CN1161478C - Optical fibre bio-sensor of acetylcholinesterase and its preparing process - Google Patents
Optical fibre bio-sensor of acetylcholinesterase and its preparing process Download PDFInfo
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- CN1161478C CN1161478C CNB021125104A CN02112510A CN1161478C CN 1161478 C CN1161478 C CN 1161478C CN B021125104 A CNB021125104 A CN B021125104A CN 02112510 A CN02112510 A CN 02112510A CN 1161478 C CN1161478 C CN 1161478C
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Abstract
The present invention discloses an acetylcholinesterase optical fiber biological sensor which is used for identifying the nature of peripheral nerve fiber. The sensor is characterized in that the sensor comprises optical fiber and a sensing membrane, wherein the sensing membrane is fixed to the surface of an optical fiber head. A preparation method of the sensor comprises the following step that the sensing membrane is fixed to the surface of the optical fiber head in a co-valent binding manner or in a physical adsorption manner. The sensing membrane can be prepared by 3 methods that 1, the sensing membrane is prepared by compounding a high-molecular membrane absorbing an oxygen indicator, choline oxidase and a high-molecular membrane; 2, the sensing membrane is prepared by compounding a high-molecular membrane absorbing an oxygen indicator, and a high-molecular membrane fixed with choline oxidase; 3, an oxygen indicator and choline oxidase are mixed with a solution of a high-molecular membrane, and the solution is concentrated so as to form a single-layer membrane. The sensor has the advantage that the live and original nature of nerve tracts at both sides of the broken end of a nerve is identified rapidly, visually and accurately without cutting the broken end of the nerve. The sensor also has the advantages of no need for dyeing, convenience, rapidness, simple operation, no toxicity, no damage to human bodies, mechanization, integration and intelligentization.
Description
One, the technical field
The invention relates to a method for manufacturing a sensor for detecting the properties of peripheral nerve tracts, in particular to a method for manufacturing an acetylcholinesterase optical fiber biosensor.
Second, background Art
In the prior art, the following methods are mainly used for judging the properties of nerve tracts:
1. the anatomical mode is as follows: the study on the location map of the nerve bundle in the nerve trunk has been carried out, and many scholars have analyzed peripheral nerves (such as median nerve, radial nerve, ulnar and radial nerve trunk, sciatic nerve trunk and the like), observed the position and the form bundle number of the nerve trunk, measured the size of the nerve trunk and drawnthe nerve bundle local location map of the nerve section.
2. Electrical stimulation method: responses are identified by electrical stimulation of the nerve bundle, with muscle contraction occurring as a motor bundle and sensory bundles not responding. However, this method requires the patient to be kept awake, is limited to fresh lesions, has large individual differences, and cannot distinguish whether the stimulation causes muscle contraction as a result of nerve conduction or current diffusion. Therefore, the clinical application value is not great.
3. The radiochemical method: the substrate of the enzyme is labeled with radioactive isotope, and the activity of the enzyme is measured according to the radioactive product produced by the enzyme reaction, so that the property of the nerve bundle is presumed, but the method involves isotope technology, has high equipment requirement, and has low practicability because radioactive substances damage patients and operators.
4. Enzyme histochemical method: based on the content difference of some bioactive substances in the motor tract and the sensory tract, as the standard for identifying the motor tract and the sensory tract, the acetylcholinesterase activity in nerve fibers of the motor tract is far higher than that in the sensory tract, so the method for identifying the motor tract and the sensory tract by using the acetylcholinesterase activity as the standard is widely accepted. At present, acetylcholinesterase is mainly detected by an enzyme histochemical staining method, and the property of nerve bundles is judged according to the principle that copper ferrocyanide (a brown precipitate) formed in the reaction appears at the enzyme activity part, the motor fibers are strong positive, most of sensory fibers are negative, and a small part of sensory fibers are weak positive. However, this method has problems: (1) long dyeing time (about 50 minutes), complex process;(2) the nerve segments at both ends need to be cut out, and the in-situ staining can not be carried out. Therefore, clinical application is limited.
5. Immunohistochemical method: the specific protein of sensory neuron is found by utilizing specific antigen-antibody reaction and observing and researching the positioning and quantitative technology of specific antigen (or antibody) of tissue cell, and the monoclonal antibody is prepared by using the protein as antigen, and the sensory fiber is identified by immunostaining, and the method is characterized by high specificity, no toxicity to human body and shortened staining time which is 25 to 30 minutes compared with the conventional method. However, in the current stage of the method, tissue fragments still need to be cut, the problems of staining specificity and time exist, the staining is not deep when the time is too short, other tissues are stained when the time is too long, the clinician is difficult to control, the operation is complex, and the practicability is not enough.
6. Chemical sensor: aiming at the defects of the prior method, the applicant carries out new exploration from 98 years, cooperates with a teaching and research room of the great biological engineering of the same-economic medical science, applies the biosensing technology to the identification of peripheral nerve motor tracts and sensory tracts, and utilizes the characteristics of sensitivity, rapidness, accuracy and strong specificity of the biosensor to prepare the chemical biosensor capable of detecting the activity of acetylcholinesterase. The sensor can detect the activity of acetylcholinesterase on each nerve bundle, the principle is that acetylcholine fixed on a sensing needle reacts with the acetylcholinesterase, potential and PH change in the reaction process, the sensing needle reflects the activity of the acetylcholinesterase by recording the change of the potential and the PH value, the nerve bundle with high activity is a motion bundle, and otherwise, the nerve bundle with high activity is a sensing bundle. The whole reaction process is tens of seconds to 3 minutes, the time is greatly shortened compared with the prior method, the use is convenient, the single nerve bundle in-situ detection can be realized, the in-situ detection is successfully realized, the clinical requirement is met, and the errors and the interference are reduced. The animal experiment shows that the medicine has high curative effect. However, the method has the problems that the sensing needle needs to be replaced after every detection, the sensing needle cannot be used repeatedly, a few minutes are needed for detecting one nerve bundle, and the time is long. Therefore, the sensor still needs to be improved to be suitable for clinical use.
Third, the invention
1. The purpose of the invention is as follows: the invention aims to provide an optical fiber biosensor system capable of quickly detecting AchE based on the content difference of acetylcholinesterase (AchE) in peripheral nerve motor tracts and sensory tracts, quickly identifying the peripheral nerve motor tracts and the sensory tracts and realizing the correct stitching of the motor tracts to the motor tracts and the sensory tracts to the sensory tracts.
2. The technical scheme is as follows: in order to achieve the purpose, the acetylcholinesterase optical fiber biosensor comprises an optical fiber and a sensing film, wherein the sensing film is a composite film formed by combining a polymer film with biocompatibility, choline oxidase and an oxygen indicator, and the sensing film is fixed on the surface of an optical fiber head of the optical fiber. The optical fiber is Y-shaped. The polymer membrane is a perfluorinated sulfonic acid proton exchange membrane (Nafion), Polydimethylsiloxane (PDMS), polyvinylalcohol (PVA) or polyvinyl chloride (PVC) produced by DuPont in the United states. The oxygen indicator is terpyridyl ruthenium (Tris (2, 2' -bipyrridl) ruthenium (ii) chloride Hexahydrate) or phenanthroline ruthenium (Ru (4,7-diphenyl-1，10-phen)3cl2)。
the method for preparing the acetylcholinesterase optical fiber biosensor is characterized in that the sensing film formed by combining the polymer film, choline oxidase and the oxygen indicator is fixed on the surface of the optical fiber head of the optical fiber in a covalent bonding or physical adsorption mode. The sensing film has three manufacturing methods, one of which is formed by compounding a polymer film adsorbing an oxygen indicator, choline oxidase and a polymer film; the second one is formed by compounding a polymer film absorbed with an oxygen indicator and a polymer film fixed with choline oxidase; and thirdly, mixing an oxygen indicator and choline oxidase in a solution of a high polymer material, and solidifying to form a single-layer film.
The working principle of the invention is as follows: the following chemical reactions occur within the nerve bundle and within the sensing membrane:
wherein: (a) chemical reactions present in the nerve bundle, and (b) and (c) reactions in the sensor head membrane.
The amount of oxygen produced in the reaction (c) is converted into the activity of acetylcholinesterase.
The choline oxidase can be directly mixed with a high polymer material and then solidified into a film, or can be covalently combined with a chain organic molecular material such as poly-hexaethylene glycol (PEG-NHS) firstly and then mixed with the high polymer material and then solidified into a film, so that the leakage of enzyme can be reduced, the service life of the sensing film can be prolonged, and the repeatability of a detection result can be improved. As the oxygen indicator, a compound of ruthenium such as Tris (2, 2' -dipyridyl) ruthenium (II) chloride hexahydrate; ru (4, 7-diphenylyl-1, 10-phen)3Cl2Etc. the fluorescent dye is sensitive to oxygen content and has fluorescence life varying with oxygen content, so that it can measure oxygen content variation not only via fluorescence intensity variation but also via measurementThe factors of the fluorescence lifetime, the astigmatism, the dye concentration and the like which restrict the intensity and the measurement accuracy influence the accurate change under various environments.
The sensing membrane can be combined by the following three methods:
the method comprises the following steps: adopts a three-layer structure, namely a polymer film modified with an oxygen indicator, choline oxidase and a polymer film protective layer.
The second method comprises the following steps: adopts a two-layer structure, namely a polymer film modified with an oxygen indicator and a polymer film fixed with choline oxidase (Cho).
The third method comprises the following steps: mixing an oxygen indicator and choline oxidase in a high polymer material solution, and solidifying to form a single-layer film.
The optical fiber adopts a Y-shaped branched optical fiber with the diameter of about 1 mm.
3. Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: the sensor applies the optical fibersensing technology which is widely applied in the fields of communication, electronics, medical treatment, environmental detection and the like. At present, the optical fiber sensor can detect electrolyte blood gas in body fluid in clinic and is widely applied to ICU ward monitors. Modern fiber optic sensors also allow single molecule detection and allow surface imaging. The data capacity is large by using the optical fiber, and the sensitivity is as high as 10-12The method has the characteristics of mol/L, low distortion rate, small volume and the like, and the acetylcholinesterase information is analyzed and processed by a computer and simulated nerve bundle section imaging is used as visual feedback so as to achieve the following effects: on the premise of not cutting off the nerve broken end, the method can visually and accurately identify the properties of nerve bundles on two sides of the nerve broken end in vivo, in situ, quickly (the whole process is completed within seconds), does not need dyeing, is convenient and quick, is simple to operate, is non-toxic and non-invasive to a human body, and realizes mechanization, integration and intellectualization.
The optical fiber biosensor for in-situ identification of peripheral nerve bundle properties in vivo provides a simple and rapid method for microsurgical repair of damaged peripheral nerves, greatly improves the functional recovery rate of the damaged peripheral nerves, and can be widely popularized in clinical application.
Description of the drawings
Fig. 1 is a schematic structural view of the present invention.
FIG. 2 is a schematic view of a first structure of a sensing film.
FIG. 3 is a schematic diagram of a second structure of a sensing film.
FIG. 4 is aschematic diagram of a third structure of a sensing membrane.
Fig. 5 is a schematic diagram of the working principle of the present invention.
Fifth, detailed description of the invention
Example 1: the diameter of the Y-shaped optical fiber 1 is 1mm, and the sensing film 2 is of a three-layer structure. The first layer is a Nafion polymer film a, the middle layer is a choline oxidase layer b, the third layer is a polymer layer modified with an oxidation indicator c, and the oxygen indicator is Tris (2, 2' -dipyridyl) ruthenaum (II) chloridehydrate. The sensing film 2 is fixed on the surface of the optical fiber head of the Y-shaped optical fiber 1 by means of covalent bonding.
Example 2: the diameter of the Y-shaped optical fiber 1 is still 1mm, and the sensing film 2 is of a two-layer structure. Choline oxidase membranes and oxygen indicator membranes. The oxygen indicator film is prepared by mixing 5mm (bpy)3 2+Dripping 1.25 wt% Nafion ethanol solution on the end face of the optical fiber, and drying at room temperature. The choline oxidase solution is prepared by dissolving Nafion solution in 90%
The ethanol was released to 1% final concentration and 3mg choline oxidase (10 units/mg) was added to 50 ul% Nafion solution. And dripping the prepared choline oxidase onto the surface of the optical fiber and drying to obtain the sensing membrane.
Example 3: the Y-shaped optical fiber 1 is 1mm in diameter, and the sensing film 2 is of a single-layer structure. And mixing the oxygen indicator C, the choline oxidase b and the PDMS high polymer material a solution, and condensing to form a single-layer film. The single-layer sensing film 2 is fixed on the surface of the optical fiber head ofthe Y-shaped optical fiber 1 in an adsorption mode.
The acetylcholinesterase optical fiber sensor prepared according to the embodiments 1 to 3, in combination with the light source 3, the detector 4, the computer 5 and the image display 6 (as shown in fig. 5), can rapidly, intuitively and accurately measure the nerve bundle properties of the nerve trunk 7. The light source may be a laser, a light emitting diode, a mercury lamp, or the like. When a light source having a wide spectrum such as a mercury lamp is used, excitation light having a desired wavelength is obtained by using a corresponding color filter, and the wavelength of the light source is selected with reference to the excitation wavelength of the oxygen indicator used. For example, Tris (2, 2' -bipyridyl) ruthenium (ii) chloride Hexahydrate is used, a light source of about 480nm is used, the light passes through an interference color filter (with a central wavelength of 470nm and a half-peak width of 30nm), and is coupled into a Y-shaped optical fiber 1 with a diameter of 1mm by a lens (f ═ 5mm), and is transmitted to a sensing membrane 2 through an optical fiber, and the excited fluorescence is transmitted to a detector by the other arm of the Y-shaped optical fiber 1, and the detector is obtained by a fiber-optic spectrometer of the marine company (Dcean electronics Inc) S2000 type. And (3) analyzing and processing the acetylcholinesterase information by a computer, simulating the imaging of the nerve bundle cross section, and performing visual feedback through a display.
During the assay, choline was diluted to different concentrations with 20mm Tris buffer (PH 8.0).
Claims (4)
1. A method for preparing acetylcholinesterase optical fiber biosensor is characterized in that a sensing membrane (2) formed by combining a polymer membrane (a), choline oxidase (b) and an oxygen indicator (c) is fixed on the surface of an optical fiber head of an optical fiber (1) in a covalent bonding or physical adsorption mode.
2. The method for preparing acetylcholinesterase optical fiber biosensor according to claim 1, wherein the sensing membrane (2) is a composite of polymer membrane (a) and choline oxidase (b) adsorbed with oxygen indicator (c).
3. The method for preparing acetylcholinesterase optical fiber biosensor according to claim 1, wherein the sensing membrane (2) is a composite of polymer membrane (a) having oxygen indicator (c) adsorbed thereon and polymer membrane (a) having choline oxidase (b) immobilized thereon.
4. The method for preparing acetylcholinesterase optical fiber biosensor according to claim 1, wherein the sensing membrane (2) is a single layer membrane formed by mixing the oxygen indicator (c) and choline oxidase (b) in the solution of polymer material (a) and coagulating.
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| CNB021125104A CN1161478C (en) | 2002-01-09 | 2002-01-09 | Optical fibre bio-sensor of acetylcholinesterase and its preparing process |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101477078B (en) * | 2009-01-20 | 2012-04-25 | 东南大学 | Detection method for acetylcholine in royal jelly |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100425976C (en) * | 2005-03-21 | 2008-10-15 | 江苏省人民医院 | Method for quick-speed in-situ authentication of peripheral nerve tracts |
| CN101246141B (en) * | 2008-02-26 | 2012-03-14 | 南京大学 | Electro-chemistry detecting method for fast identifying peripheral nerve bundle nature |
| CN104614367B (en) * | 2015-02-10 | 2017-12-12 | 华中科技大学 | A kind of Multi-channel optical detecting system |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101477078B (en) * | 2009-01-20 | 2012-04-25 | 东南大学 | Detection method for acetylcholine in royal jelly |
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