CN106568974A - Pituitary adenylate cyclase-activated peptide protein chip, preparation and application thereof - Google Patents

Pituitary adenylate cyclase-activated peptide protein chip, preparation and application thereof Download PDF

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Publication number
CN106568974A
CN106568974A CN201610954668.9A CN201610954668A CN106568974A CN 106568974 A CN106568974 A CN 106568974A CN 201610954668 A CN201610954668 A CN 201610954668A CN 106568974 A CN106568974 A CN 106568974A
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detection
adenylate cyclase
pituitary adenylate
pacap
activating peptide
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李莹
钟金钢
赵明路
齐攀
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Jinan University
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Jinan 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
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
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  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
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  • General Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a pituitary adenylate cyclase-activated peptide protein chip, and the preparation and application thereof, and relates to the field of biochips and detection methods based on biochips. According to the technical scheme of the invention, the pituitary adenylate cyclase-activated peptide protein chip is applied to the PACAP detection and the kinetic study. The experiment on the quantitative detection of the concentration level of PACAP is conducted, and the detection limit of the direct detection of PACAP can reach 0.5 mg/L. Meanwhile, the affinity and the kinetic reaction process of the PACAP and a receptor PAC1R are studied. Compared with the traditional biochemical analysis method, the SPR biochip-based detection and research of PACAP is free of labels and simple in sample treatment. Moreover, a quantitative result can be quickly obtained, and the reaction kinetics is studied. Equipment is portable and low in cost. The on-site quick detection can be realized. The apparatus and the method can be applied to the PACAP detection for a large number of samples, the basic research on the affinity of PACAP and receptors thereof, and the like.

Description

A kind of pituitary adenylate cyclase activating peptide protein chip and its preparation and application
Technical field
The present invention relates to biochip field and the detection method based on it, more particularly to a kind of pituitary adenylate cyclase Activating peptide (PACAP) protein chip and preparation method and application.
Background technology
Pituitary adenylate cyclase activating peptide (Pituitary Adenylate Cyclase-Activating Polypeptide, PACAP) it is initially a kind of rat pituitary cell that can activate culture separated from sheep hypothalamuses The neuropeptide of adenyl cyclase activity, with two kinds of activity forms of PACAP-38 and PACAP-27.In different tissues or system In, PACAP can play neurotransmitter/biological function such as quenched, neurotrophic factor and immune system.For example, exist In early days or in ripe brain, PACAP passes through the type (Pituitary of pituitary adenylate cyclase activating peptide 1 to fetal development Adenylate Cyclase-Activating Polypeptide Receptor 1, PAC1) receptor, it is right to reduce or reduce The damage of neuron, promotes the survival of neuron, plays its nutrition or protective effect to neuron.PACAP adjusts lipopolysaccharide Dendritic cell (Den-Dritic Cell, the DC) immunologic function of (Lipopolysaccharide, LPS) activation can improve sense The occurrence and development of infectious diseases and rheumatism etc., so as to play therapeutical effect.Therefore, to PACAP contents in biofluid Detection and the immunoreation of PACAP and its receptor carry out research and will be helpful to play it in the industries such as pharmacy industry, clinical treatment Important function.Traditional PACAP detections research method has radio immunoassay (Radioimmunoassay, RIA), indirectly Immunofluorescence assay method (Indirect Immunofluorescent Antibody Method, IFA), enzyme linked immunological skill Art (Enzyme Linked Immunosorbent Assay, ELISA) etc., the test limit of these methods is low, but instrument price is high Expensive, sample handling procedure complicated (generally needing to sample labelling, antibody purification etc.), detection cycle length, complex operation, it is difficult to full The requirement of sufficient quick detection research, therefore, quick, special, sensitive, easy, inexpensive PACAP detections research side is set up in research Method is extremely necessary.
Surface plasma body resonant vibration (Surface Plasmon Resonance, SPR) biosensor is by immunoreation High specific combines with the high sensitivity of surface plasma body resonant vibration Photoelectric Detection, it is not necessary to which labelling and dedicated test set Standby, sensitivity is high, high specificity, easy to operate, sample pre-treatments are simple, be widely used in biology, medical science, chemistry, Many fields such as drug screening and environmental monitoring.
Surface plasma body resonant vibration (SPR) effect is a kind of physical light occurred on electrolyte and thin metal layer separating surface Learn phenomenon.SPR effects are very sensitive to the dielectric variations in refractive index being attached on noble metal film surface, and refractive index is institute There is the inherent feature of material so that the biochemical analysises based on SPR effects need not be to sample labelling, so that it may by detecting sample folding The minor variations of rate are penetrated, the biochemical analysises to sample are realized.
When carrying out biochemical analysises experiment using SPR, biochip fixing biological probe (antigen or antibody) after modification, When the sample of the receptor containing matching flows through chip surface, compatible reaction captures analyte (receptor) on film surface, causes The increase of refractive index, the resonance state of reflected light also can accordingly change.By the way that injection is containing material to be detected and does not contain to be checked Survey material buffer, can real-time monitoring analyte combine and dissociate process.With the change of response time (abscissa), Testing sample refractive index produces change, so as to cause the change of new resonant wavelength or resonance angle (vertical coordinate).Therefore, may be used Detected with the relation of metal film surfaces liquid refractivity according to lambda1-wavelength during surface plasma body resonant vibration or angle Interaction between biomolecule.
The content of the invention
For existing pituitary adenylate cyclase activating peptide (PACAP) detection research method behaviour in order to overcome prior art The shortcoming for making that complicated, instrument is valuable, cost is high etc. with it is not enough, the primary and foremost purpose of the present invention is to provide a kind of hypophysis adenylic acid ring Change the preparation method of enzyme activition peptides proteins chip.
Another object of the present invention is to provide the pituitary adenylate cyclase prepared by above-mentioned preparation method swash Peptides proteins chip living.
Another object of the present invention is to provide the application of above-mentioned pituitary adenylate cyclase activating peptide protein chip.
It is still another object of the present invention to provide a kind of detected using pituitary adenylate cyclase activating peptide protein chip The SPR detection methods of pituitary adenylate cyclase activating peptide.
Surface plasma body resonant vibration (SPR) pituitary adenylate cyclase activating peptide protein chip is voluntarily have developed, is applied Detection and dynamics research in PACAP.Using direct method detectable concentration be respectively 0.5mg/L, 1mg/L, 2mg/L, 5mg/L, The PACAP samples of 8mg/L and 10mg/L and the immunoreaction process of receptor PCA1R.Test result indicate that, direct method detection The test limit of PACAP is up to 0.5mg/L.It has been experimentally confirmed the feasibility of the apparatus and method.Based on surface plasma The PACAP detection methods of resonance are simple, and instrument is portable, low cost, are capable of achieving field quick detection.
The purpose of the present invention is achieved through the following technical solutions：
A kind of preparation method of pituitary adenylate cyclase activating peptide protein chip, comprises the steps：
Surface plasma body resonant vibration (SPR) response is produced using golden film, it is fixed in solid phase carrier (sheet glass of gold-plated film) Receptor (PAC1R) is used as bioprobe on cell membrane, and detection object is pituitary adenylate cyclase activating peptide (PACAP), inclined by P The light that shakes detects the reaction of biochip probe and sample.
In order to further realize the present invention：
When there is the material with probe matching in detection sample, resonance angle change, SPR detectors record testing result, profit Detection process is controlled with the software voluntarily worked out and analyze experimental result.
Biological chip modifying, chip activation, bioprobe the process such as fix, inactivate for the preparation process of protein chip, Concrete operation method is：With the HS (CH of 1mmol/L2)10COOH (mercaptoundecylic acid) and HS (CH2)6The ethanol of OH (mercaptohexanoic acid) Solution, mass ratio (M/M) is 1：9, to golden film (the circular glass piece surface of diameter 20mm thickness 1mm deposits the gold of 50nm) surface Carry out self-assembled modified 2h；With cedar oil (refractive index is 1.51～1.52) sensing chip after modification is fitted as matching fluid On SPR instrument cylindrical prisms (K9 glass), flow path system is installed；Arranging SPR sweep parameters is：- 1 ° of sweep starting point, scans model 2 ° are enclosed, 0.01 ° of scanning step；PBS (2mmol/L NaH are pumped into chip surface2PO4,2mmol/L Na2HPO4, 150mmol/L NaCl, pH7.4), start recording SPR response value, the resonance angle using now PBS is used as benchmark；Baseline stability The EDC mixed liquors (1 of the NHS and 0.1mol/L of 0.1mol/L are added afterwards：1, V/V), chip surface 15min, SPR at this moment are activated Response value is raised；Rinsed after 2min with PBS, be receptor on the cell membrane of 300ppm in biochip surface fixed concentration (PAC1R) as bioprobe, now SPR response values are significantly raised, PBS is passed through after 30min and rinses 2min, and response value only has little Width declines, and illustrates that probe fixed effect is preferable；The remaining ester bond of ethanolamine (pH8.5) closing inactivation of addition 1mol/L, 5～ After 7min, PBS are rinsed, protein chip is prepared and completed, and can be used for the immune detection of next step.
A kind of pituitary adenylate cyclase activating peptide protein chip, is prepared by above-mentioned preparation method.
Described pituitary adenylate cyclase activating peptide protein chip is in detection pituitary adenylate cyclase activating peptide Application.
A kind of utilization pituitary adenylate cyclase activating peptide protein chip detection pituitary adenylate cyclase activating peptide SPR detection methods, comprise the steps：
Receptor on cell membrane (PAC1R) is fixed on into biochip surface, detection object is that pituitary adenylate cyclase swashs Peptide (PACAP) living, is first rinsed with PBS after sample detection is complete, then pass to SDS-HCl make antibody-antigen conjugates from Chip surface dissociates, and realizes that chip regenerates, and SPR response values drop back to baseline can be continued to detect next sample.
The present invention mechanism be：This research is activated using independently developed angle scanning type SPR pituitary adenylate cyclase Peptides proteins chip, using the specificity of immunoreation, studies the detection of PACAP-38, analyzes the dynamic of PACAP and receptor PAC1R Mechanics course of reaction.Compared with traditional PACAP detection research methoies, this research instrument is portable, easy to operate, without the need for labelling, Low cost, can carry out field quick detection.
Fig. 1 is SPR biochip test process schematics.After biochip is successfully prepared, buffer is injected into chip list Face, records SPR responses now and conduct detection baseline；By the injection chip surface of the sample containing variable concentrations analysans, Analyte is combined with the bioprobe for being fixed on chip surface, and the more high speed that then combines of analyte concentration is faster, so as to cause folding The change for penetrating rate is faster, is presented as the quick increase of SPR response values；Absorption of the analyte on chip is reached after saturation, injection Buffer, unconjugated test analyte will be from chip surface eluting；Injection actified solution, chip is regenerated, SPR response weights Detection baseline is newly returned to, can continue to detect analyte.
The present invention has the following advantages and effect relative to prior art：
The angle scanning type SPR pituitary adenylate cyclase activating peptide protein chip voluntarily developed is applied to into PACAP Detection and dynamics research.The detection by quantitative experiment of PACAP levels is carried out, direct detection PACAP test limit is reachable 0.5mg/L；Have studied the affinity and kinetic reaction process of PACAP and receptor PAC1R.With conventional biochemical analysis method phase Than, SPR biochip test research PACAP have label-free, sample treatment it is simple, can rapidly quantitative result, research be instead Answer the series of advantages such as kinetics.The apparatus and method detection of PACAP, PACAP and its receptor suitable for a large amount of samples Basic research of PCA1R affinitys etc..
Description of the drawings
Fig. 1 is SPR biochip immunoreation detection process schematic diagrams.
Fig. 2 is the experimentation recording curve of SPR biochip tests PACAP.
Fig. 3 is the kinetic curve that direct method detects PACAP.
Fig. 4 is the standard curve of direct detection PACAP.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited In this.
This institute is given with pituitary adenylate cyclase activating peptide by bioengineered pharmaceuticals key lab of Ji'nan University； Mercaptoundecylic acid [HS (CH2)10COOH], mercaptohexanoic acid [HS (CH2)6OH], ethanolamine (Eth), sodium lauryl sulphate (SDS), N-hydroxy-succinamide (N-Hydroxysuccinimide, NHS), carbodiimide [N-ethyl-N'- (dimethylaminopropyl) carbodiimide, EDC] it is purchased from Sigma Co., USA；Other reagents are purchased from Beijing chemistry Reagent Company.The buffer of immunoreation is PBS (2mmol/L NaH2PO4, 2mmol/L Na2HPO4, 150mmol/L NaCl, pH7.4).
Embodiment 1
1. prepared by biochip
(1) with the HS (CH of 1mmol/L2)10COOH (mercaptoundecylic acid) and HS (CH2)6The ethanol of OH (mercaptohexanoic acid) is molten Liquid, mass ratio (M/M) is 1：9, golden film (the circular glass piece surface of diameter 20mm thickness 1mm deposits the gold of 50nm) surface is entered The self-assembled modified 2h of row；
(2) sensing chip after modification is fitted in into SPR as matching fluid with cedar oil (refractive index is 1.51～1.52) On instrument cylindrical prism (K9 glass), flow path system is installed；
(3) arranging SPR sweep parameters is：- 1 ° of sweep starting point, 2 ° of sweep limitss, 0.01 ° of scanning step；
(4) PBS (2mmol/L NaH are pumped into chip surface2PO4, 2mmol/L Na2HPO4, 150mmol/L NaCl, pH7.4), start recording SPR response value, using the resonance angle of now PBS as benchmark (see the stage 1 in Fig. 2)；
(5) the EDC mixed liquors (1 of the NHS and 0.1mol/L of 0.1mol/L are added after baseline stability：1, V/V), chip is activated Surface 15min, SPR response values at this moment are raised (see the stage 2 in Fig. 2)；
(6) rinsed after 2min with PBS, be receptor on the cell membrane of 300ppm in biochip surface fixed concentration (PAC1R) as bioprobe, now SPR response values significantly raised (see stage 4 in Fig. 2)；
(7) PBS is passed through after 30min and rinses 2min, response value only slightly declines (see the stage 5 in Fig. 2), illustrates that probe is consolidated Determine effect preferable；
(8) the remaining ester bond (see the stage 6 in Fig. 2) of ethanolamine (pH8.5) closing inactivation of addition 1mol/L, 5～7min, After PBS is rinsed, biochip is prepared and completed, and can be used for the immune detection of next step.
2. protein chip detects pituitary adenylate cyclase activating peptide
(1) concentration is fixed on into biochip surface for receptor (PAC1R) on the cell membrane of 300ppm, detection object is vertical Body adenyl cyclase activating peptide (PACAP)；
(2) PACAP concentration is respectively 0.5mg/L, 1mg/L, 2mg/L, 5mg/L, 8mg/L and 10mg/L.Stage 8 in Fig. 2, 10th, 12,14,16 and 18 have recorded the dynamic detection that the PACAP samples of above-mentioned 6 concentration and receptor PAC1R immunoreation are combined Curve, each sample detection about 200s；
(3) after each sample detection is complete first with PBS rinse, then passing to SDS-HCl makes antibody-antigen conjugates From chip surface dissociation, realize that chip regenerates, SPR response values drop back to baseline can be continued to detect next sample.7 in Fig. 2,9, 11st, 13,15,17 and 19 records is chip regenerative process.
(4) PACAP is individually drawn with the kinetic curve of receptor PAC1R immunoreaction process, as shown in Figure 3.By Fig. 3 It can be seen that, the immunoreation speed of variable concentrations PACAP and receptor PAC1R is all first quick and back slow, gradually to tend to saturation, course of reaction In approximate log relation, meet immunoreation rule.And raise with PACAP concentration in sample, immunoreation speed speeds.Each Sample detection about 200s, with the carrying out of immunoreation, biochip surface antibody-antigen conjugates increase, and SPR response values increase Greatly.If the experiment of this group extends the response time, curve will tend towards stability, and the trend of immunoreation saturation can become apparent from.
3. standard curve
Fig. 4 is the standard curve of direct detection PACAP, room temperature (20 ± 1) DEG C, to be passed through during sample immunoreation 200s Relative response is vertical coordinate, in sample the concentration of antibody be abscissa, it is seen that the sample SPR response values of 0～5mg/L concentration with Concentration is substantially linear, and by the testing sample SPR response values of unknown concentration, query criteria curve can draw sample The concentration of middle PACAP.The method is applied to PACAP antibody screenings, research affinity of antibody and immunoreaction kineties.Directly examine PACAP test limits are surveyed up to 0.5mg/L.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment Limit, other any spirit without departing from the present invention and the change, modification, replacement made under principle, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

Claims (6)

1. a kind of preparation method of pituitary adenylate cyclase activating peptide protein chip, it is characterised in that comprise the steps：
Using golden film produce surface plasma resonance response, solid phase carrier fix cell membrane on receptor as bioprobe, Detection object is pituitary adenylate cyclase activating peptide, and by P polarization light the reaction of biochip probe and sample is detected.
2. the preparation method of pituitary adenylate cyclase activating peptide protein chip according to claim 1, its feature exists In：Also comprise the steps：
When there is the material with probe matching in detection sample, resonance angle change, SPR detectors record testing result, using certainly The software control detection process of row establishment simultaneously analyzes experimental result.
3. the preparation method of pituitary adenylate cyclase activating peptide protein chip according to claim 1 and 2, its feature It is：
Described solid phase carrier is the sheet glass of gold-plated film.
4. a kind of pituitary adenylate cyclase activating peptide protein chip, it is characterised in that by any one of claims 1 to 3 institute The preparation method stated is prepared.
5. the pituitary adenylate cyclase activating peptide protein chip described in claim 4 swashs in detection pituitary adenylate cyclase Application in peptide living.
6. a kind of utilization pituitary adenylate cyclase activating peptide protein chip detects the SPR of pituitary adenylate cyclase activating peptide Detection method, it is characterised in that comprise the steps：
Receptor on cell membrane is fixed on into biochip surface, detection object is pituitary adenylate cyclase activating peptide, and sample is examined After having surveyed first with PBS rinse, then pass to SDS-HCl make antibody-antigen conjugates from chip surface dissociate, realize core Piece regenerates, and SPR response values drop back to baseline and continue to detect next sample.
CN201610954668.9A 2016-10-27 2016-10-27 Pituitary adenylate cyclase-activated peptide protein chip, preparation and application thereof Pending CN106568974A (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101634630A (en) * 2009-08-18 2010-01-27 暨南大学 Two-dimensional scanning detection method and device of surface plasma resonance biochemical analysis
CN101650370A (en) * 2008-08-13 2010-02-17 中国科学院电子学研究所 Integrated microfluidic sensing chip and method for detecting microfluid
CN103792368A (en) * 2014-01-27 2014-05-14 暨南大学 Surface plasma resonance immunosense chip as well as preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101650370A (en) * 2008-08-13 2010-02-17 中国科学院电子学研究所 Integrated microfluidic sensing chip and method for detecting microfluid
CN101634630A (en) * 2009-08-18 2010-01-27 暨南大学 Two-dimensional scanning detection method and device of surface plasma resonance biochemical analysis
CN103792368A (en) * 2014-01-27 2014-05-14 暨南大学 Surface plasma resonance immunosense chip as well as preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
K. NAKAMURA ET AL.: "Distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) in the human testis and in testicular germ cell tumors", 《ANDROLOGIA》 *
潘延凤 等: "慢性肝炎患者血浆PACAP检测及意义", 《山东医药》 *

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Application publication date: 20170419