CN103196970A - Beta2 receptor biosensor as well as preparation method and application thereof - Google Patents
Beta2 receptor biosensor as well as preparation method and application thereof Download PDFInfo
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- CN103196970A CN103196970A CN 201310094697 CN201310094697A CN103196970A CN 103196970 A CN103196970 A CN 103196970A CN 201310094697 CN201310094697 CN 201310094697 CN 201310094697 A CN201310094697 A CN 201310094697A CN 103196970 A CN103196970 A CN 103196970A
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Abstract
The invention relates to a beta2 acceptor biosensor, as well as a preparation method and an application thereof. The beta2 receptor biosensor is a three-electrode system sensor, wherein a counter electrode is a platinum electrode, a reference electrode is a saturated calomel electrode, and a work electrode is a gold electrode. The beta2 acceptor biosensor is characterized in that a recombinant beta2 epinephrine purinergic receptor is modified on the gold electrode, and the recombinant beta2 epinephrine purinergic receptor is a base sequence as described in SEQ ID NO: 1. After the beta2 acceptor biosensor is used, the test result that the electrochemical biosensor has false negative can be reduced, the test sensitivity can be further improved, the urgent need to the test of a beta2 agonist field in the field can be preferably met, and a novel technical platform is provided for the food safety test; and therefore, the beta2 acceptor biosensor can be used for the field test for the import and export inspection and quarantine, the food hygiene supervision, a livestock rearing farm and the like, thereby being wide in market application prospect, and relatively good in economic benefit and social benefit.
Description
Technical field
The present invention relates to a kind of electrochemica biological sensor, preparation method and application thereof, particularly a kind of beta 2 receptor biology sensor, preparation method and application thereof.
Background of invention
Beta 2-adrenergic activator (be called for short β2Ji Dongji or β 2 parts, be commonly called as clenbuterol hydrochloride) is the artificial synthetic hormone class of class medicine, and the family member reaches tens of kinds more than.Its acting in conjunction mechanism is: β2Ji Dongji at first is incorporated into the beta 2-adrenergic receptor (abbreviation beta 2 receptor) on the cell membrane, by the cAMP signal transduction pathway of the G albumen coupling physiological effect of bringing into normal play, also has enhancing muscle simultaneously, reduce the physiological action of fat, made growth promoter to improve domestic animal lean meat percentage, such medicament residue serious harm people's health in the meat product by abuse.
Existing detection technique can't be contained the abuse of β2Ji Dongji at all.
Bioelectrochemical sensor is mainly constituted by biomolecule identification and information translation parts two parts.To be determinand convert the non-electrical signal of perceived material to measurable telecommunications breath by the biomolecule identification component to its design concept, handles through amplifying signal again, carries out signal output.Electrochemical system is realized inputing or outputing of electric energy by electrode, thereby obtains the electric signal of electrode face finish material, and commonly used is three-electrode system.Three-electrode system comprises working electrode, auxiliary electrode (also claiming electrode) and contrast electrode, flow through working electrode and to electrode of electric current.Electrochemical method has advantages such as highly sensitive, quick, simple and efficient as a kind of analyzing detecting method.
Be that nowadays the receptor biosensor technology that base growth is got up has received great concern with galvanochemistry.Electrochemical receptor biosensor is with the receptor protein cell membrane of this receptor albumen (or be loaded with) thereby being fixed in the metal surface as molecular recognition elements makes up working electrode, when when ligand analysis thing generation affinity to be determined is combined, can be with this combination conversion, amplification and output electrochemical signals in three-electrode system.The electrochemical receptor biosensor technological synthesis modern biology and microelectronics two big technology, both had the high degree of specificity of the former receptor-ligand combination and the characteristics of broad spectrum activity, have again latter's sensitivity, fast, operate easy characteristics, this remarkable advantages is expected to develop into strong novel detection technique
Summary of the invention
One of purpose of the present invention is to provide a kind of galvanochemistry beta 2 receptor biology sensor that detects β2Ji Dongji, and this sensor is realized the detection for β2Ji Dongji by the specific bond of ligand-receptor.
Two of purpose of the present invention is to provide the preparation method of this sensor.
Three of purpose of the present invention is to provide the application of this sensor in detecting β2Ji Dongji.
For achieving the above object, the present invention adopts following technical scheme:
A kind of beta 2 receptor biology sensor, be the three-electrode system sensor, be platinum electrode to electrode, contrast electrode is saturated calomel electrode, working electrode is gold electrode, it is characterized in that being modified with the reorganization beta 2-adrenergic receptor at described gold electrode, this reorganization beta 2-adrenergic receptor of this reorganization beta 2-adrenergic receptor is the base sequence shown in the SEQ ID NO:1.Its amino acid sequence is shown in the SEQ ID NO:2
A kind of method for preparing above-mentioned beta 2 receptor biology sensor is characterized in that the concrete steps of this method are:
A. gold electrode that pre-service is good immerses to modify in the 1 mM mercaptopropionic acid MPA solution and spends the night;
B. the gold electrode of step a gained is immersed and contains 400 mM 1-ethyls-3-(3-dimethylamino propionic acid) 10 min in the aqueous solution of carbodiimide hydrochloride EDC, 200 mM N-hydroxy-succinamide NHS, ultrapure water thoroughly cleans the back and immerses 10 min in the aqueous solution that contains 100 mM histidines, and ultrapure water cleans;
C. step b gained gold electrode is immersed 10 min in nickel oxide (NiO) the nano particle aqueous solution of 1 μ g/mL, ultrapure water cleans;
D. with 10～30 min in the solubilising beta 2 receptor protein solution of step c gained gold electrode immersion 0.27mg/mL, clean the gold electrode that obtains through the beta 2 receptor modification through ultrapure water;
E. steps d gained gold electrode is formed the beta 2 receptor biology sensor of three-electrode system as working electrode with platinum electrode and saturated calomel electrode.
The preparation method of above-mentioned increase-volume beta 2 receptor albumen is:
A. the recombination engineering cell membrane with the beta 2 receptor gene carries out the solubilising processing with 1% DDMN-dodecyl-β-D-maltoside-PBS damping fluid, 0 C 45min, and the centrifugal 20min of 18000g gets the solubilising beta 2 receptor protein solution that supernatant is homogeneous;
B. step a gained solubilising beta 2 receptor solution is added Ni
2+-NTA affinity column absorption, with 200mM imidazoles high-salt buffer wash-out, part is collected and is namely obtained beta 2 receptor albumen after 50mM imidazoles high-salt buffer is washed post three times.
A kind of method that detects β2Ji Dongji adopts above-mentioned beta 2 receptor biology sensor to detect, and it is characterized in that the concrete steps of this method are:
A. working electrode is immersed in and contains in the testing sample, 0 C, 30 min, ultrapure water cleans;
B. under 20 C temperature, the working electrode behind step a is immersed in 5.0 mL detects in the buffer solution, described detection buffer solution is that concentration is the K of 0.5 M
3Fe (CN)
6/ K
4Fe (CN)
6Phosphate buffer, its pH is 7.4.
C. cyclic voltammetry is carried out galvanochemistry scanning; Parameter arranges: sweep limit-0.1 ~ 0.55 V, sweep speed 100 mV/s.
The present invention at first utilizes beta 2 receptor to make " target ", set up a kind of receptor-ligand detection system, can detect all β2Ji Dongji in theory, simultaneously because the β2Ji Dongji functionating is to mediate by beta 2 receptor, therefore this system had both had the technique effect of high flux and broad spectrum activity " biochip ", also had the implication that biological function detects simultaneously.This receptor-part detection system has following advantage: 1. the combination of high specific-acceptor and part has strict three-dimensional conformation complementarity.2. high-affinity-receptor-ligand system affinity generally is higher than the Ag-Ab system.3. high flux-can only detect a kind of β2Ji Dongji based on the detection technique of Ag-Ab system, and can once detect the β2Ji Dongji of all kinds based on the detection technique of receptor-ligand system, it " is caught all in one draft ".
Utilize gene recombination technology to efficiently express the beta 2 receptor gene at yeast cells, the expression product beta 2 receptor is positioned cell membrane, as the memebrane protein functionating." target " in beta 2 receptor-part detection system both can be the film of this genetic recombination cell, also can be the receptor protein of separation and purification, and the two respectively has characteristics.The former preparation technology is simple, but because the cell membrane component complexity causes detection system specificity and insufficient sensitivity height; Latter preparation technology elaborate, but composition is single, and the detection system specificity of structure and sensitivity are all than the former height.
Outstanding feature of the present invention mainly is, based on the cyclic voltammetric technology, modify the beta 2 receptor albumen of purifying solubilising at working electrode, made up the galvanochemistry beta 2 receptor biology sensor of three-electrode system as molecular recognition elements with this, the system of modifying working electrode (" a kind of electrochemical receptor biosensor and application thereof " (application number 200910196550.4)) with the cell membrane of previous application compares, the present invention is further by separating, purification technique, make up model electrochemical beta 2 receptor biology sensor with the beta 2 receptor albumen that obtains as molecular recognition elements, had higher specificity and sensitivity.Can better satisfy at present to on-the-spot the pressing for of detecting of β2Ji Dongji, for food safety detection provides new technology platform, can be used for importing and exporting on-the-spot detection such as inspection and quarantine, food hygiene supervision, livestock rearing field, have wide market application prospect and bigger economical, societal benefits.
Description of drawings
Fig. 1 carries out independent loops voltammetry scanning result for the electrochemica biological sensor that adopts unloaded host cell membrane working electrode (contrast 2) and form to the CLE of six kinds of series concentration.（a）0，（b）1，（c）10?，（d）100?，（e）1000，（f）10000。
Fig. 2 carries out independent loops voltammetry scanning result for the electrochemica biological sensor that adopts beta 2 receptor cell membrane working electrode (contrast 1) and form to the CLE of six kinds of series concentration.（a）0，（b）1，（c）10?，（d）100?，（e）1000，（f）10000。
Fig. 3 is for adopting beta 2 receptor albumen working electrode; The electrochemica biological sensor that the concentration of CLE (pM) is formed carries out independent loops voltammetry scanning result to the CLE of six kinds of series concentration.（a）0，（b）1，（c）10?，（d）100?，（e）1000，（f）10000。
Fig. 4 is the relation curve of three kinds of electrochemica biological sensor peak point current changing values and CLE concentration logarithm among Fig. 1.(A) unloaded host cell membrane working electrode (contrast 2); (B) beta 2 receptor cell membrane working electrode (contrast 1); (C) beta 2 receptor albumen working electrode; The concentration of CLE (pM): (a) 0, (b) 1, (c) 10, (d) 100, (e) 1000, (f) 10000.
Embodiment
Embodiment one: the purifying of solubilising beta 2 receptor and preparation
Clone, the expression of people's beta 2 receptor gene in Pichia pastoris, and the preparation of recombination engineering cell membrane sees that " research of setting up a kind of new detection system of β 2-activator " (wear small peak, the Master degree candidate of Shanghai University paper, 2004), " a kind of electrochemical receptor biosensor and application thereof " (Chen Yuguang etc., application for a patent for invention numbers 200910196550.4,2009).
The preparation of solubilising beta 2 receptor albumen: with above-mentioned recombination engineering cell membrane with 1% DDM(N-dodecyl-β-D-maltoside)-the PBS damping fluid carries out solubilising to be handled, 0 C 45min, the centrifugal 20min of 18000g gets the solubilising beta 2 receptor albumen that supernatant is homogeneous.
The partial purification of solubilising beta 2 receptor albumen: above-mentioned solubilising beta 2 receptor solution is added Ni
2+-NTA affinity column absorption, with 200mM imidazoles high-salt buffer wash-out, part is collected and is namely obtained beta 2 receptor albumen after 50mM imidazoles high-salt buffer is washed post three times.
Embodiment two: the preparation of beta 2 receptor working electrode
The pre-service of gold electrode: gold electrode is at first polished at 5000 purpose abrasive paper for metallograph, use the suspension polishing of the alumina powder of the descending 1.0 μ m of particle diameter, 0.3 μ m, 0.05 μ m again, make gold electrode surfaces become smooth mirror surface, take out ultrasonic 5min in absolute ethyl alcohol and ultrapure water respectively, then gold electrode is put into the H of 0.5 M
2SO
4In carry out cyclic voltammetry scan (0-1.5 V voltage range), sweep speed and be set to 100 mV/s, about 20 the circle reach stable.In nitrogen, dry up electrode then, obtain the naked gold electrode of cleaning surfaces.
The preparation of beta 2 receptor working electrode:
A. above-mentioned naked gold electrode is immersed to modify in 1 mM mercaptopropionic acid (MPA) solution and spend the night;
B. above-mentioned (a) modified gold electrode is immersed and contains 400 mM EDC(1-ethyls-3-(3-dimethylamino propionic acid) carbodiimide hydrochloride), 200 mM NHS(N-N-Hydroxysuccinimide) aqueous solution in 10 min, ultrapure water thoroughly cleans the back and immerses 10 min in the aqueous solution that contains 100 mM histidines, and ultrapure water cleans;
C. above-mentioned (b) modified gold electrode is immersed 10 min in nickel oxide (NiO) the nano particle aqueous solution of 1 μ g/mL, ultrapure water cleans;
D. above-mentioned (c) modified gold electrode is immersed purifying solubilising beta 2 receptor solution (0.27mg/mL) 10-30 min, clean through ultrapure water and obtain the beta 2 receptor working electrode;
E. steps d gained beta 2 receptor working electrode is formed the galvanochemistry beta 2 receptor biology sensor of three-electrode system with platinum electrode and saturated calomel electrode.
Embodiment three: galvanochemistry beta 2 receptor biosensor application is in the detection of β2Ji Dongji (Clenbuterol)
Running program: in the system of this three-electrode electro Chemical sensor
A. above-mentioned gained beta 2 receptor working electrode is immersed in the testing sample solution or negative control solution that contains 100 μ L β2Ji Dongji, 0 C, 30 min, ultrapure water cleans;
B. the reacted beta 2 receptor working electrode of above-mentioned a. is immersed 5.0 mL and detect buffer solution (20 C), detection buffer solution is: the phosphate buffer of 0.5 M K3Fe (CN) 6/ K4Fe (CN) 6 (50 mM, pH 7.4).
C. cyclic voltammetry is carried out galvanochemistry scanning; Parameter arranges: sweep limit-0.1 ~ 0.55 V, sweep speed 100 mV/s.
The detected characteristics of β2Ji Dongji (Clenbuterol):
The six kinds of series concentration (0,1,10,100,1000,10000 pM) that prepare Clenbuterol (CLE) respectively.
Prepare three kinds of working electrodes respectively, i.e. beta 2 receptor albumen working electrode, beta 2 receptor cell membrane working electrode (contrast 1), unloaded host cell membrane working electrode (contrast 2).These three kinds of working electrodes have been formed three kinds of biology sensors.
The electrochemica biological sensor that uses these three kinds of working electrodes to form carries out the scanning of independent loops voltammetry to the CLE of six kinds of series concentration respectively.The result shows:
1, linearity test 1.0 * 10
-12M to 1.0 * 10
-8M 5 number order magnitude range (Fig. 3, Fig. 4 (C));
2, the detectable concentration lower limit reaches 0.1nM(0.03ppb) (Fig. 3, Fig. 4 (C));
3, response is quick, highly sensitive, and the beta 2 receptor biology sensor improves nearly ten times than beta 2 receptor cell membrane working electrode (contrast 1), referring to table 1.
Studied the electrochemical response of variable concentrations CLE in this experiment.Shown in Fig. 1, Fig. 4 (A), the working electrode of having modified unloaded host cell membrane when carrying out cyclic voltammetry scanning, when increasing progressively along with the concentration of part CLE, [Fe (CN)
6]
3-/4-The redox current peak does not change basically or is not regular and changes, when even the concentration of CLE reaches 10 nM, the redox current peak does not clearly change yet, this is because what modify is unloaded host cell membrane on the control experiment group electrode, can't identify the little molecule of CLE, can not hinder [Fe (CN)]
3-/4-Electronics transmits, and the redox peak point current changes very little.
Shown in Fig. 2, Fig. 4 (B), working electrode surface is modified with the beta 2 receptor cell membrane, can specific identification β
2Activator CLE forms the receptor-ligand compound at electrode surface, has hindered [Fe (CN)]
3-/4-Probe and gold electrode surfaces are carried out the electronics transmission, reduce [Fe (CN)]
3-/4-The redox peak point current, and along with the increase of CLE concentration, peak current is downward trend gradually.
Shown in Fig. 3, Fig. 4 (C), working electrode surface is modified with purified beta 2 receptor albumen, can specific identification CLE, form the receptor-ligand compound at electrode surface, and hindered [Fe (CN)]
3-/4-Probe and gold electrode surfaces are carried out the electronics transmission, reduce [Fe (CN)]
3-/4-The redox peak point current, along with the increase of CLE concentration, peak current is downward trend gradually.And the peak current downtrending obviously is better than Fig. 2 (10 more than the order of magnitude), and hence one can see that, by purifying β
2The beta 2 receptor biology sensor of receptor protein working electrode preparation obtains to increase substantially for detection of the sensitivity of CLE.
Claims (4)
1. beta 2 receptor biology sensor, be the three-electrode system sensor, be platinum electrode to electrode, contrast electrode is saturated calomel electrode, working electrode is gold electrode, it is characterized in that being modified with the reorganization beta 2-adrenergic receptor at described gold electrode, this reorganization beta 2-adrenergic receptor is the base sequence shown in the SEQ ID NO:1.
2. method for preparing beta 2 receptor biology sensor according to claim 1 is characterized in that the concrete steps of this method are:
A. gold electrode that pre-service is good immerses to modify in the 1 mM mercaptopropionic acid MPA solution and spends the night;
B. the gold electrode of step a gained is immersed and contains 400 mM 1-ethyls-3-(3-dimethylamino propionic acid) 10 min in the aqueous solution of carbodiimide hydrochloride EDC, 200 mM N-hydroxy-succinamide NHS, ultrapure water thoroughly cleans the back and immerses 10 min in the aqueous solution that contains 100 mM histidines, and ultrapure water cleans;
C. step b gained gold electrode is immersed 10 min in nickel oxide (NiO) the nano particle aqueous solution of 1 μ g/mL, ultrapure water cleans;
D. with 10-30 min in the solubilising beta 2 receptor protein solution of step c gained gold electrode immersion 0.27mg/mL, clean the gold electrode that obtains through the beta 2 receptor modification through ultrapure water;
E. steps d gained gold electrode is formed the beta 2 receptor biology sensor of three-electrode system as working electrode with platinum electrode and saturated calomel electrode.
3. method according to claim 2 is characterized in that the preparation method of described increase-volume beta 2 receptor albumen is:
A. the recombination engineering cell membrane with the beta 2 receptor gene carries out the solubilising processing with 1% DDMN-dodecyl-β-D-maltoside-PBS damping fluid, 0 C 45min, and the centrifugal 20min of 18000g gets the solubilising beta 2 receptor protein solution that supernatant is homogeneous;
B. step a gained solubilising beta 2 receptor solution is added Ni
2+-NTA affinity column absorption, with 200mM imidazoles high-salt buffer wash-out, part is collected and is namely obtained beta 2 receptor albumen after 50mM imidazoles high-salt buffer is washed post three times.
4. a method that detects β2Ji Dongji adopts beta 2 receptor biology sensor according to claim 1 to detect, and it is characterized in that the concrete steps of this method are:
A. working electrode is immersed in and contains in the testing sample, 0 C, 30 min, ultrapure water cleans;
B. under 20 C temperature, the working electrode behind step a is immersed in 5.0 mL detects in the buffer solution, described detection buffer solution is that concentration is the K of 0.5 M
3Fe (CN)
6/ K
4Fe (CN)
6Phosphate buffer, its pH is 7.4.
C. cyclic voltammetry is carried out galvanochemistry scanning; Parameter arranges: sweep limit-0.1 ~ 0.55 V, sweep speed 100 mV/s.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104634847A (en) * | 2013-11-14 | 2015-05-20 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Electrochemical immunosensing method for detecting Listeria monocytogenes |
| CN106353376A (en) * | 2016-08-30 | 2017-01-25 | 南京师范大学 | Beta-adrenal agonists residue detection electrochemical sensor and detection methods thereof |
| CN115096961A (en) * | 2022-06-27 | 2022-09-23 | 福建省妇幼保健院 | Biosensor for detecting estrogen-related receptor alpha and detection method thereof |
-
2013
- 2013-03-22 CN CN 201310094697 patent/CN103196970A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104634847A (en) * | 2013-11-14 | 2015-05-20 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Electrochemical immunosensing method for detecting Listeria monocytogenes |
| CN106353376A (en) * | 2016-08-30 | 2017-01-25 | 南京师范大学 | Beta-adrenal agonists residue detection electrochemical sensor and detection methods thereof |
| CN115096961A (en) * | 2022-06-27 | 2022-09-23 | 福建省妇幼保健院 | Biosensor for detecting estrogen-related receptor alpha and detection method thereof |
| CN115096961B (en) * | 2022-06-27 | 2023-04-21 | 福建省妇幼保健院 | Biosensor for detecting estrogen-related receptor alpha and detection method thereof |
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Application publication date: 20130710 |