibodies' mixed solution.
1st, Quality Control of the goldleaf chip of succinyl-beta-cyclodextrin modification to different proteins compatibility is tested
Embodiment 1 is completed to the goldleaf chip of surface chemical modification as solid phase carrier, take four different succinyl-β- Cyclodextrin modified goldleaf core, numbering is 1,2,3,4 respectively, and No. 1 chip point adds Incubating Solution 1；No. 2 chip points add Incubating Solution 2；3 Number chip point adds Incubating Solution 3；No. 4 chip points add Incubating Solution 4；Each gradient concentration sets two repetitions, room temperature condition (25 DEG C) It is lower to be incubated 2 hours, cleaned after taking-up with PBST 3 times, 2 minutes every time, nitrogen drying was obtained and examined for Lyme disease immunoserology Disconnected protein-chip.
It is again 50 μ g/mL rabbit-anti VlsE IgG antibodies, rabbit-anti Flagellin IgG antibodies, rabbit-anti OspC IgG by concentration Point sample has VlsE (numbering is 1), Flagellin (numbering is 3), OspC (numbering is 4) to resist to antibody-solutions in above-mentioned incubation respectively On the protein-chip of former probe, concentration is incubated for 2.5 μ g/ml Cy3 mark donkey anti-human IgG antibodies point samples in above-mentioned in addition On the protein-chip for giving birth to human IgG antibody's probe (numbering is 2).Under the conditions of room temperature (25 DEG C), wet box is incubated 1 hour.Take Go out to be cleaned with PBST 3 times, 2 minutes every time, nitrogen drying was stand-by.
Concentration is had to the volume of antibody for 2.5 μ g/mL Cy3 mark goat anti-rabbit IgG antibody solution point samples with above-mentioned incubation Number be respectively 1,3, on april protein chip, lucifuge is incubated 0.5 hour under the conditions of room temperature (25 DEG C).Taking-up cleans 3 with PBST It is secondary, 2 minutes every time.Nitrogen is dried up.
Use chip scanner (Beijing Bo Ao companies, model：Brilliant core Luxscan 10K-A) to above protein-chip point It is not scanned, is as a result shown in Fig. 5 A.As shown in Figure 5A, the VlsE protein-chips for being 1 for numbering, from left from the right side, the 1st It is respectively 0.5 μ g/ml, 0.25 μ g/ml, 0.125 μ g/ml, 0.0625 μ g/ml to arrange to the 11st VlsE concentration that represents respectively of row, 0.0313 μ g/ml, 0.0156 μ g/ml, 0.0078 μ g/ml, 0.0039 μ g/ml, 0.0019 μ g/ml, 0.00095 μ g/ml, 0.000475 μ g/ml, rightmost one is arranged, that is, is arranged from left from the right side the 12nd, is PBST-BSA, negative control, antigen concentration is zero； The IgG protein-chips for being 2 for numbering, from left from the right side, the IgG concentration that the 1st row are represented respectively to the 11st row is respectively 0.5 μ G/ml, 0.25 μ g/ml, 0.125 μ g/ml, 0.0625 μ g/ml, 0.0313 μ g/ml, 0.0156 μ g/ml, 0.0078 μ g/ml, 0.0039 μ g/ml, 0.0019 μ g/ml, 0.00095 μ g/ml, 0.000475 μ g/ml, rightmost one is arranged, that is, from left from right the 12 row, are PBST-BSA, negative control, antibody concentration is zero；The Flagellin protein-chips for being 3 for numbering, from a left side certainly The right side, from left from right, the Flagellin that the 1st row are represented respectively to the 11st row concentration is respectively 0.5 μ g/ml, 0.25 μ g/ml, The μ of the μ of 0.125 μ g/ml, 0.0625 μ g/ml, 0.0313 g/ml, 0.0156 μ g/ml, 0.0078 g/ml, 0.0039 μ g/ml, 0.0019 μ g/ml, 0.00095 μ g/ml, 0.000475 μ g/ml, rightmost one is arranged, that is, is arranged from left from the right side the 12nd, is PBST-BSA, cloudy Property control, antigen concentration is zero；For numbering the OspC protein-chips for being 4, from left from the right side, the 1st row to the 11st arrange generation respectively The OspC of table concentration is respectively 0.5 μ g/ml, 0.25 μ g/ml, 0.125 μ g/ml, 0.0625 μ g/ml, 0.0313 μ g/ml, 0.0156 μ g/ml, 0.0078 μ g/ml, 0.0039 μ g/ml, 0.0019 μ g/ml, 0.00095 μ g/ml, 0.000475 μ g/ml, most The right one is arranged, that is, is arranged from left from the right side the 12nd, is PBST-BSA, negative control, antigen concentration is zero.
Fig. 5 B are gained fluorescence intensity curves figure when being detected using various concentrations antigen coat：By each row in Fig. 5 A 2 fluorescence intensities obtained by same concentration are averaged, and using antigen concentration as abscissa, mean value of fluorescence intensity is ordinate, is painted Fluorescence intensity curves figure processed, as a result as shown in Figure 5 B.As can be seen from Figure 5B：In each Lyme disease antigen I gG antibody of rabbit-anti and In the case that Cy3 mark goat anti-rabbit IgG antibody incubation conditions are constant, it is equal to when VlsE recombinant antigens incubation concentration is respectively greater than During 0.000475 μ g/ml, produced fluorescence signal intensity has obvious poor with the fluorescence signal intensity that negative control group is produced It is different.Therefore the optimal concentration that display is incubated VlsE recombinant antigens should be in more than 0.000475 μ g/ml.And human IgG, Flagellin, The Percentage bound for the protein-chip that OspC recombinant antigens are modified with succinyl-beta-cyclodextrin is very poor.Absolutely prove, succinyl-β- The combination of cyclodextrin and protein is that have certain selectivity, and not all albumen can be pasted with succinyl-β-ring Essence is combined well, and realizes detection.
2nd, antigen incubation temperature-time Quality Control experiment
Concentration is completed into surface succinyl-beta-cyclodextrin for 0.0156 μ g/ml VlsE antigenic solutions point to embodiment 1 On the goldleaf chip solid phase carrier for learning modification, respectively at 37 DEG C, incubation 24 hours under the conditions of room temperature (25 DEG C) and 4 DEG C, 12 hours, 6 hours, 3 hours, 1 hour and 0.5 hour, taking-up was cleaned 3 times with PBST, 2 minutes every time, nitrogen drying；It is again 50 μ by concentration G/ml rabbit-anti VlsE antigen Is gG antibody-solutions point sample has on the solid phase carrier of VlsE antigen probes in incubation, room temperature (25 DEG C) under the conditions of be incubated 1 hour, taking-up clean 3 times with PBST, 2 minutes every time, and nitrogen is dried up.It is again 2.5 μ g/ by diluted concentration Ml Cy3 mark goat anti-rabbit IgG antibodies are dissolved in PBST-BSA solution point sample to be had on the solid phase carrier of antibody in incubation, dark Incubation at room temperature 0.5 hour, taking-up is cleaned 3 times with PBST-BSA, 2 minutes every time, nitrogen drying.
Use chip scanner (Beijing Bo Ao companies, model：Brilliant core Luxscan 10K-A) to above protein-chip point It is not scanned, as the Quality Control experiment of antigen incubation temperature-time, as a result shows as shown in Figure 6.Same concentrations are obtained first VlsE antigens fluorescent scanning figure obtained by different time and different temperatures is coated with chip；Then calculated according to fluorescent scanning figure Obtain VlsE antigens as shown in Figure 6 and fluorescence intensity curves figure obtained by different time and different temperatures is coated with chip.
It can be known according to the Quality Control of antigen incubation temperature-time experiment：
(1) fluorescence intensity extends with the extension of incubation time, when incubation time be more than 1 it is small when, fluorescence intensity with The extension change of incubation time is not apparent.Therefore in actually detected, in order to shorten incubation time, it is proposed that probe manufacturing is incubated Educate the time be more than can meet demand within 1 hour.
(2) incubation temperature, fluorescence intensity is better than the fluorescence intensity under the conditions of 37 DEG C and 4 DEG C under the conditions of room temperature (25 DEG C). Therefore in actually detected, it is proposed that incubation experiment is carried out under the conditions of room temperature (25 DEG C).
3rd, rabbit-anti VlsE antigen Is gG antibody concentrations gradient is incubated Quality Control experiment
Embodiment 1 is completed to the goldleaf chip of surface chemical modification, and to be dipped in concentration as solid phase carrier be 0.0156 μ g/ Among ml VlsE recombinant antigen solution, it is incubated 2 hours under the conditions of room temperature (25 DEG C), taking-up is cleaned 3 times, every time 2 points with PBST Clock, nitrogen drying；The rabbit-anti VlsE antigen Is gG antibody-solutions point sample of Incubating Solution 5 there are into VlsE, recombinant antigen probe in incubation again Chip on, be incubated 1 hour under the conditions of room temperature (25 DEG C), taking-up clean 3 times with PBST, 2 minutes every time, nitrogen is dried up；Again Plus 2.5 μ g/mL Cy3 mark goat anti-rabbit IgG antibodies, it is dark, be incubated 0.5 hour under the conditions of room temperature (25 DEG C), take out and use PBST is cleaned 3 times, 2 minutes every time, nitrogen drying.
Chip is scanned using chip scanner, as rabbit-anti VlsE antigen Is gG antibody concentrations gradient is incubated Quality Control Experiment, as a result shows as shown in Figure 7 A.As shown in Figure 7 A, for VlsE protein-chips：From left from right, the 1st row to the 11st row Represent respectively, the concentration of rabbit-anti VlsE antigen I gG antibody is respectively 50 μ g/ml, 25 μ g/ml, 12.5 μ g/ml, 6.25 μ g/ml, The μ of the μ of the μ of 3.13 μ g/ml, 1.56 μ g/ml, 0.78 g/ml, 0.39 μ g/ml, 0.19 g/ml, 0.095 μ g/ml, 0.0475 g/ml, it is most right Bian Yilie, that is, arranged from left from the right side the 12nd, it is PBST-BSA, negative control, antibody concentration is 0.
Fig. 7 B are gained fluorescence intensity curves figure when being checked using various concentrations antibody bag：By each row in Fig. 7 A 4 fluorescence intensities obtained by same concentration are averaged, and using antibody concentration as abscissa, mean value of fluorescence intensity is ordinate, is painted Fluorescence intensity curves figure processed.
As can be seen from Figure 7B：In VlsE recombinant antigens and the constant feelings of Cy3 mark goat anti-rabbit IgG antibody incubation conditions Under condition, when rabbit-anti VlsE antigen I gG antibody incubation concentration is respectively greater than 0.39 μ g/mL, produced fluorescence signal intensity with The fluorescence signal intensity that negative control group is produced has cognizable difference.Illustrate this modification chip can detect naked eyes can Depending on rabbit-anti VlsE antigen I gG antibody concentrations in g/mL grades of μ.
The repeatability experiment of embodiment 3
Table 1 is repeatability experiment in group, and every 16 holes are as one group, totally three groups, are incubated interior on the same day according to step The rabbit-anti VlsE antigen I gG antibody and concentration that concentration is 0.0156 μ g/ml VlsE antigens, concentration is 50 μ g/ml are 2.5 μ g/ Ml Cy3 mark goat anti-rabbit IgG antibodies.
Table 1
Table 2 is repeatability experiment between group, and every 24 holes three groups, were incubated totally in three days according to step respectively as one group It is 2.5 μ to educate concentration is 0.0156 μ g/ml VlsE antigens, concentration is 50 μ g/ml rabbit-anti VlsE antigen I gG antibody and concentration G/ml Cy3 mark goat anti-rabbit IgG antibodies.
Table 2
In table 1,2, SD is standard deviation, and CV is the coefficient of variation, and both indexs can reflect biochip in anti-albumen sample Sequence table reaches the stability in E protein antibody test and is not in big deviation.
As shown in Figure 8 A：Three groups of fluorescence results are not limited by different loading wells and different time in repeated experiment in group System, preferably, stability is higher for repeatability.
As shown in Figure 8 B, each group fluorescence results are not by different loading wells and different time during repeatability is tested between group Limitation, preferably, stability is higher for repeatability.
In summary, the result of present protein chip detection VlsE antigen I gG antibody not by different loading wells and The limitation of different time, preferably, stability is higher for repeatability.
The serum dilution of embodiment 4 is tested
Any take clinical has made a definite diagnosis the Lyme disease patients serum of B. barrgdorferi infection and clinical definite is uninfected by Bai Shi Each 3 of the Healthy Human Serum of Borellia.
By every serum with PBST-BSA according to gradient 1:2.5,1:5,1:10,1:20,1:40,1:80,1:160；1: 320,1:640,1:1280.1:2560 dilutions.Then it is according to from left free order successively point sample to containing incubation concentration On the chip of the probe of 0.0156 μ g/ml primary VlsE antigens, remaining 6 null point sample liquid is PBST-BSA solution.That is, for Detect the VlsE protein-chips of positive serum：From left from the right side, the 1st row to the 11st row are represented respectively, serum-dilution ratio difference For 1:2.5,1:5,1:10,1:20,1:40,1:80,1:160；1:320,1:640,1:1280.1:2560, rightmost one is arranged, also Arranged from left from the right side the 12nd, be PBST-BSA, negative control, serum-concentration is 0.VlsE protein for detecting negative serum Chip：From left from the right side, the 1st row to the 11st row are represented respectively, and serum-dilution ratio is respectively 1:2.5,1:5,1:10,1:20,1: 40,1:80,1:160；1:320,1:640,1:1280.1:2560, rightmost one is arranged, that is, is arranged from left from the right side the 12nd, is PBST- BSA, negative control, serum-concentration is 0.
It is incubated 1 hour, is cleaned after taking-up with PBST 3 times, 2 minutes every time, nitrogen drying under normal temperature condition.Again plus 2.5 μ g/ Ml Cy3 mark donkey anti-human IgG antibodies, it is dark, it is incubated 0.5 hour under the conditions of room temperature (25 DEG C), taking-up is cleaned 3 times with PBST, 2 minutes every time, nitrogen drying.
Chip is scanned using chip scanner, as a result shown as shown in Figure 9 A, above 3 rows be positive serum group (appoint take 3 positive serum groups), below 3 rows be negative serum group (appoint and take 3 negative serum groups).Fig. 9 B：Calculate positive serum Organize the ratio A of fluorescence average value and corresponding dilution factor negative serum control group fluorescence average value.Using serum dilution as abscissa, Mean value of fluorescence intensity A is ordinate, draws fluorescence intensity curves figure.We set 3 serum fluorescent values of positive serum group group The ratio between average value and 3 serum fluorescent values of negative serum group A be more than 4 dilution factor and can be used for experiment detection, institute for The scope of the serum dilution of detection is (1:10)~(1:160).
The Lyme disease patients serum's pattern detection of embodiment 5
Method according to embodiment 1 prepares the goldleaf chip of succinyl-beta-cyclodextrin modification, as solid phase carrier, will incubate VlsE antigenic solutions difference point sample that concentration is 0.0156 μ g/ml is educated on each hole of solid phase carrier, room temperature condition (25 DEG C) It is lower to be incubated 2 hours, cleaned after taking-up with PBST 3 times, 2 minutes every time, nitrogen drying was obtained and examined for Lyme disease immunoserology Disconnected protein-chip.
Such as Figure 10 A and as shown in Figure 10 B, 188 clinics have been diagnosed as to the lime patient of B. barrgdorferi infection Serum distinguishes point sample on wherein 188 holes of shown protein-chip, and 2 hole sampling liquids are that clinical definite is uninfected by primary in addition The Healthy Human Serum of borrelia burgdorferi, remaining 2 hole sampling liquid are PBST-BSA solution, are incubated 1 hour under normal temperature condition, are taken out Cleaned afterwards with PBST 3 times, 2 minutes every time, nitrogen drying.The point sample of Incubating Solution 11 is had to the chip of patients serum's antibody in incubation again On, it is dark, it is incubated 0.5 hour under the conditions of room temperature (25 DEG C), taking-up is cleaned 3 times with PBST, 2 minutes every time, nitrogen drying.
Chip is scanned using chip scanner, Figure 10 A and Figure 10 B is as a result respectively indicated as.Wherein, Figure 10 A are IgM detections figure in Lyme disease patients serum；Figure 10 B are IgG detection figures in Lyme disease patients serum.As can be seen from the figure： Actually detected clinic has been diagnosed as in the serum of lime patient, the fluorescence intensity of the serum of positive patient and the blood of healthy population Obvious otherness is there is between the clear and fluorescence intensity of PBST-BSA point samples.
Counted according to fluoroscopic examination result, protein-chip (Biochip) and the tradition side of the present invention is respectively adopted Method ELISA method has made a definite diagnosis 188 clinics Lyme disease patients serum and has carried out Comparison between detecting methods, as a result as shown in table 3：
Table 3
“+”：positive；“-”：negtive
*:χ2=0.44, p>0.05
Carry out Chi-square Test：Biochip vs.ELISA, p>0.05, illustrate that chip method is examined with traditional ELISA method Surveying Lyme disease does not have significant difference.
Calculated according to the result of table 3：The positive rate of Biochip methods detection is 45.2% (85), ELISA method detection Positive rate be 43.6% (82).It can thus be concluded that, the positive rate of Biochip methods detection Lyme disease is higher than ELISA method.
In summary, protein-chip of the invention can detect that anti-VlsE antigen Is gG and IgM in patient's body resist respectively Body, protein-chip method can substitute traditional ELISA method detection Lyme disease as a kind of, especially in a large amount of examination samples In have great advantage.
It is described above, only presently preferred embodiments of the present invention, it is not any to the present invention in form and substantial limitation, It should be pointed out that for those skilled in the art, on the premise of the inventive method is not departed from, can also make Some improvement and supplement, these are improved and supplement also should be regarded as protection scope of the present invention.All those skilled in the art, Without departing from the spirit and scope of the present invention, when made using disclosed above technology contents it is a little more Dynamic, modification and the equivalent variations developed, are the equivalent embodiment of the present invention；Meanwhile, all substantial technologicals pair according to the present invention The variation, modification and evolution for any equivalent variations that above-described embodiment is made, still fall within the scope of technical scheme It is interior.

Claims (6)

1. a kind of protein-chip detected for Lyme disease, including solid phase carrier and the capture being fixed on the solid phase carrier Molecule, the capture molecule contains the sequence table of Borrelia burgdoyferi variable major protein sample up to E protein (VlsE)；The solid phase is carried Body is the goldleaf chip that surface is modified with 16- amino -1- hexadecanethiols and succinyl-beta-cyclodextrin successively；Succinyl-β-ring In the 16- amino -1- hexadecanethiols modified on dextrin on the succinimido and solid phase carrier of activated carboxylic Amino combine；The sequence table of Borrelia burgdoyferi variable major protein sample reaches E protein (VlsE) and succinyl-beta-cyclodextrin knot Close.
2. protein-chip according to claim 1, it is characterised in that Borrelia burgdoyferi variable on the solid phase carrier The sequence table of major protein sample is up to the density range of E protein (VlsE)：3.9x10-4ng/mm2~0.41ng/mm2。
3. it is used for solid phase carrier in the protein-chip that Lyme disease is detected according to claim 1~2 any claim Preparation method, including step：
(1) it is succinimido by the activated carboxylic on succinyl-beta-cyclodextrin, the succinyl-β after being activated-ring paste Essence；
(2) 16- amino -1- hexadecanethiols are modified on goldleaf chip, the gold of 16- amino -1- hexadecanethiols modification is obtained Paper tinsel chip；
(3) activated in step (2) on the goldleaf chip of gained 16- amino -1- hexadecanethiols modification obtained by modification step (1) Succinyl-beta-cyclodextrin afterwards, obtains solid phase carrier.
4. it is used for the construction method for the protein-chip that Lyme disease is detected according to claim 1~2 any claim, bag Include step：Capture molecule is diluted, capture molecule solution is obtained；Capture molecule solution is distinguished by dot matrix using conventional method In the diverse location of surface of solid phase carriers, and fix.
5. the protein-chip for being used for Lyme disease detection according to claim 1~2 any claim is dredged in preparation Bai Shi Purposes in conveyor screw detection kit.
6. a kind of Borrelia burgdoyferi detection kit, the kit is included as described in claim 1~2 any claim The protein-chip detected for Lyme disease.
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