CN102212609A - Method for detecting lipase heterogeneous catalytic reaction with biosensor - Google Patents
Method for detecting lipase heterogeneous catalytic reaction with biosensor Download PDFInfo
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- CN102212609A CN102212609A CN2011100815070A CN201110081507A CN102212609A CN 102212609 A CN102212609 A CN 102212609A CN 2011100815070 A CN2011100815070 A CN 2011100815070A CN 201110081507 A CN201110081507 A CN 201110081507A CN 102212609 A CN102212609 A CN 102212609A
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- lipase
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- pbs
- catalytic reaction
- immobilized lipase
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Abstract
The invention relates to a biosensor research method of the lipase heterogeneous catalytic reaction. The electron transfer of the immobilized lipase catalytic substrate is converted to current or electric resistance to output. The method comprises the following steps: first connecting device according to the appended drawings of the description, then placing PBS (Poly(butylene succinate) in an anode chamber; placing the mixed solution of PBS and potassium ferricyanide supersaturated solution in a cathode chamber; and mixing substrate and immobilized lipase rapidly and evenly, and placing the mixture in the cathode chamber to detect. Liquid chromatography is adopted to measure the contents of fatty acid and ester and the measured relative error is only 4.7%, which proves that the method can be used to measure the ester bond breaking level in the reaction process of the enzyme and the substrate rapidly and accurately. In the method, immobilized lipase is used as the biological element, the specificity, sensitivity and electrics of lipase can be combined conveniently, rapidly and cleverly, electrical signals can be output accurately, the dead weight of the reaction substrate can be calculated and the reaction end point can be determined.
Description
Technical field:
The present invention relates to a kind of method that detects immobilized lipase heterogeneous catalytic reaction process with biosensor.
Background technology:
Along with science and technology development, biosensor has become the requisite a kind of advanced person's of biomass technology detection method and monitoring method, also is quick, the trace analysis method of material molecule level, has higher selectivity and sensitivity
Biosensor is a kind of special device or device, what use is biomechanism, compare with off-line analysis is backward in technique with traditional chemical sensor, have highly selective, highly sensitive, preferably stability, can in complex system, carry out advantage such as quick on-line monitoring.Basic functional principle is: specific reaction that biosensor is taken place and signal are via physical component---transverter, change easily detection signal such as light, electricity, sound into, thereby the reflection degree can be expressed with discrete or successive numerary signal, be known that indirectly determinand for information about.Biosensor is mainly used in food, agricultural, biotechnology, medicine and military field at present.But also do not utilize biosensor to detect the method for lipase-catalyzed reaction process at present.
Summary of the invention:
The invention provides a kind of method that detects lipase heterogeneous catalytic reaction process with biosensor, at present also do not utilize biosensor to detect the problem of the method for lipase-catalyzed reaction process to solve, convert the electronic transfer process that fixed lipase catalyzed substrate produced to electric current or resistance and output, thereby judge the terminal point of lipase-catalyzed reaction.
Use the device of the inventive method to comprise damping fluid, reometer 8, resistance 9, micro-chip 10, replicating machine 11, PC 12, circuit card 13,5V power supply 14 and platinum filament 15 in the interior buffered soln of thermostat water bath 1, open glass container 2, cylindrical graphite body 3, platinized platinum 4, sheet glass, ion-exchange membrane 5, cathode compartment 7 and cathode compartment, anolyte compartment 6 and the anolyte compartment; Open glass container 2 places thermostat water bath 1, cylindrical graphite body 3 and platinized platinum 4 place cathode compartment 7 and cathode compartment 6 respectively, cut off by sheet glass between cathode compartment 7 and the anolyte compartment 6, and be connected by the ion-exchange membrane 5 that is installed on the sheet glass between cathode compartment 6 and the cathode compartment 7, be connected by platinum filament 15 between cylindrical graphite body 3 and the platinized platinum 4, reometer 8 and resistance 9 are connected on the platinum filament 15, the centre tap of resistance 9 is connected on the signal input part of micro-chip 10, a communication port of replicating machine 11 connects micro-chip 10, another communication port of replicating machine 11 connects PC 12, and 5V power supply 14 provides power supply for micro-chip 10;
Step of the present invention is as follows:
Step 1: bath temperature: 65 ℃, adopt water bath with thermostatic control control;
Step 2: produce anolyte PBS: take by weighing potassium primary phosphate and Sodium phosphate dibasic and be respectively 1.36g and 1.42g, be mixed with the buffered soln that concentration is 0.1mol/L (PBS) with the 200ml deionized water;
Step 3: produce catholyte: with deionized water the Tripotassium iron hexacyanide is made into supersaturated solution, catholyte is the mixed solution of Tripotassium iron hexacyanide supersaturated solution and PBS;
Step 4: test: the pH of buffered soln is transferred to 6,7,8 respectively, with Novozym 435 immobilized lipase 0.09g and the quick mixing of triglyceride level, places in the cathode compartment again;
Step 5: record current intensity: adopt sensitive reometer respectively the strength of current under three kinds of different pH environment to be tested, every numerical value of half a minute record;
Step 6: the mixed solution of getting the differential responses time in the experimentation five carries out centrifugation, and the upper strata liquid that obtains is measured the content of lipid acid and ester with liquid chromatography, thus the corresponding relation of content between verificating current value and lipid acid and the ester.
The present invention is as biological elements with immobilized lipase, with the triglyceride level is reaction substrate, design, developed the electrochemica biological sensor that the transfer transport that lipase-catalyzed substrate produced can be converted to the output of electric current or resistance, and studied the influence of pH variation its output data.The immobilized lipase enzyme biologic sensor can combine to second the easy, rapid of specificity, susceptibility and electricity of lipase ingeniously, can be in the system of a complexity, not disturbed by other materials, measure the content of some material fast and accurately, and can measure the degree of enzyme-to-substrate reaction.
Description of drawings
Fig. 1 is the structural representation of the device used of the present invention.
Embodiment:
Embodiment one: in conjunction with Fig. 1 explanation, present embodiment is as follows:
Use the device of the inventive method to comprise damping fluid, reometer 8, resistance 9, micro-chip 10, replicating machine 11, PC 12, circuit card 13,5V power supply 14 and platinum filament 15 in the interior buffered soln of thermostat water bath 1, open glass container 2, cylindrical graphite body 3, platinized platinum 4, sheet glass, ion-exchange membrane 5, cathode compartment 7 and cathode compartment, anolyte compartment 6 and the anolyte compartment; Open glass container 2 places thermostat water bath 1, cylindrical graphite body 3 and platinized platinum 4 place cathode compartment 7 and cathode compartment 6 respectively, cut off by sheet glass between cathode compartment 7 and the anolyte compartment 6, and be connected by the ion-exchange membrane 5 that is installed on the sheet glass between cathode compartment 6 and the cathode compartment 7, be connected by platinum filament 15 between cylindrical graphite body 3 and the platinized platinum 4, reometer 8 and resistance 9 are connected on the platinum filament 15, the centre tap of resistance 9 is connected on the signal input part of micro-chip 10, a communication port of replicating machine 11 connects micro-chip 10, another communication port of replicating machine 11 connects PC 12, and 5V power supply 14 provides power supply for micro-chip 10;
Step 1: the bath temperature in the thermostat water bath 1 is 65 ℃, adopts water bath with thermostatic control control;
Step 2: produce anolyte PBS: take by weighing potassium primary phosphate and Sodium phosphate dibasic and be respectively 1.36g and 1.42g, be mixed with the buffered soln that concentration is 0.1mol/L (PBS) with the 200ml deionized water;
Step 3: produce catholyte: with deionized water the Tripotassium iron hexacyanide is made into supersaturated solution, catholyte is the mixed solution of Tripotassium iron hexacyanide supersaturated solution and PBS;
Step 4: test: the pH of solution is transferred to 6,7,8 respectively, with immobilized lipase 0.09g and the quick mixing of triglyceride level, places in the cathode compartment again;
Step 5: record current intensity: adopt sensitive reometer respectively the strength of current under three kinds of different pH environment to be tested, every numerical value of half a minute record;
Step 6: the mixed solution of getting the differential responses time in the experimentation five carries out centrifugation, and the upper strata liquid that obtains is measured the content of lipid acid and ester with liquid chromatography, thus the corresponding relation of content between verificating current value and lipid acid and the ester.
Embodiment two: present embodiment and embodiment one difference are that the temperature of water bath with thermostatic control in the step 1 is 50～70 ℃, and other composition is identical with embodiment one with step.
Embodiment three: present embodiment and embodiment one difference are that the concentration of PBS in the step 2 is 0.01～1mol/L, and other composition is identical with embodiment one with step.
Embodiment four: present embodiment and embodiment one difference are that the pH in the step 4 is 5～9, and other composition is identical with embodiment one with step.
Embodiment five: present embodiment and embodiment one difference are that step 4 immobilized lipase add-on is 0.1～0.3% of a triglyceride level, and other composition is identical with embodiment one with step.
Embodiment six: present embodiment and embodiment one difference are that be 1～60 second writing time in the step 5.Other composition is identical with embodiment one with step.Step 1: bath temperature: 65 ℃, adopt water bath with thermostatic control control;
Step 2: anolyte PBS: take by weighing potassium primary phosphate and Sodium phosphate dibasic and be respectively 1.36g and 1.42g, be mixed with the buffered soln liquid (PBS) that concentration is 0.1mol/L with the 200ml deionized water;
Step 3: catholyte: with deionized water the Tripotassium iron hexacyanide is made into supersaturated solution, catholyte is the mixed solution of Tripotassium iron hexacyanide supersaturated solution and PBS;
Step 4: ion-exchange membrane: between anolyte compartment and the cathode compartment ion-exchange membrane is installed;
Step 5: accompanying drawing ligation device to specifications;
Step 6: test: the pH of solution is transferred to 6,7,8 respectively, with immobilized lipase 0.09g and the quick mixing of triglyceride level 3ml, places in the cathode compartment again, respectively the strength of current under three kinds of different pH environment is tested;
Step 7: record current intensity: adopt sensitive reometer that the strength of current in the circuit is tested, every numerical value of 30 seconds records;
Step 8: in the experimentation mixed solution is carried out centrifugation, the upper strata liquid that obtains is measured the content of lipid acid and ester with liquid chromatography, thus the conclusion of confirmatory experiment.
The major equipment that adopts in the present embodiment is: thermostat water bath.
Embodiment two: present embodiment and embodiment one difference are that the temperature of water bath with thermostatic control in the step 1 is 50～70 ℃, and other composition is identical with embodiment one with step.
Embodiment three: present embodiment and embodiment one difference are that the concentration of PBS in the step 2 is 0.01～1mol/L, and other composition is identical with embodiment one with step.
Embodiment four: present embodiment and embodiment one difference are that the pH in the step 4 is 5～9, and other composition is identical with embodiment one with step.
Embodiment five: present embodiment and embodiment one difference are that step 4 immobilized lipase add-on is 0.1～0.3% of a triglyceride level, and other composition is identical with embodiment one with step.
Embodiment six: present embodiment and embodiment one difference are that be 1～60 second writing time in the step 5.Other composition is identical with embodiment one with step.
Claims (6)
1. method that detects lipase heterogeneous catalytic reaction process with biosensor, it is characterized in that in the mixed solution of the damping fluid Tripotassium iron hexacyanide supersaturated solution of cathode compartment and PBS, fixed lipase catalyzed substrate produces ion, ion enters the anolyte compartment by ion-exchange membrane, between graphite and platinized platinum, formed circulation circuit, therefore, electronic transfer process converts electric current or resistance output to.Present method realizes based on following apparatus: a kind of on-line monitoring lipase heterogeneous catalytic reaction biosensor arrangement, it is characterized in that: described a kind of biosensor arrangement of monitoring the immobilized lipase hydrolytic process, the open glass container is placed thermostat water bath, and cylindrical graphite and platinized platinum place cathode compartment and anolyte compartment respectively, and be connected by ion-exchange membrane between cathode compartment and the anolyte compartment, be connected by platinum filament between graphite and the platinized platinum, resistance R and power supply are connected with it.Connect a micro-chip on resistance R, micro-chip is connected last replicating machine respectively at 5V power supply, circuit card and replicating machine again and is connected on the PC.
This process realizes by following steps:
Step 1: bath temperature: 65 ℃, adopt water bath with thermostatic control control;
Step 2: anolyte PBS: take by weighing potassium primary phosphate and Sodium phosphate dibasic and be respectively 1.36g and 1.42g, be mixed with the buffered soln liquid (PBS) that concentration is 0.1mol/L with the 200ml deionized water;
Step 3: catholyte: with deionized water the Tripotassium iron hexacyanide is made into supersaturated solution, catholyte is the mixed solution of Tripotassium iron hexacyanide supersaturated solution and PBS;
Step 4: ion-exchange membrane: between anolyte compartment and the cathode compartment ion-exchange membrane is installed;
Step 5: accompanying drawing ligation device to specifications;
Step 6: test: the pH of solution is transferred to 6,7,8 respectively, with immobilized lipase 0.09g and the quick mixing of triglyceride level, places in the cathode compartment again, respectively the strength of current under three kinds of different pH environment is tested;
Step 7: record current intensity: adopt sensitive reometer that the strength of current in the circuit is tested, every numerical value of half a minute record;
Step 8: in the experimentation mixed solution is carried out centrifugation, the upper strata liquid that obtains is measured the content of lipid acid and ester with liquid chromatography, thus the conclusion of confirmatory experiment.
2. a kind of method with biosensor detection immobilized lipase heterogeneous catalytic reaction process according to claim 1, the temperature that it is characterized in that water bath with thermostatic control in the step 1 is 50～70 ℃.
3. a kind of method with biosensor detection immobilized lipase heterogeneous catalytic reaction process according to claim 1, the concentration that it is characterized in that PBS in the step 2 is 0.1～1mol/L.
4. a kind of method with biosensor inspection immobilization survey lipase heterogeneous catalytic reaction process according to claim 1 is characterized in that the pH in the step 6 is 5～9.
5. a kind of method with biosensor detection immobilized lipase heterogeneous catalytic reaction process according to claim 1 is characterized in that step 6 immobilized lipase add-on is 0.01～0.3% of a triglyceride level.
6. a kind of method with biosensor detection immobilized lipase heterogeneous catalytic reaction process according to claim 1 is characterized in that be 1～60 second writing time in the step 7.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104597100B (en) * | 2014-07-24 | 2021-11-05 | 南京工业大学 | Method for detecting triglyceride in serum |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101801985A (en) * | 2007-06-19 | 2010-08-11 | 霍夫曼-拉罗奇有限公司 | redox mediators |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101801985A (en) * | 2007-06-19 | 2010-08-11 | 霍夫曼-拉罗奇有限公司 | redox mediators |
Non-Patent Citations (2)
| Title |
|---|
| 刘滨城: "《生物传感器在脂酶酯化反应过程中的应用》", 《CNKI HTTP://WWW.CNKI.NET/KCMS/DETAIL/11.1759.TS.20110314.1030.007.HTML》, 14 March 2011 (2011-03-14), pages 1 - 6 * |
| 王永生 等: "《甘油三酯酶传感器的研制及应用》", 《生物化学与生物物理进展》, vol. 26, no. 2, 31 December 1999 (1999-12-31), pages 144 - 146 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104597100B (en) * | 2014-07-24 | 2021-11-05 | 南京工业大学 | Method for detecting triglyceride in serum |
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Application publication date: 20111012 |