CN105486738A - Multi-channel composite magnetic control electrochemical immunosensor and preparation method and application thereof - Google Patents
Multi-channel composite magnetic control electrochemical immunosensor and preparation method and application thereof Download PDFInfo
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Abstract
A multi-channel composite magnetic control electrochemical immunosensor and a preparation method and application thereof are disclosed, the immunosensor comprises a polytetrafluoroethylene outer chamber, a magnet control means, a magnet control means limit device, a working electrode and a counter electrode, the working electrode and the counter electrode are arranged on one end of the polytetrafluoroethylene outer chamber, the immunosensor controls the distance between a magnet control connecting rod and the surface of the working electrode to achieve electrochemical detection and cleaning to detect a next sample without regeneration, the immunosensor can meanwhile achieve multi-sample detection, is rapid, high in sensitivity and low in detection cost, and can meet modern clinical rapid testing requirements.
Description
Technical field
The invention belongs to electrochemical field, be specifically related to the composite magnetic controlled electrochemical immunosensor of hyperchannel, also relate to the preparation method and application of the composite magnetic controlled electrochemical immunosensor of hyperchannel.
Background technology
At present, electrochemical immunosensor is due to its detection speed is fast, highly sensitive, low cost and other advantages has been applied to pathogen early diagnosis research.But, traditional electrochemistry immuno-sensing working electrode detected in the three-electrode system used only has a workplace, its modification is complicated and the reproducibility of electrode is poor, electrode surface of polishing is needed after detection, and carry out loaded down with trivial details process, length consuming time, and can not multisample detection be carried out, the requirement that modern clinic detects fast can not be met.
Therefore, be badly in need of a kind of simple to operate, detect quick, highly sensitive, that testing cost is low electrochemical immunosensor.
Summary of the invention
In view of this, the object of the present invention is to provide the composite magnetic controlled electrochemical immunosensor of hyperchannel; Two of object of the present invention is the preparation method providing the composite magnetic controlled electrochemical immunosensor of hyperchannel; Three of object of the present invention is to provide the composite magnetic controlled electrochemical immunosensor of described hyperchannel detecting the application in biological specimen; Four of object of the present invention is to provide the method utilizing the composite magnetic controlled electrochemical immunosensor of described hyperchannel to detect biological sample.
For achieving the above object, the invention provides following technical scheme:
The composite magnetic controlled electrochemical immunosensor of hyperchannel, comprises teflon exocoel, the stop means of magnet control device, magnet control device and be arranged at tetrafluoroethene exocoel one end working electrode and to electrode;
Described magnet control device comprises magnet control linkage bar and is arranged at the permanent magnet of magnet control linkage bar one end; Described stop means is arranged at the inwall of the nearly working electrode end of teflon exocoel; Described working electrode is provided with at least one workplace and is connected with external power source binding post by wire, and described workplace is golden material.
Preferably, described stop means comprises spacing pad groove and spacing mat.
Preferred, working electrode comprises 3 workplaces.
Preferred, described teflon exocoel wall thickness is 1 ~ 2mm, and external diameter is 10mm, is highly 80mm.
Most preferred, the external diameter of described golden material is 2mm, and thickness is 1.5mm.
2, the preparation method of the composite magnetic controlled electrochemical immunosensor of described hyperchannel, comprises the steps:
(1) polytetrafluoroethylmaterial material is adopted to prepare electrode outer space cavity layer;
(2) one end end face installment work electrode of outer space cavity layer and glass-carbon electrode, the workplace of described working electrode is made up of golden material, is then connected with external power source binding post by wire by golden material;
(3) inwall of cavity layer nearly working electrode end installs the stop means be made up of spacing pad groove and spacing mat outside, then above stop means, install the magnet control device be made up of with the permanent magnet being positioned at magnet control linkage bar one end magnet control linkage bar.
3, the composite magnetic controlled electrochemical immunosensor of described hyperchannel is detecting the application in biological specimen.
4, utilize the composite magnetic controlled electrochemical immunosensor of described hyperchannel to detect the method for biological sample, comprise the steps:
(1) Streptavidin is modified in magnetic particle sub-surface, then the primary antibodie of at least one biotin modification is fixed, form nano combined magnetic bead, then nano combined magnetic bead is hatched with detection two is anti-, form the immune complex of the anti-shape of nano combined magnetic bead-detection two;
(2) permanent magnet of composite magnetic controlled for hyperchannel electrochemical immunosensor is moved to stop means place, and electrode front end is placed in hatches solution, electrifying electrodes, the immune complex of the anti-shape of nano combined magnetic bead-detection two can be absorbed and fixed at the electrode surface of sensor;
(3) composite magnetic controlled for the hyperchannel of absorption electrochemical immunosensor is carried out voltammetry detection to pH=7.0, PBS containing in the detection solution of biological sample to be checked, the current value that before and after record immune response, peak current declines, the current value of decline is directly proportional to the amount of biological sample to be checked.
Preferably, also comprise the steps: composite magnetic controlled for the hyperchannel of absorption electrochemical immunosensor to become in the detection solution of concentration gradient to the biological sample to be checked of pH=7.0, PBS respectively after step (3) to carry out voltammetry detection, the current value that before and after record peak immune response, peak current declines, then according to current value and the biological sample concentration drawing standard curve of peak current decline, the biological sample concentration to be checked of the current value of the finally then corresponding decline of typical curve calculation procedure (3).
Preferred, after detection, the permanent magnet of composite magnetic controlled for hyperchannel electrochemical immunosensor is shifted out stop means place, magnetic disappears, PBS flushing is carried out to the electrode face of sensor, the immune complex of the anti-shape of nano combined magnetic bead-detection two departs from from the electrode face of sensor, namely can be used for the detection of biological sample next time.
Beneficial effect of the present invention is: the composite magnetic controlled electrochemical immunosensor of hyperchannel disclosed by the invention, the detection of target molecule can be effectively applied to, overcome that Conventional electrochemical immunosensor exists regenerability restriction, working electrode can not reuse simply and effectively, the defect of the aspects such as complicated process of preparation.Key technical problem of the present invention is to be modified by magnetic particle functionalization and composite magnetic controlled electrode achieves convenient, effective preparation and the regeneration of electrochemical immunosensor, achieve reusing of electrochemical immunosensor, can fast, high sensitivity, low testing cost realize detection of biological samples.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:
Fig. 1 is hyperchannel composite magnetic controlled electrochemical immunosensor structural drawing (A: the composite magnetic controlled electrochemical immunosensor general assembly drawing of hyperchannel; B: hyperchannel composite magnetic controlled electrochemical immunosensor teflon outer chamber side view; C: the inner key element of the composite magnetic controlled electrochemical immunosensor of hyperchannel divides zero structure figure; 1-teflon exocoel; 2-magnet control linkage bar; 3-permanent magnet; The spacing pad groove of 4-; The spacing mat of 5-; 6-electrode).
Fig. 2 is the composite magnetic controlled electrochemical immunosensor chart at the bottom of of hyperchannel.
Fig. 3 is that the composite magnetic controlled electrochemical immunosensor of hyperchannel carries out biology sample detection procedure chart.
Fig. 4 is that the composite magnetic controlled electrochemical immunosensor of hyperchannel detects O157:H7 result figure.
Fig. 5 is the testing result of O157:H7 and the Cholera pathogen of the composite magnetic controlled immunosensor of hyperchannel.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, the usually conveniently conditioned disjunction condition of advising according to manufacturer.
The composite magnetic controlled electrochemical immunosensor of embodiment 1, hyperchannel
The composite magnetic controlled electrochemical immunosensor of hyperchannel, structure as shown in Figure 1.The composite magnetic controlled electrochemical immunosensor of hyperchannel comprises teflon exocoel 1, magnet control device, the stop means of magnet control device and be arranged at tetrafluoroethene exocoel 1 one end working electrode and to electrode, described magnet control device comprises magnet control linkage bar 2 and is arranged at the permanent magnet 3 of magnet control linkage bar one end, the stop means of magnet control device comprises spacing pad groove 4 and spacing mat 5, and be positioned at the inwall of the nearly working electrode end of teflon exocoel 1, described working electrode is provided with multiple workplace, be preferably 3 workplaces, described workplace is golden material, and be connected with external power source binding post by wire, described glass-carbon electrode is preferably to electrode.
In the present invention, teflon (PTFM) the material wall thickness of the outer cavity of electrode is preferably 1 ~ 2mm, and external diameter is preferably 10mm, is highly preferably 80mm; The external diameter of piece structure is preferably 2mm, and thickness is preferably 1.5mm, and adopts wire to make it be connected with binding post.
In the present invention, magnet control linkage bar is provided with the projection protruding from teflon exocoel 1, controls the position of adjustable magnetic member control apparatus.
The composite magnetic controlled electrochemical immunosensor of hyperchannel by arranging 3 workplaces (Fig. 2) on electrode, each workplace adopts 9999 proof gold materials, and adsorbs detected material by regulating the magnet control linkage bar of magnet control device to regulate the distance of permanent magnet and working electrode surface to realize Electrochemical Detection with removal.When magnet control device is limited, functional Fe_3 O_4 magnetic particles in liquid to be detected can be adsorbed on the workplace of working electrode by the permanent magnet of electrode interior, realizes Electrochemical Detection; When magnet control device leaves position limiting structure, permanent magnet 3 increases with the workplace distance of working electrode, functional Fe_3 O_4 magnetic particles being adsorbed in the workplace of working electrode comes off and is not adsorbed the electrode of detection thing, and does not need regeneration can detect other liquid to be measured.
The preparation method of the composite magnetic controlled electrochemical immunosensor of embodiment 2, hyperchannel
The preparation method of the composite magnetic controlled electrochemical immunosensor of hyperchannel, concrete steps are as follows:
(1) teflon (PTFM) material is adopted to prepare electrode outer space cavity layer, wherein wall thickness 1 ~ 2mm, external diameter 10mm, height 80mm;
(2) one end end face installment work electrode of outer space cavity layer and glass-carbon electrode, the workplace of working electrode is made up of golden material, is then connected with external power source binding post by wire by golden material;
(3) cavity layer inwall in outer space installs the stop means be made up of spacing pad groove and spacing mat, then above stop means, install the magnet control device be made up of with the permanent magnet being positioned at magnet control linkage bar one end magnet control linkage bar.
In the present invention, teflon (PTFM) the material wall thickness of the outer cavity of electrode is preferably 1 ~ 2mm, and external diameter is preferably 10mm, is highly preferably 80mm; The external diameter of piece structure is preferably 2mm, and thickness is preferably 1.5mm.
Embodiment 3, the composite magnetic controlled electrochemical immunosensor of employing hyperchannel detect biological sample
Composition graphs 3 illustrates the process adopting the composite magnetic controlled electrochemical immunosensor of the hyperchannel obtained by the present invention to detect biological sample, specific as follows:
(1) Streptavidin is modified in magnetic particle sub-surface, then the primary antibodie of fixed biologically element modification, form nano combined magnetic bead, it be coupled with O157:H7 standard items and cadmium acetate two anti-(Cd-anti-O157) are hatched jointly, incubation time 20min;
(2) magnet of composite magnetic controlled for hyperchannel electrode is moved to stop means place, and electrode front end is placed in hatches solution, be subject to magnetic field suction, the immune complex of the anti-formation of nano combined magnetic bead-O157:H7 pathogen-Cd-anti-O157 two can be absorbed and fixed at magnetic control electrode surface;
(3) after absorption, moved to by magnetic control electrode and detect in solution (pH=7.0PBS detects liquid), carry out anodic stripping voltammetry detection, peak current is directly proportional to the amount of absorption pathogen, and it the results are shown in Figure 4.
(4) after detecting, magnet is moved to magnetic control electrode rear end, magnetic disappears, and magnetic particle can be resolved from magnetic control electrode surface under PBS washing action, and flows away along with solution.Afterwards, the detection of sample next time can be proceeded.
Embodiment 4, O157:H7 and Cholera pathogen parallel detection based on the composite magnetic controlled immunosensor of hyperchannel
1. the preparation method of the composite magnetic controlled immunosensor of hyperchannel
After O157:H7 and the Cholera pathogen cultivated respectively by 96 orifice plates carries out high-temperature inactivation, be diluted to 3.6cfu/mL respectively, 3.6 × 10
2cfu/mL, 3.6 × 10
3cfu/mL, 3.6 × 10
4cfu/mL, 3.6 × 10
5cfu/mL, 3.6 × 10
6cfu/mL, 3.6 × 10
7cfu/mLL; 4.2cfu/mL, 4.2 × 10
2cfu/mL, 4.2 × 10
3cfu/m, 4.2 × 10
4cfu/mL and 4.2 × 10
5cfu/mL, 4.2 × 10
6cfu/mL, 4.2 × 10
7cfu/mL.
The magnetic control electrochemical immunosensor prepared by embodiment 1 and described testing process thereof detect the standard solution of above-mentioned concentration respectively, and the current value that oxidation peak current declines before and after immune response is as quantitative basis.To each concentration standard pathogen duplicate detection three times, using its mean value as current value corresponding to this pathogen concentration, and the current value corresponding with it to antigen concentration carries out linear regression analysis, and it the results are shown in Figure 5.
Result shows, O157:H7 standard concentration is 3.6 × 10
2cfu/mL ~ 3.6 × 10
6within the scope of cfu/mL, magnetic control electrochemical immunosensor peak current drop-out current value and antigen concentration are good linear relationship.Cholera standard concentration is 4.2 × 10
2cfu/mL ~ 4.2 × 10
6within the scope of cfu/mL, magnetic control electrochemical immunosensor peak current drop-out current value and antigen concentration are good linear relationship.
The magnetic control electrochemical immunosensor prepared by embodiment 2 and described testing process thereof are applied to the detection of clinical sample, and its testing result is in table 1 and table 2.
The detection of table 1, O157:H7 clinical samples
| O157:H7 | 1 | 2 | 3 | 4 | 5 |
| This method | 3.5×10 3 | 7.6×10 3 | 2.6×10 4 | 7.4×10 4 | 1.9×10 5 |
| Slat chain conveyor | 3.6×10 3 | 7.9×10 3 | 2.5×10 4 | 7.2×10 4 | 1.8×10 5 |
The detection of table 2, Cholera clinical samples
| Cholera | 1 | 2 | 3 | 4 | 5 |
| This method | 2.0×10 3 | 4.4×10 3 | 9.9×10 3 | 4.1×10 4 | 8.2×10 4 |
| Slat chain conveyor | 2.1×10 3 | 4.2×10 3 | 9.6×10 3 | 4.2×10 4 | 8.4×10 4 |
Result shows that magnetic control immunosensor testing result of the present invention and plating method testing result have good consistance.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (10)
1. the composite magnetic controlled electrochemical immunosensor of hyperchannel, is characterized in that: comprise teflon exocoel, the stop means of magnet control device, magnet control device and be arranged at tetrafluoroethene exocoel one end working electrode and to electrode;
Described magnet control device comprises magnet control linkage bar and is arranged at the permanent magnet of magnet control linkage bar one end; Described stop means is arranged at the inwall of the nearly working electrode end of teflon exocoel; Described working electrode is provided with at least one workplace and is connected with external power source binding post by wire, and described workplace is golden material.
2. the composite magnetic controlled electrochemical immunosensor of hyperchannel according to claim 1, is characterized in that: described stop means comprises spacing pad groove and spacing mat.
3. the composite magnetic controlled electrochemical immunosensor of hyperchannel according to claim 1, is characterized in that: working electrode comprises 3 workplaces.
4. the composite magnetic controlled electrochemical immunosensor of hyperchannel according to claim 1, is characterized in that: described teflon exocoel wall thickness is 1 ~ 2mm, and external diameter is 10mm, is highly 80mm.
5. the composite magnetic controlled electrochemical immunosensor of hyperchannel according to claim 1, is characterized in that: the external diameter of described golden material is 2mm, and thickness is 1.5mm.
6. the preparation method of the composite magnetic controlled electrochemical immunosensor of hyperchannel described in any one of Claims 1 to 5, is characterized in that: comprise the steps:
(1) polytetrafluoroethylmaterial material is adopted to prepare electrode outer space cavity layer;
(2) one end end face installment work electrode of outer space cavity layer and platinum electrode, the workplace of described working electrode is made up of golden material, is then connected with external power source binding post by wire by golden material;
(3) inwall of cavity layer nearly working electrode end installs the stop means be made up of spacing pad groove and spacing mat outside, then above stop means, install the magnet control device be made up of with the permanent magnet being positioned at magnet control linkage bar one end magnet control linkage bar.
7. the composite magnetic controlled electrochemical immunosensor of hyperchannel described in any one of Claims 1 to 5 is detecting the application in biological specimen.
8. utilize the composite magnetic controlled electrochemical immunosensor of hyperchannel described in any one of claim 1 ~ 5 to detect the method for biological sample, it is characterized in that, comprise the steps:
(1) Streptavidin is modified in magnetic particle sub-surface, then the primary antibodie of at least one biotin modification is fixed, form nano combined magnetic bead, then nano combined magnetic bead is hatched with detection two is anti-, form the immune complex of the anti-type of nano combined magnetic bead-detection two;
(2) permanent magnet of composite magnetic controlled for hyperchannel electrochemical immunosensor is moved to stop means place, and electrode front end is placed in hatches solution, electrifying electrodes, the immune complex of the anti-shape of nano combined magnetic bead-detection two can be absorbed and fixed at the electrode surface of sensor;
(3) composite magnetic controlled for the hyperchannel of absorption electrochemical immunosensor is carried out voltammetry detection to pH=7.0, PBS containing in the detection solution of biological sample to be checked, the current value that before and after record immune response, peak current declines, the current value of decline is directly proportional to the amount of biological sample to be checked.
9. method according to claim 8, it is characterized in that, also comprise the steps: composite magnetic controlled for the hyperchannel of absorption electrochemical immunosensor to become in the detection solution of concentration gradient to the biological sample to be checked of pH=7.0, PBS respectively after step (3) to carry out voltammetry detection, the current value that before and after record peak immune response, peak current declines, then according to current value and the biological sample concentration drawing standard curve of peak current decline, the biological sample concentration to be checked of the current value of the finally then corresponding decline of typical curve calculation procedure (3).
10. method according to claim 8, it is characterized in that: after detection, the permanent magnet of composite magnetic controlled for hyperchannel electrochemical immunosensor is shifted out stop means place, magnetic disappears, PBS flushing is carried out to the electrode face of sensor, the immune complex of the anti-shape of nano combined magnetic bead-detection two departs from from the electrode face of sensor, namely can be used for the detection of biological sample next time.
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| CN117368289A (en) * | 2023-12-07 | 2024-01-09 | 广州市赛特检测有限公司 | Method for coupling protein aptamer on surface of carbon electrode, carbon electrode and electrochemical immunosensor |
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