CN201177630Y - Acetylcholine esterase piezoelectric biosensor for checking nerve-tract nature - Google Patents
Acetylcholine esterase piezoelectric biosensor for checking nerve-tract nature Download PDFInfo
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- CN201177630Y CN201177630Y CNU200820032593XU CN200820032593U CN201177630Y CN 201177630 Y CN201177630 Y CN 201177630Y CN U200820032593X U CNU200820032593X U CN U200820032593XU CN 200820032593 U CN200820032593 U CN 200820032593U CN 201177630 Y CN201177630 Y CN 201177630Y
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Abstract
The utility model discloses an acetylcholine esterase piezoelectric bio-sensor for detecting the property of nerve tracts, which comprises a piezoelectric crystal probe, an oscillation circuit, a differential frequency circuit, a frequency counter and a computer, wherein the piezoelectric crystal probe is a quartz crystal chip combined with glutaraldehyde and marked with an acetylcholine esterase antibody. The oscillation circuit is connected with the piezoelectric crystal; and one end of the frequency counter is connected with the oscillation circuit through the differential frequency circuit and the other end is connected with the computer. The piezoelectric bio-sensor can rapidly detect an acetylcholine esterase piezoelectric bio-sensing system and rapidly identify motor tracts and sensory tracts of peripheral nerves according to the difference in the acetylcholine esterase content in the motor tracts and the sensory tracts of the peripheral nerves, thereby achieving accurate suture of the motor tracts and the sensory tracts. The acetylcholine esterase piezoelectric bio-sensor can be widely popularized in later clinical application.
Description
Technical field
The utility model relates to a kind of sensor that detects peripheral nerve bundle nature, specifically a kind of acetylcholinesterase piezoelectric biological sensor that is used to detect nerve tract character.
Background technology
In existing technology, nerve tract character is carried out judgment mode mainly contains following several:
1, anatomy mode; Location collection of illustrative plates to the nerve tract in the nerve cord is studied, a lot of scholars with peripheral nerve (as median nerve, nervus radialis, chi nervus radialis do, sciatic nerve-trunk etc.) analyze respectively, position, form, the bundle number of nerve cord have been observed, measure its size, drawn the nerve tract local positioning figure of neural section, but in clinical, often easily be distorted because of two broken ends of fractured bone after the neurotrosis, changed original normal anatomy position, so restriction is received in clinical practice.
2, electrostimulation: differentiating by the reaction that the electrical stimulation bundle is produced, is motor tract then if contraction of muscle appears in electro photoluminescence, and sensory tract is then reactionless.But this method need keep patient clear-headed, and is limited to fresh damage, and individual difference is big, and stimulation causes that contraction of muscle is due to nerve conduction result or electric current spread, can not distinguish.So clinical value is little.
3, radiation biochemistry method: with the substrate of labelled with radioisotope enzyme, measure the content of enzyme again according to the radioactive product that enzyme reaction produced, infer the character of nerve tract, but this method relates to isotope technology, the equipment requirements height, and radiating matter all has infringement to patient and patient, so practicality is little.
4, enzyme histochemistry's method: the content difference that contains quantity of material according to some biology in motor tract and the sensory tract, as the standard of differentiating motor tract and sensory tract, far above sensory tract, serves as that the method for differentiating motor tract and sensory tract is extensively approved with acetylcholinesterase content as acetylcholinesterase content in the motor tract nerve fibre therefore.Detect acetylcholinesterase at present and be mainly enzyme staining's method, form copper ferrocyanide (a kind of brown precipitate) according to reaction and come across enzyme content position, motor fiber is strong positive, and the sensory fibre large part is negative, small part is p+ principle, and nerve tract character is judged.But these method existing problems are: (1) dyeing time long (about 50 minutes), process complexity; (2) two severed nerve fragments need be cut, original position dyeing can not be carried out.So certain restriction is also received in clinical practice.
5, SABC method: utilize specific antigen-antibody reaction, the location and the quantitative technique of observational study histocyte specific antigen (or antibody), found the differential protein of sensory neuron, and with this albumen as antigen preparation monoclonal antibody, differentiate sensory fibre by immunostaining, be characterized in the specificity height, nontoxic to human body, dyeing time shortens than previous methods, needs 25 to 30 minutes.But this method present stage still needs to cut tissue fragment, and has dyeing specificity and matter of time, and the time, too short dyeing was not dark, and long then other is organized and also catches look, and the clinician is difficult to control, and the operation more complicated, and practicality is not enough.
At present, biology sensor has obtained widespread use in biomedicine, the main performance in the following areas:
1, immunity biosensor
Piezoelectric immunosensor is used the extensive of ten minutes in field of biology, sensing range is constantly widened, can be used for sulphur phosphorus agricultural chemicals in the testing environment, the organophosphorus nerve gas, cocaine, Candida albicans, Escherichia coli, salmonella, herpesviral, the hepatitis A hepatitis B, the detection of pathogenic microorganisms such as comma bacillus, people's complement, protein analyses such as enzyme, thyroxine, the human chorionic gonadal hormone, hormones such as cortisol detect, red blood cell in the blood, T cell and granulocyte assay, can be used for immunoreactive dynamic analysis in addition, can monitor in real time immunoreaction process.
2, DNA biology sensor
Be used to measure the reaction of RNA/DNA molecular hyridization at first, can be used for molecule crosslinked to DNA and protein now, the hydrolysis of DNase, RNase, dna ligase, polymerization, linkage function in real time, in-situ monitoring detects and the dynamic analysis of molecular hyridization process, the health detection of food etc.
3, bionical sensing
Can be used for the anthropomorphic dummy or biological smell, sense of taste sense organ cell, early stage mainly is to adopt lipoid or the polymer substance that is not only hydrophilic but also lipophilic that function is close with it to modify the piezoelectric crystal surface, thereby fragrance or odoring substance, bitter taste or confectionery is realized the judgement of type of goods, concentration and the mechanism of action; Be used to realize simultaneous determination of multiponents recently, adopt molecular imprinting to improve sensor selectivity and serviceable life, chemometrics methods such as employing artificial neural network are used for bionical sensing and can carry out the intellectuality processing to data.
In addition, enzyme, cell fixation be can be made into enzyme, cell sensor in plane of crystal, except using single enzyme, adopting plurality of enzymes to constitute complex enzyme sensor also has report.Up to the present, do not see report relevant for the piezoelectric biological sensor that detects acetylcholinesterase content.
Summary of the invention
The purpose of this utility model provides a kind of acetylcholinesterase piezoelectric biological sensor that is used to detect nerve tract character.The difference of acetylcholinesterase (AchE) content of this biology sensor from peripheral nerve motor tract and sensory tract is started with, but the piezoelectric biological sensor-based system of fast detecting AchE, identify peripheral nerve motor tract and sensory tract fast, realize that motor tract correctly sews up sensory tract motor tract, sensory tract.
The purpose of this utility model is achieved through the following technical solutions:
A kind of acetylcholinesterase piezoelectric biological sensor that is used to detect nerve tract character, it is characterized in that: it comprises detects piezoelectric crystal probe, base pressure electric crystal probe, detection oscillatory circuit, reference oscillation circuit, differential frequency circuit, frequency counter and computing machine, is fixed with cholinesterase antibody on detection piezoelectric crystal probe; Detect oscillatory circuit and be connected with detection piezoelectric crystal probe, reference oscillation circuit is connected with base pressure electric crystal probe; Article two, oscillatory circuit all is connected with differential frequency circuit; One end of frequency counter is connected with differential frequency circuit, and the other end is connected with computing machine.
In the utility model, described piezoelectric crystal is the quartz crystal chip, on the quartz crystal chip in conjunction with glutaraldehyde, and mark acetylcholinesterase antibody.
In the utility model, use the acetylcholinesterase piezoelectric biological sensor when detecting nerve tract character, may further comprise the steps:
1) on the quartz crystal chip in conjunction with glutaraldehyde;
2) mark acetylcholinesterase antibody on the quartz crystal chip;
3) the neural segment of clip 1～2mm is put into the vibration of 200ul improvement cell pyrolysis liquid for several times;
4) the improvement cell pyrolysis liquid after will vibrating adds sample holes, and the content of observing acetylcholinesterase on monitor just changes, and the nerve tract that content is high is a motor tract, otherwise then is sensory tract.After waiting for 180 seconds, on monitor, observe the content of acetylcholinesterase.
The utility model reacts by acetylcholinesterase contained in acetylcholinesterase antibody fixing on quartz crystal and the neural sample to be measured, and the change of quartz crystal oscillation frequency takes place in the course of reaction.Detection signal and reference signal send differential frequency circuit to through behind the oscillatory circuit, and detect and reflect the content height of acetylcholinesterase by computer monitoring system by frequency counter, the nerve tract to be measured that content is high is a motor tract, and the nerve tract to be measured that content is low then is a sensory tract.
Use the flow process of acetylcholinesterase piezoelectric biological sensor in detecting nerve tract character to see Fig. 1.
Improvement cell pyrolysis liquid described in the utility model is a 0.5%Triton X-100 cell lysis buffer solution, and it is as follows to fill a prescription:
Triton?X-100 0.5％
Tris 5mmol/L
EDTA 2mmol/L
Sucrose 0.3mol/L
Regulate pH to 7.4 ,-70 ℃ of preservations are faced with before adding
PMSF(100mmol/L) 5μl/ml
Na3VO4(200mmol/L) 5μl/ml
Cocktail 10μl/ml
Calyculin?A 1.0μg/ml
The utility model is by cholinesterase antibody and the glutaraldehyde cross-linking that is combined in the quartz crystal chip surface, at the plane of crystal bag by cholinesterase antibody, in the neural sample to be measured if corresponding with it acetylcholinesterase antigen arranged, then combine, increased the quality of crystal and changed oscillation frequency with the specificity of acetylcholinesterase in the neural sample to be measured.The concentration of contained acetylcholinesterase antigen is proportional in the variation of frequency and the nerve to be detected, and this variation can reflect from monitor intuitively by electric signal, not only accurately but also convenient.
1. the even pass that adds quality Δ M of adsorbing of Sauerbrey derivation piezoelectric quartz crystal frequency change Δ F and plane of crystal is:
ΔF＝-F
2ΔM/(ANp)
In the formula, Δ F is frequency, the F of quartz crystal
2Be that resonance frequency, Δ M are that deposition quality, the A of plane of crystal is that area, the N that participates in crystal oscillator is that frequency constant, p are crystalline densities.
2. by experimental formula Δ F=c
1d
1/2+ c
2η
1/2-c
3ε-c
4κ+c
0
On behalf of density of liquid, η, d represent viscosity, ε to represent specific inductive capacity, κ to represent conductivity, c in the formula
1, c
2, c
3, c
4, c
0It is respectively constant by crystal and circuit decision.Can get: quartz crystal has stable oscillation frequency in liquid phase.
This shows that the variation of its quality is directly proportional in change of resonance frequency when foreign matter is deposited on the quartz crystal surface.
The utility model adopts the glutaraldehyde cross-linking method: at first setting up one deck inert hydrophobic material on the crystalline metal surface, mainly is polyethyleneimine and 3-aminopropyl-triethoxysilane, and then with glutaraldehyde as cross-linking reagent acetylcholinesterase antibodies.Glutaraldehyde is a kind of bifunctional agent commonly used, is easy to make protein cross, this crosslinkedly undertaken by lysine residue in the protein.
The utility model adopts and uses fixing means comparatively widely at present.Before the immobilization, at first on stromal surface, set up layer of polyethylene imines (PEI) or r-aminopropyl triethoxysilane (3-APTES) inert hydrophobic aqueous substance, connect acetylcholinesterase antibody with cross-linking reagent then.Glutaraldehyde is a double-functional group cross-linking reagent commonly used, very easily with albumen in lysine residue crosslinked.Concrete grammar is as follows:
1. PEI fixation: be coated on the electrode of crystal with the PEI methyl alcohol 5ml that contains 4% (V/V) under the room temperature, dry back washed with methanol crystal, immersing concentration then is 30min in 0.4mol/L glutaraldehyde (PH=7) solution, be coated in the crystal both sides with the low concentration antigenic solution then, the hydration aldehydes that does not react uses the glycocoll of 0.5mol/L (in 20mmol/L PBS, pH7) PBS and distilled water flushing are used in sealing subsequently, do bath in the air.
2. silane (APTES) solidification method: crystal is immersed 1h in the acetone soln of APTES of 35mmol/L, vacuum drying under 70 ℃ of conditions, wash several times with acetone, in the glutaraldehyde (pH=7) of 0.6mol/L, hatch 1h, with the distillation washing, be coated in the both sides of crystal again with the 5mg/ml antigen liquid of 5 μ l, dry 30 ~ 45min then, crystal is with after PBS and the distilled water flushing, at air drying.
The utility model is applied to the piezoelectric biological sensor technology discriminating of peripheral nerve motor tract and sensory tract, utilize the biology sensor sensitivity, fast, accurately, the characteristics of high specificity, made the piezoelectric biological sensor that can detect acetylcholinesterase content.This sensor can detect the content of acetylcholinesterase on the every nerve tract, principle is that acetylcholinesterase contained in cholinesterase antibody fixing on quartz crystal and the neural sample reacts, the change of quartz crystal oscillation frequency is arranged in the course of reaction, then detect and reflect the content height of acetylcholinesterase by computer monitoring system, the nerve tract that content is high is a motor tract, otherwise then is sensory tract.Entire reaction course has shortened the time greatly than previous methods within 3 minutes, easy to use, has satisfied clinical requirement and has reduced error and interference.Obtained effect preferably by zoopery.
Compared with prior art, remarkable advantage of the present utility model is:
1. the simple and sensitivity of pick-up unit is up to 10
-12Mol/L, so sample do not need special processing, and only need cut few tissue and just can detect clearly difference;
2. detection efficiency is very high, and a sample only needs 3 minutes;
3. because to detect material in the sample be form combination by antigen and antibody, so specificity is very high;
4. this device needs the material that detects in can the detection sample of real-time quantitative, can be visual in image show on computers;
5. detection material does not need to do in advance any physics, chemical modification;
6. chip can the repeated multiple times application etc. advantage.Therefore repacking is put and can be accomplished fast, identify accurately, intuitively nerve tract ground, neural broken ends of fractured bone both sides character, and the method simple and fast need not chemical reagent, and is harmless, realizes mechanization, integrated and intelligent.
The piezoelectric biological sensor of this discriminating peripheral nerve bundle nature, will for after the peripheral nerve injury Microsurgical repair provide a kind of method efficiently and effectively, the functional rehabilitation rate after improving peripheral nerve injury greatly and repairing.Help extensively being promoted in the clinical practice afterwards.
Description of drawings
Fig. 1 is to use the process flow diagram of acetylcholinesterase piezoelectric biological sensor in detecting nerve tract character;
Fig. 2 is the zoopery result curve figure that adopts the utility model to obtain.
Fig. 3 is a structural representation of the present utility model.
Embodiment
A kind of acetylcholinesterase piezoelectric biological sensor that is used to detect nerve tract character, it comprises detects piezoelectric crystal probe 11, base pressure electric crystal probe 12, detection oscillatory circuit 21, reference oscillation circuit 22, differential frequency circuit 3, frequency counter 4 and computing machine 5, detecting piezoelectric crystal probe 11 is the quartz crystal chip, on the quartz crystal chip in conjunction with glutaraldehyde, and mark acetylcholinesterase antibody.Detect oscillatory circuit 21 and be connected with detection piezoelectric crystal probe 11, reference oscillation circuit 22 is connected with base pressure electric crystal probe 12; Article two, oscillatory circuit all is connected with differential frequency circuit 3; One end of frequency counter 4 is connected with differential frequency circuit 3, and the other end is connected with computing machine 5.
The utility model reacts by acetylcholinesterase contained in cholinesterase antibody fixing on quartz crystal and the neural sample to be measured, the change of quartz crystal oscillation frequency takes place in the course of reaction, then detect and reflect the content height of acetylcholinesterase by computer monitoring system, the nerve tract to be measured that content is high is a motor tract, and the nerve tract to be measured that content is low then is a sensory tract.Use the flow process of acetylcholinesterase piezoelectric biological sensor in detecting nerve tract character to see accompanying drawing 1.
At first on the quartz crystal chip in conjunction with glutaraldehyde; Mark cholinesterase antibody on the quartz crystal chip then; The neural segment of clip 1～2mm is put into the vibration of 200ul improvement cell pyrolysis liquid for several times; Improvement cell pyrolysis liquid after the vibration is added sample holes, and after waiting for 180 seconds, the content of observing acetylcholinesterase on monitor just changes, and the nerve tract that content is high is a motor tract, otherwise then is sensory tract.
The utility model is applied to the piezoelectric biological sensor technology discriminating of peripheral nerve motor tract and sensory tract, utilize the biology sensor sensitivity, fast, accurately, the characteristics of high specificity, made the piezoelectric biological sensor that can detect acetylcholinesterase content.This sensor can detect the content of acetylcholinesterase on the every nerve tract, and entire reaction course has shortened the time greatly than previous methods within 3 minutes, easy to use, has satisfied clinical requirement and has reduced error and interference.Obtained effect preferably by zoopery.
Zoopery
Material: (Nanjing Medical University antibody center provides the cholinesterase monoclonal antibody, dog sciatic nerve, cervical plexus nerve, facial nerve, cell pyrolysis liquid, phosphate buffer, 1M Glycine, 10% glutathione solution.Equipment: ADS plus Affinity Sensor piezoelectric biological sensor (ANT company in Taiwan produces, and inferior new science and technology incorporated company provides).
Experimental procedure:
1,10% glutathione solution is hatched chip 10min.
2, antibody is hatched chip 10min with phosphate buffer dilution back (1: 1000).
3,1M Glycine is hatched chip 1min.
4, get dog sciatic nerve, arm nerve respectively from, facial nerve broken ends of fractured bone 1mm, put into cell pyrolysis liquid, concussion 5-10s puts into ADS plus Affinity Sensor piezoelectric biological sensor, reading of data.Result such as Fig. 2.
(1) kinesitherapy nerve 1
(2) kinesitherapy nerve 2
(3) kinesitherapy nerve 3
(4) the sensorimotor composite nerve 1
(5) the sensorimotor composite nerve 2
(6) kinesitherapy nerve 4
(7) kinesitherapy nerve 4
(8) kinesitherapy nerve 5
(9) sensory nerve 1
(10) sensory nerve 1
(11) sensory nerve 1
(12) sensory nerve 1
Remarks: GA: the sample source that the glutathione sensation is different with the representative of kinesitherapy nerve numbering.
Conclusion; Sensation, motion and composite nerve, the content difference of its cholinesterase presents different curves by this method on the display screen of sensor.Obvious difference, and good reproducibility.This law has good feasibility.
Claims (2)
1, a kind of acetylcholinesterase piezoelectric biological sensor that is used to detect nerve tract character, it is characterized in that: it comprises detects piezoelectric crystal probe (11), base pressure electric crystal probe (12), detection oscillatory circuit (21), reference oscillation circuit (22), differential frequency circuit (3), frequency counter (4) and computing machine (5), is fixed with cholinesterase antibody on detection piezoelectric crystal probe (11); Detect oscillatory circuit (21) and be connected with detection piezoelectric crystal probe (11), reference oscillation circuit (22) is connected with base pressure electric crystal probe (12); Detecting oscillatory circuit (21) all is connected with differential frequency circuit (3) with reference oscillation circuit (22); One end of frequency counter (4) is connected with differential frequency circuit (3), and the other end is connected with computing machine (5).
2, the acetylcholinesterase piezoelectric biological sensor that is used to detect nerve tract character according to claim 1, it is characterized in that: described detection piezoelectric crystal probe (11) is the quartz crystal chip, on the quartz crystal chip in conjunction with glutaraldehyde, and mark acetylcholinesterase antibody.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102692513A (en) * | 2012-06-26 | 2012-09-26 | 长沙理工大学 | Piezoelectric immunosensing chip and device for detecting oncogene C-myc recombinant protein |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102692513A (en) * | 2012-06-26 | 2012-09-26 | 长沙理工大学 | Piezoelectric immunosensing chip and device for detecting oncogene C-myc recombinant protein |
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Granted publication date: 20090107 Termination date: 20110221 |