CN106645352B - One kind is for detecting bilharzial nano antibody composition, immunosensor and its preparation method and application - Google Patents

One kind is for detecting bilharzial nano antibody composition, immunosensor and its preparation method and application Download PDF

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CN106645352B
CN106645352B CN201710047340.3A CN201710047340A CN106645352B CN 106645352 B CN106645352 B CN 106645352B CN 201710047340 A CN201710047340 A CN 201710047340A CN 106645352 B CN106645352 B CN 106645352B
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antibody composition
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陆绍红
孔庆明
陈睿
郑斌
楼涤
付益修
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Zhejiang Academy of Medical Sciences
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Abstract

The invention discloses one kind for detecting bilharzial nano antibody composition, immunosensor and its preparation method and application.The nano antibody composition, including two kinds of nano antibodies, amino acid sequence is respectively as shown in SEQ ID NO.1 and SEQ ID NO.2.The immunosensor is using the graphene oxide film after restoring as working electrode, in one layer of gold nano grain of electrode reaction area deposition, the nano antibody composition is then passed through into strong be integrated in the graphene oxide electrode of gold nano particle modification of golden ammonia and is obtained.It is to be immunized obtained by camel by the soluble egg antigen of Schistosoma japonicum that the present invention, which is used to detect bilharzial nano antibody composition, nano antibody good hydrophilic property, molecular weight are small, the immunosensor prepared using nano antibody composition of the present invention is for when detecting blood fluke, high sensitivity, sensitivity can achieve 0.01ng/mL, significantly larger than conventional ELISA method；Specificity is good, is not susceptible to accidentally survey between the polypide of different plant species.

Description

One kind is for detecting bilharzial nano antibody composition, immunosensor and its system Preparation Method and application
Technical field
The present invention relates to biomedical or biotechnology detection technique fields, more particularly to one kind for detecting blood fluke Nano antibody composition, immunosensor and its preparation method and application.
Background technique
Snail fever is a kind of infecting both domestic animals and human parasitic disease for seriously endangering human health, and schistosomiasis control is still It is an important task.Circulating antigen of schistosome (Circulating antigen, CAg) can be used as Current Infection and curative effect The important indicator of examination, conventional immunological method is more difficult to detect the lower CAg of content in body fluid, particularly with low infectiosity Or the crowd of the slight Endemic Area of snail fever.Therefore, a kind of sensitive, special CAg detection technique is established, for snail fever Prevention and treatment is of great significance.
Biosensor converts electric signal for biochemical signals by cured biological identification molecule and energy converter and reaches straight The purpose for connecing detection, compared to its accuracy of conventional method and high sensitivity, continuous mode is simple, convenient for popularizing.With The addition of nano material, sensor has further promotion in the sensitivity and specificity in terms of checkout and diagnosis, and has become Function detects bacterium, virus, helminth, protein.
In recent years, antibody technique has been widely used in the diagnosis and treatment field of disease, and novel gene engineered antibody constantly goes out Existing, antibody miniaturization is one of main direction of studying of antibody genetic engineering.Newest single domain antibody (Single domain Antibody, SdAb) refer to the VH small antibody fragments for remaining preferable antigen binding capacity and specificity, structure letter It is single, it is easy the external scale of progress and is combined to express.SdAb small volume, have stronger tissue penetration, be conducive to they into Enter fine and close tissue and penetrates blood-brain barrier.This antigen table for making it provide easy access to the ditch on target surface, stitch or be hidden Position, identifies the unrecognized antigen of many conventional antibodies.However, SdAb exposes surface hydrophobicity group, non-specific adsorption increases Add, easily condense and stick, must could be used after structure of modification.
In camel body there are a kind of functional heavy chain antibody of natural light chain missing (Heavy chain antibodies, HCAb), the variable region for cloning the heavy chain antibody obtains the smallest antigen-binding fragment, i.e. nano antibody (Nanobody, Nb).It receives The sharpest edges of meter Kang Ti are the small in size of it.Its molecular weight only has 15kDa, insufficient conventional antibodies (160KDa) ten/ One, also much smaller than single domain antibody (55KDa).The more common antibody of Nb also has high water soluble and conformational stability, stronger anti- The advantages that former affinity and excellent tissue penetration, easy vivoexpression and humanization modified modification, the above characteristic of Nb It is set to show wide application prospect in diagnosis detection field.
Graphene is a kind of novel carbon nanomaterial, has biggish specific surface area and efficient electronic conduction ability, Its unique two-dimensional layered structure assigns graphene largely electroactive distribution sites again, can load a large amount of large biological molecule Substance is to itself carrying out modifying and do not change activity itself with good biocompatibility, while graphene has directly perception With the ability of amplification boundary material variation, these characteristics are all conducive to the identification of biological substance and the conversion of signal.Therefore, graphite Alkene also becomes the ideal material of biosensor.This research establishes a kind of micro-nano based on novel nano-material graphene and Nb Immunosensor, and the detection of Preliminary Applications CAg in Sera from Schistosomiasis cases.
Summary of the invention
In view of the deficiencies of the prior art, the present invention provides a kind of for detecting bilharzial nano antibody composition, benefit The immunosensor prepared with the composition is good with high sensitivity, specificity for detecting blood fluke.
One kind is for detecting bilharzial nano antibody composition, including two kinds of nano antibodies, amino acid sequence difference As shown in SEQ ID NO.1 and SEQ ID NO.2.
The nano antibody composition encodes the nucleotide sequence of described two nano antibodies respectively such as SEQ ID Shown in NO.3 and SEQ ID NO.4.
The ratio of described two nano antibodies are as follows: according to the molar ratio 1: 0.5~2.
Invention further provides application of the nano antibody composition in preparation blood fluke detection kit.
The present invention also provides one kind for detecting bilharzial immunosensor, combines comprising the nano antibody Object.
The application in blood fluke detection that the present invention also provides the immunosensors for the purpose of non-disease diagnoses.
The present invention also provides the preparation methods of the immunosensor, comprising the following steps:
(1) graphene oxide film after restoring is as working electrode, in one layer of gold nano of electrode reaction area deposition The graphene oxide electrode of particle acquisition gold nano particle modification；
(2) the nano antibody composition is passed through into the strong graphene oxide electrode for being integrated to gold nano particle modification of golden ammonia It is upper to obtain the immunosensor.
The preparation method, gold nano grain deposit to working electrode conversion zone by electrochemical deposition method, plating Liquid uses 1% HAuCl4Solution, electroplating voltage are -0.66V, reaction time 100s.
The preparation method is closed after nano antibody composition combines using BSA.After nano antibody combines, still There can be the exposed gold nano grain for being not bound with antibody, need to be closed using other albumen.Certainly, in addition to using BSA also can be used other albumen and be closed, for example milk powder is also common confining liquid.
The present invention is to be exempted from by the soluble egg antigen of Schistosoma japonicum for detecting bilharzial nano antibody composition Obtained by epidemic disease camel, nano antibody good hydrophilic property, molecular weight are small, the immunosensor prepared using nano antibody composition of the present invention When for detecting blood fluke, high sensitivity, sensitivity can achieve 0.01ng/mL, significantly larger than conventional ELISA method；It is special It is anisotropic good, it is not susceptible to accidentally survey between the polypide of different plant species.
Detailed description of the invention
Fig. 1 is the glue testing result figure for the total serum IgE that immune rear camel blood sample extracts, and wherein M is DNA Marker DL2000 Swimming lane 1 is total serum IgE.
Fig. 2 is nested PCR product glue testing result figure, wherein M is DNA Marker DL2000.
Fig. 3 is the bacterium colony PCR qualification result figure of initial libraries, wherein M is DNA Marker DL2000, and swimming lane 1-18 is 18 single colonie PCR products of random picking, PC is positive control.
Fig. 4 is indirect Phage-ELISA qualification result figure, and wherein 1-48 is the phage clone of random picking, and PC is sun Property control, NC is negative control.
Fig. 5 is the testing result figure of purifying gained Nb recombinant expression, wherein figure A is SDS-PAGE testing result figure, schemes B For the DAB colour developing figure of Western Blot result.
Fig. 6 is the atomic force microscope phenogram of graphene film.
Fig. 7 is the scanning electron microscope phenogram of graphene film, wherein figure A is cross-sectional structure, figure B is surface shape Looks
Fig. 8 be it is gold-plated after graphene membrane electrode surface scanning electron microscope phenogram.
Fig. 9 is the Raman spectrogram of graphene oxide film and graphene film.
Figure 10 is 6 middle impedance test chart of embodiment, wherein a: graphene bare electrode；B: the electricity after gold nano particle modification Pole；C: the electrode after nano antibody is fixed；Electrode after d:BSA closing；Electrode after e:SEA combination.
Figure 11 is immunosensor specific detection result figure prepared by embodiment 2, wherein figure A is nano antibody modification The impedance diagram of electrode detection difference parasite antigen；Figure B is the impedance that nano antibody modified electrode detects different parasite antigens Changing value histogram, * represent significant difference；Figure C is the impedance diagram that antiserum modified electrode detects different parasite antigens；Figure D is the impedance variations value histogram that antiserum modified electrode detects different parasite antigens.
Detection curve figure when Figure 12 is the immunosensor sensitivity technique of the preparation of embodiment 2 under difference SEA concentration, In, curve a~h concentration is respectively 10-8mg/mL、10-7mg/mL、10-6mg/mL、10-5mg/mL、10-4mg/mL、10-3mg/mL、 10-2mg/mL、10-1mg/mL。
Figure 13 be embodiment 2 prepare immunosensor sensitivity technique when electronics transfer resistance and SEA concentration it is linear Curve graph.
Specific embodiment
Main agents:
0.1% chlorauric acid solution, graphite (spectroscopic pure) are purchased from Chinese pharmaceutical chemistry reagent Co., Ltd；HI solution, 95% Ethyl alcohol is purchased from raw work biology Shanghai Co., Ltd；Graphene oxide solution passes through JY92- by graphite oxide (preparation of Hummers method) II ultrasonic cell disruptor (being purchased from NingBo XinZhi Biology Science Co., Ltd) degradation preparation；0.45 μm of nitrocellulose Film is purchased from Bio-Rad company；Ag/Agcl reference electrode is purchased from the limited public affairs of Shanghai Chen Hua instrument to electrode, electrochemical reaction cell Department；1%BSA；PBS solution (pH=7.4 contains 0.01% polysorbas20)；5.0mM K containing 0.1M KCl3Fe(CN)6/K4Fe (CN)6(1: 1) mixed solution；Distilled water；Other chemical reagent are that analysis is pure.
Key instrument:
Vacuum pump (GM-0.33A, Tian Jinjin rise)；Bottle (sartorius, Germany) is filtered by vacuum in 1000mL；Electrochemical operation It stands (CHI660E, Shanghai Chen Hua)；1260 type impedance analyzers (Solartron Analytical, Farmbrough, Britain)； The defeated power forceful electric power chem workstation of 1287 types (Solartron Analytical, Farmbrough, Britain)；Ultra-55 type Flied emission Scanning electron microscope (Zeiss, Germany)；Micro-Raman spectroscopy (JobinYvonLabRam HRUV, France).
Samples sources:
The normal human serum patients serum positive with the inspection of Schistosoma japonicum excrement is provided by schistosomiasis in Zhejiang centre of prevention and cure.
Embodiment 1
(1) camel is immune
Camel is immunized with 600 μ g Schistosoma japonicum soluble egg antigens (soluble egg antigen, SEA) every time, First immunisation is complete Freund's adjuvant, and booster immunization cannots be used up full Freund's adjuvant, altogether booster immunization 3 times, 2 Zhou Houjian of final immunization Survey the titer level of serum antibody.It is divided between each secondary immunization time 3 weeks.
(2) antibody titer detects
Bioactivity: the SEA wrapper sheet of 10 μ g/mL, the closing of 4% skimmed milk power when antiserum thinner ratio is 1: 51200, are surveyed Obtaining OD490nm is 0.188 (P/N≤2), meets requirement for construction data base.
(3) library is built
The camel blood sample collection for building library standard will be met in 50mL centrifuge tube, dispense and extract after isometric PBS is added RNA has two gem-pure bands, respectively 28S rRNA and 18S rRNA as shown in Figure 1 in swimming lane 1, show extraction Total serum IgE quality is good, there is no significantly degrading, can be used for the building in following nano antibody library.
Using total serum IgE as template, cDNA is synthesized through two-step reaction using reverse transcription PCR.Then using this cDNA as template, carry out Nest-type PRC (Nest PCR), amplification have obtained the heavy chain variable region gene VHH (Fig. 2) of about 500bp.
Double digestion is carried out using amplified production and phagemid vector pHEN 4 of the restriction enzyme to Nest PCR, it will be pure Digestion products after change by after optimization VHH insertion sub-piece and carrier molecule molar ratio 3: 1 be attached, electrotransformation, through flat Plate, which counts, measures constructed library clump count up to 2.8 × 108It is a.18 lists of random picking from the plate that library bacterium colony counts Bacterium colony carries out the insertion rate of bacterium colony PCR identification VHH gene.Bacterium colony PCR result as shown in figure 3, positive control has obvious band, Credible result, 15 single colonies have amplified the band of 500-750bp, positive rate 83.3%, it follows that initially in experimental group The actual storage capacity in library is 2.3 × 108cfu。
(4) antibody screening
Using Schistosoma japonicum SEA antigen as target, two kinds of bacteriophages of product competitive elution are eluted and marked in conjunction with Gly-HCl and are washed Off-square formula carries out the affine elutriation of three-wheel to nano antibody phage display non-immune libraries.As shown in Table 1, in preceding two-wheeled Gly-HCl It elutes in elutriation, positive bacteriophage has obtained effective enrichment.And in third round competitive elution elutriation, bacteriophage quantum of output compared with The first round increases two orders of magnitude, shows that specific bacteriophage has obtained effective enrichment, bites for subsequent screening specificity Thallus provides guarantee.
Table 1
Elutriation wheel number SEA concentration (μ g/mL) Starting quantity (cfu) Elutriation quantity (cfu) Degree of enrichment
1 500 2.80×1011 1.06×106 2.64×105
2 50 3.11×1011 5.04×107 6.17×103
3 20 2.84×1011 6.14×108 4.63×102
Random 48 bacteriophage monoclonals of picking from the eluate titer determination plate of third round elutriation, using biting indirectly Thallus ELISA (phage ELISA) carries out positive-selecting to 48 phage clones, as a result as shown in figure 4,48 bacteriophages gram It is grand to have very strong combination with detection antigen, tentatively show 48 phage clone positive rates up to 100%.Extract 27 specificity The DNA of phage clone simultaneously send biological order-checking company to be sequenced.
Sequencing result is analyzed, nano antibody has easy solubility expression, production in the expression thallus such as Escherichia coli It measures the features such as high, the DNA sequence dna of positive phage clones is translated as amino acid by this research, according to homology difference and phagocytosis Body positive strength two indices, (nucleotide sequence is such as by selection VHH2 (nucleotide sequence is as shown in SEQ ID NO.3) and VHH48 Shown in SEQ ID NO.4) gene is subcloned to prokaryotic expression carrier pALTER (purchased from Addgene), with for pure on carrier The His label of change, is named as pALTER-VHH2 and pALTER-VHH48.
(5) nano antibody recombinantly expresses
Recombinant vector is converted respectively to Bacillus coli cells E.coli BL21 (DE3) pLysS, the IPTG through 0.3mM, 30 DEG C induction 12h after, thalline were collected by centrifugation and separates soluble nano antibody, after ni-sepharose purification, obtain size be about 17kDa's Expression product after purification is carried out SDS-PAGE and immunoblotting assay by 2 kinds of nano antibodies, and immunoblot results use DAB It develops the color, as a result as shown in figure 5, obtaining the nano antibody of about 15kD size.
Embodiment 2
Immunosensor preparation process is as follows:
Graphene oxide film (GO film, GOP) is to utilize nitrocellulose filter (diameter 47mm, 0.45 μm of aperture) and GO Solution is filtered by vacuum filtration method, is removed from nitrocellulose filter after natural drying using absolute alcohol, the thickness of GOP It can be depending on the volume by controlling certain density GO solution.At room temperature, it is restored using the GOP that HI solution will acquire 6h after the completion of reduction, impregnates rinsing for several times to there is no HI residual, naturally dries, after successfully preparing reduction using 95% ethyl alcohol Graphene oxide film (rGOP).
RGOP is cut out the electrode (can according to require to determine suitable size) that length and width dimensions specification is 20mm × 5mm, Obtained electrode is fixed to and on plastic bottom board and determines that the response area of electrode is 5mm × 5mm, is marked using insulating tape solid It is fixed, it obtains graphene oxide electrode (rGOPE).Electrode reaction region is then placed in 1% HAuCl4Electrification is utilized in solution It learns sedimentation and gold nano grain is deposited into electrode reaction region surface to obtain the graphene oxide of gold nano particle modification Electrode (rGOPE/AuNPs) is rinsed and dry with nitrogen using distilled water, and voltage is selected as -0.66V, reaction time when plating 100s。
By the 6 μ L of solution of nano antibody VHH2 and VHH48 equal proportion mixing (molar ratio 1: 1), (concentration of antibody is equal later For 1mg/mL, potency is 1: 51200) being titrated to electrode reaction region, be incubated overnight under the conditions of 4 DEG C, use 1mM after staying overnight PBS solution rinses and uses nitrogen dry.Then 1%BSA is added dropwise and is incubated at room temperature 30min, is rinsed later using 1mM PBS solution And it is dry using nitrogen, to be used to close the site that gold nano grain surface is not bound with antibody, reduce nonspecific interference.
Sample to be tested finally is added dropwise in electrode surface, is incubated at room temperature after 30min and reuses PBS solution and clean and do It is dry.The detection that electrochemical impedance value is carried out to the immunoelectrode being incubated for, it is real by the linear relationship of impedance value and concentration of specimens Now to sample to be tested quantitative detection purpose.
Embodiment 3 and 4
Immunosensor preparation, nano antibody VHH2 and VHH48 mixing ratio it is as shown in table 2, nano antibody concentration and Potency and remaining step are the same as embodiment 2.
Table 2
VHH2: VHH48 (molar ratio)
Embodiment 3 1∶0.5
Embodiment 4 1∶2
Embodiment 5
Using instruments such as atomic force microscope, scanning electron microscope, Raman spectrometers in the graphene film in embodiment 2 External structure, hydroiodic acid reduction front and back graphene film and it is gold-plated after electrode surface etc. characterized.
Atomic force microscope characterization result illustrates that graphite oxide is complete as shown in fig. 6, graphene sheet layer thickness is about 1nm Fully stripped is at single-layer graphene.Fig. 7 A is shown: prepared graphene film is accumulated by the assembling of graphene monolithic layer, it Electron transmission between internal lamellar structure is in close relations to the electric conductivity of graphene itself.As can be seen from Figure 7B, have perhaps Mostly random pleated structure is distributed in this special construction in graphene film surface and is conducive to Nb solid phase binding exists to high-density Surface.The flexural property of graphene is the major reason that film surface irregular fold is formed.
Graphene membrane electrode surface after gold-plated can see gold particle deposition (Fig. 8) not of uniform size, by golden former The golden ammonia key formed between son and protein amino realizes the solid phase binding of Schistosoma japonicum nano antibody and graphene film.
The oxygen-containing group that hydroiodic acid can remove inside graphene film increases its electric conductivity, with graphene film thickness Degree increases, and reduction can become inadequate, and then influence its electric conductivity.Forward and backward graphene film is restored to hydroiodic acid (HI) It carries out Raman analysis to learn (Fig. 9), the ratio of the ID/IG of graphene oxide film is the graphene oxide after 1.34, HI reduction ID/IG ratio becomes 1.86, illustrates that hydroiodic acid has carried out abundant reduction to graphene oxide.
Embodiment 6
The variation feelings of electrode surface impedance in immunosensor preparation process in embodiment 2 are indicated using electrochemical impedance Condition.One complete impedance curve is made of two parts, i.e. one section of semi-circular portions and one section of straight line portion, they are respectively represented instead The transfer and diffusion process of electrode surface electronics during answering.Wherein, the diameter of semicircle represents the electronics of electrode surface The size of transfer resistance.
Three traditional electrode test systems are selected in experiment measurement, and the immunosensor prepared is as working electrode, auxiliary Electrode and reference electrode are separately connected platinum electrode and Ag/AgCl reference electrode, and reaction buffer is to contain 5mM K3Fe(CN)6/ K4Fe(CN)6The mixed solution of (1: 1) and 0.1mM KCl.Test equipment is 1260 type impedance analyzer (Solartron Analytical, Farmbrough, Britain) and 1287 types defeated power forceful electric power chem workstation (Solartron Analytical, method grace Greensboro, Britain), test analysis software is Zview and Zplot, and measurement frequency range is respectively set to 1Hz with disturbance voltage is exchanged ~100kHz and 5mV.
The results are shown in Figure 10, wherein curve a~e is respectively graphene bare electrode (a), and graphene/gold nano grain is repaired The electrode (b) of decorations, electrode (c) after antibody is fixed close after non-specific sites electrode (e) after electrode (d), with antigen binding The testing impedance curve spectrum of electrode.It can be seen from the figure that bigger (the curve of impedance spectrum electronics transfer resistance of bare electrode A), show that the resistance of bare electrode is bigger, the rate of electron transmission is slow；When gold particle be electrodeposited into Graphene electrodes it (curve b), half circular diameter are substantially reduced, and the Au particle for showing to modify on the electrode has good electron transmission effect, are made afterwards The transfer impedance for obtaining the electronics of electrode is substantially reduced；Continue modification antibody on the electrode (curve c), to seal by bovine serum albumin Closing non-specific sites, (curve d), (the impedance spectrum result of curve e), electrode are aobvious after being detected in conjunction with specific antigen Show, the impedance of immunoelectrode is incrementally increased with the gradually modification to electrode, this is because when antibody, bovine serum albumin and After electrode surface, the electron transport rate of electrode surface can be decreased gradually this kind of large biological molecule substance modification of antigen, After the ability of electrodes transfer electronics is suppressed, electronics transfer resistance just be will increase.Pass through the modification to immunoelectrode gradually Reaction, each component materials success walked in reaction are successively assembled in electrode surface, make the impedance of electrode that significant change occur, this One the result shows that immunosensor successful preparation
Embodiment 7
The specificity of the immunosensor prepared in embodiment 2 is detected.Choose Schistosoma japonicum (S.japonicum) antigen and other six kinds of polypides: toxoplasma (T.gondii), Schistosoma mansoni (S.mansoni), people's spine jaw Mouth nematode (G.spinigerum), anisakis simplex (A.simplex), clonorchis sinensis (C.sinensis), Wei Shi are simultaneously grown The antigen of fluke (P.westermani) is 0.1mg/mL as test object, the concentration of determined antigen.
Specific detection process is identical as method in embodiment 6.It as a result as shown in figure 11, can be with by Figure 11 A and Figure 11 B Find out, after being incubated for the immunosensor combination antigen of Schistosoma japonicum nano antibody, in conjunction with the immunoelectrode of Schistosoma japonicum SEA Impedance amplification is up to 646.88 Ω, much higher than the changing value of blank value and heterogenetic antigen.
(preparation method: blood about 3ml is taken to be used for from auricular vein before the injection with common Schistosoma japonicum antiserum simultaneously Prepare negative control sera.The antigenic solution that 2ml is emulsified with Freund's complete adjuvant, it is soluble containing about 500 μ g Schistosoma japonicum Egg antigen is injected in well-developed purebred new zealand white rabbit body by the method for subcutaneously combining muscle multi-point injection.Base 14 days progress booster immunizations after plinth is immune.The injection interval period is 14 days, and antigenic solution Freund used in booster shots is endless Full adjuvant is emulsified, and booster immunization 3 times, is taken a blood sample from rabbit ear central vein and is separated serum, measured using indirect ELISA method Antiserum titre is up to 1: 16000.) nano antibody of the present invention is replaced, common blood, which is prepared, in method same as Example 2 inhales The immunosensor of worm antiserum modification is as control.Testing result is as shown in Figure 11 C and Figure 11 D, although the value-added inspection of impedance It surveys result and is higher than blank value, but there is no significance difference to the measurement result of blood fluke SEA and other six kinds of non-target antigens Not, the variation of impedance spectrum does not have an apparent discrimination, and the measurement amplification of both clonorchis sinensis and Paragonismus westermani is high In Schistosoma japonicum.
By the above result shows that: nano antibody of the present invention is significantly larger than common in the binding ability with specific antigen Serum antibody, the immunosensor based on nano antibody and graphene composite material have good specificity.
Embodiment 8
The sensitivity of the immunosensor prepared in embodiment 2 is detected.Using the sensor to various concentration day Japonicum SEA (10-8~10-1Mg/mL it) is detected.
Specific detection process is identical as method in embodiment 6.As a result as shown in Figure 12 and Figure 13, inhaled in minimum blood Worm antigen concentration 10-8(in curve a) test, the detection amplification of nano antibody has reached 87.092 Ω to mg/mL；In maximum concentration 10-1(in curve h) testing result, the test increment of the immunosensor of nano antibody modification is then 139.93 Ω to mg/mL, is shown With successively increasing for test antigen concentration, the impedance value of immunosensor is also successively increased, and hinders in different antigen diluent degree Anti- value has apparent discrimination (Figure 12) with blank control.And within the scope of the detectable concentration, immunosensor have compared with Good linear relationship, related coefficient are 0.9868 (Figure 13).2002, (Mao Yafei opened and realizes clear Pan Jing grass magnetic Mao Yafei etc. Pearl-ELISA detects the research China's parasitology and parasitic disease magazine 20,372-373 of Schistosoma japonicum circulation egg antigen (2002)) report that MB-ELISA detection method is 2.9ng/mL, conventional sandwich method to the minimal detectable concentration of blood fluke SEA ELISA is 5.8ng/mL to the minimal detectable concentration of SEA.And the minimum concentration that immunosensor of the present invention detects is 0.01ng/mL.The above result shows that the sensor has preferable sensitivity.
Embodiment 9
Positive and negative human serum sample is examined to excrement using the immunosensor prepared in embodiment 2 and carries out Schistosoma japonicum The detection of circulating antigen.
Specific detection process is identical as method in embodiment 6.
The results are shown in Table 3, and the testing result of immunosensor of the present invention is consistent with excrement inspection result.In addition, in same Under part, sample is carried out repeating to detect three times, there is preferable reproducibility.
Table 3
Sample Excrement examines result Immunosensor result
1 It is positive It is positive
2 It is positive It is positive
3 It is positive It is positive
4 It is negative It is negative
5 It is negative It is negative
SEQUENCE LISTING
<110>Zhejiang Academy of Medical Sciences
<120>a kind of for detecting bilharzial nano antibody composition, immunosensor and preparation method thereof and answering With
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 124
<212> PRT
<213>two-humped camel (Camelus bactrianus)
<400> 1
His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Ala Ile Asn Lys
20 25 30
Arg Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ala Gly Thr Tyr Arg Leu Thr Gly Asp Thr Ala Tyr Ala His Ser Val
50 55 60
Lys Gly Arg Phe Thr Leu Ser Gln Asp Thr Ala Lys Thr Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Ala Val Trp Thr Gly Gly Val Trp Tyr Asp Ala Arg Phe Tyr Lys
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 2
<211> 124
<212> PRT
<213>two-humped camel (Camelus bactrianus)
<400> 2
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Ala Arg Asn Lys
20 25 30
Arg Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val
35 40 45
Ala Gly Ile Tyr Arg Leu Thr Gly Asp Thr Ala Tyr Ala Asn Ser Val
50 55 60
Lys Gly Arg Phe Thr Leu Ser Gln Asp Thr Ala Lys Thr Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ser Tyr Tyr Cys
85 90 95
Ala Ala Val Trp Thr Gly Gly Val Trp Tyr Asp Ala Arg Met Tyr Lys
100 105 110
Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 3
<211> 372
<212> DNA
<213>two-humped camel (Camelus bactrianus)
<400> 3
catgtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctctggact taccgccatt aacaagcgca tggcctggtt ccgccaggct 120
ccagggaagg agcgcgaggg agtcgcaggt acttatcggc ttactggtga cacagcctat 180
gcccactccg tgaagggccg attcaccctc tcccaagata ccgccaagac tacgctatat 240
ctgcagatga acagcctgaa acctgaggac actgccatgt actactgtgc ggcagtctgg 300
actggcggtg tctggtatga tgcgcgtttc tataagtcct ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210> 4
<211> 372
<212> DNA
<213>two-humped camel (Camelus bactrianus)
<400> 4
caggtgcagc tggtggagtc tgggggaggc tcggtgcagg ctggagggtc tctgagactc 60
tcctgtgcag cctctggact taccgcccgt aacaagcgca tggcctggtt ccgccaggct 120
ccagggaagg agcgcgaggc agtcgcaggt atttatcggc ttactggtga cacagcctat 180
gccaactccg tgaagggccg attcaccctc tcccaagaca ccgccaagac tacgctatat 240
ctgcagatga acagcctgaa gcctgaggac actgcctcgt actactgtgc ggcagtctgg 300
actggcggtg tttggtatga tgcgcgtatg tataagtcct ggggccaggg gaccctggtc 360
accgtctcct ca 372

Claims (9)

1. one kind is for detecting bilharzial nano antibody composition, including two kinds of nano antibodies, amino acid sequence is respectively such as Shown in SEQ ID NO.1 and SEQ ID NO.2.
2. nano antibody composition as described in claim 1, which is characterized in that encode the nucleotide of described two nano antibodies Sequence is respectively as shown in SEQ ID NO.3 and SEQ ID NO.4.
3. nano antibody composition as described in claim 1, which is characterized in that the ratio of described two nano antibodies are as follows: press Molar ratio computing is 1: 0.5~2.
4. application of the nano antibody composition in preparation blood fluke detection kit as described in claims 1 to 3 is any.
5. one kind is for detecting bilharzial immunosensor, which is characterized in that comprising as described in claims 1 to 3 is any Nano antibody composition.
6. application of the immunosensor as claimed in claim 5 in blood fluke detection for the purpose of non-disease diagnoses.
7. the preparation method of immunosensor as claimed in claim 5, which comprises the following steps:
(1) graphene oxide film after restoring is as working electrode, in one layer of gold nano grain of electrode reaction area deposition Obtain the graphene oxide electrode of gold nano particle modification；
(2) the nano antibody composition is integrated in the graphene oxide electrode of gold nano particle modification by golden ammonia key Obtain the immunosensor.
8. preparation method as claimed in claim 7, which is characterized in that gold nano grain deposits to work by electrochemical deposition method Make electrode reaction region, electroplate liquid uses 1% HAuCl4Solution, electroplating voltage are -0.66V, reaction time 100s.
9. preparation method as claimed in claim 7, which is characterized in that after nano antibody composition combines, sealed using BSA It closes.
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