CN101526490B - Thermal biosensor based on reversible immobilized enzyme - Google Patents
Thermal biosensor based on reversible immobilized enzyme Download PDFInfo
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Abstract
The invention relates to a thermal biosensor combined with reversible immobilized enzyme. A system comprises a constant flow pump, an injection valve, a thermostat, a microreactor, a detector, a circulating flow line, etc. The system is characterized in that the injection valve not only can be used for combining and controlling enzyme liquid, zymolyte, carrying flow, reagent and the like, but also can realize circulation mode, namely, the liquid flowing out of the microreactor enters the thermostat again to circulate through the microreactor so as to realize reversible immobilization. The thermal biosensor combined with reversible immobilized enzyme technology can realize the reactor loading (immobilization) of enzyme and reactor unloading (reversible immobilization), so that the sorts of the measured maters and enzymes can be switched in an on-line way without replacing carriers. A step of prefabricating and immobilizing enzyme can be omitted, so that the time and the economic cost can be saved, interference can be reduced, and the system is especially flexible and convenient when being applied to the tests of multiple samples and combinations. The invention can improve the degree of integration and automation, and enhance and expand the functions and reaction modes of the traditional thermal biosensor.
Description
Technical field
Patent of the present invention relates to a kind of calorimetric biosensor in conjunction with reversible enzyme immobilization technology, can be applied in on-the-spot and online many samples and the combined test and the analysis in fields such as food, medicine, biochemistry, environment and chemical industry.
Background technology
At present, analytical equipment develops towards accuracy, direction integrated and robotization.Require can be enough lower consumption, easier method and apparatus, faster analysis speed provide in real time, accurately, comprehensive information.
Calorimetric biosensor is the biology sensor of analyzing by the thermal change of measuring the biochemical reaction process generation.The calorimetric biosensor highly versatile is applicable to most biological samples.It is not subjected to the influence of sample attributes such as color, muddiness and galvanochemistry in addition, is particularly suitable for detecting undressed actual sample and on-the-spot, online combinatory analysis detection.Calorimetric method is easy, reliable, quick, highly sensitive, and helps the exploitation of microsystem.But the shortcoming of calorimetric biosensor is baseline stability time length, is subject to disturb.
Consideration based on stability and recycling, traditional calorimetric biosensor is often in conjunction with immobilised enzymes, and adopt method pre-preparation such as crosslinked, embedding, its step is loaded down with trivial details, the increase cost, and guarantee that immobilised enzymes keeps homogeneous, stable activity in preparation for a long time, storage, transportation and testing process, being the standardization of immobilised enzymes, is a complicated problems.The particularly important is,, can not reuse, additionally increased cost for carrying out irreversible inhibitor (as noxious materials such as agricultural chemicals, heavy metals) when detecting.In addition, on-the-spot and online many samples and combinatory analysis test request are frequently changed reagent and enzyme class, will artificially introduce interference, and this has influenced calorimetric biosensor greatly and has reached efficient, the stable use in the on-line testing at the scene.
Reversible enzyme immobilization technology is to utilize characteristic conditions such as functional group, pH, temperature, and the control enzyme is fixed on the reversible process on the carrier.In conjunction with the calorimetric biosensor of reversible enzyme immobilization technology online loading enzyme on carrier very easily, i.e. immobilization also can online unloading enzyme, promptly contrary immobilization.Need not to change the kind of just changeable tester of carrier and immobilised enzymes, flexible and convenient operation also can reduce interference.Simultaneously, omitted the process of pre-preparation immobilised enzymes, asked during saving and financial cost, and can reclaim enzyme, to the enzyme of costliness, particularly important.
Summary of the invention
The objective of the invention is by providing a kind of energy to control the calorimetric biosensor of the reversible immobilization process of enzyme by modes such as cross reaction, pH, temperature, it is flexible, applicability is strong, is particularly useful for scene, online many samples and combined test.
The technical solution adopted for the present invention to solve the technical problems is: a kind of calorimetric biosensor system based on reversible immobilised enzymes, comprise constant flow pump, fillup valve, thermostat, microreactor, detecting device and circulation stream etc., be characterized in the power that constant flow pump provides the constant flow of current-carrying, enzyme liquid, substrate and reagent to carry; Fillup valve is realized current-carrying, enzyme liquid, substrate and combination of agents and control, and changeable mode of operation, be open circuit mode and circulation pattern, the former discharges system with the effluent of microreactor, as waste liquid or recycling, the latter enters microreactor by the thermostat circulation again with the effluent of microreactor, carries out the immobilization of enzyme; Thermostat makes being combined in of current-carrying, enzyme liquid, substrate and reagent etc. enter the preceding constant design temperature that reaches of microreactor, and can change temperature of reaction by regulating design temperature; The inner reversible immobilized function carrier of enzyme (sorbing material, macromolecular material, gel etc.) of filling of microreactor; The thermal sensor that is used for detecting device detects the variation of microreactor heat, and its output terminal is connected with signal Processing and display unit or computing machine respectively, carries out signal Processing, and shows; Thermostat, microreactor and connecting line and thermal sensor all are placed in the adiabatic environment.Calorimetric biosensor based on reversible immobilised enzymes adopts following testing process: the first step, prepare and stabilization process, and in microreactor, to pile up naturally and fill function carrier, total system feeds current-carrying and to system stability, obtains steady baseline; In second step, enzyme loads (immobilization) process, switches fillup valve, is operated in circulation pattern, and current-carrying, enzyme liquid and reagent mixture constant flow circular flow are crossed microreactor at this moment, and process continues setting-up time as the set time, and enzyme is fixed on the function carrier; In the 3rd step, reaction and testing process are switched fillup valve, be operated in open circuit mode, substrate is introduced into system, carries out enzyme reaction on the function carrier in microreactor, the enzyme reaction liberated heat changes in the omnidistance monitoring of the thermal sensor microreactor, and its signal variation has reflected the enzyme reaction degree; In the 4th step, uninstall process is contrary immobilization process, changes the controlled condition of enzyme immobilization, and enzyme breaks away from from function carrier, and the effluent of microreactor can be used as waste liquid, also can be used to reclaim enzyme; The 5th step fed current-carrying, got rid of the raffinate in microreactor and the stream, carried out next test.Described system flow, corresponding respectively corresponding program of per step and control realize operations such as flow velocity and switching.
The invention has the beneficial effects as follows the link of saving prefabricated immobilised enzymes based on reversible immobilized calorimetric biosensor, saved time and financial cost, can change tester or enzyme class flexibly, easily and quickly, simplify step, reduce and disturb.Improve the robotization and the integrated degree that detect, expanded and optimized the function and the reaction pattern of traditional calorimetric biosensor.
Fig. 1 is the calorimetric biosensor system based on reversible immobilised enzymes
Fig. 2 is many samples of the calorimetric biosensor combinatory analysis synoptic diagram based on reversible immobilised enzymes
The present invention does an explanation in conjunction with the accompanying drawings, as shown in Figure 1 based on the calorimetric biosensor system of reversible immobilised enzymes.Wherein C is a current-carrying; E is an enzyme liquid; S is a substrate; R is a reagent; P is a constant flow pump; V is a fillup valve; T is a thermostat; M is a microreactor; D is a detecting device; C﹠amp; D is signal Processing and display unit; PC is a computing machine; W is a waste liquid.Constant flow pump P realizes the transmission of the setting flow of current-carrying C, enzyme liquid E, substrate S and reagent R etc., and constant current has guaranteed the reappearance and the precision of system; By switching and the independent assortment between fillup valve V realization current-carrying C, enzyme liquid E, substrate S and reagent R, finish regularly, quantitatively inject, realize operations such as programmed control and auto injection, and can realize the switching of open circuit mode and circulation pattern, open circuit mode makes the effluent that flows out microreactor M discharge system, as waste liquid or recycling, circulation pattern is exactly that effluent cycles through microreactor M by thermostat T again; Thermostat T carries out constant temperature to the potpourri of the current-carrying C, enzyme liquid E, substrate S and the reagent R that enter microreactor, can guarantee to be reflected at the reappearance under the optimum temperature condition; The inner function carrier (sorbing material, macromolecular material, gel etc.) of filling of microreactor M is used for the reversible immobilization of enzyme; Obtain reaction heat signal among the microreactor M as the thermal sensor of detecting device D, and through signal Processing and display unit C﹠amp; D, or be connected with computer PC carries out signal Processing and shows; The adiabatic environment of thermostat T, microreactor M and link to each other pipeline and detecting device D outside can effectively be eliminated thermal loss, improves precision.Use the concrete enforcement testing process of above-mentioned bio-sensor system based on reversible immobilised enzymes to be: the first step, prepare and stabilization process, in microreactor M, pile up naturally and fill function carrier, open constant flow pump P and thermostat T, feed current-carrying C to system stability, obtain steady baseline.Second step, switch fillup valve V, the potpourri constant flow circular flow of enzyme liquid E, reagent R and current-carrying C is crossed microreactor M, is fixation procedure, continues setting-up time as the set time, and at this moment, enzyme is fixed on the fixation support; The 3rd step, reaction and testing process, switch fillup valve V, the potpourri constant flow of introducing substrate S, reagent R and current-carrying C flows through microreactor M, and carry out enzyme reaction on the function carrier therein, the enzyme reaction liberated heat changes in the omnidistance monitoring of the thermal sensor microreactor, and its signal changes by signal Processing and display unit C﹠amp; D or computer PC are handled the back and are shown; The 4th step, uninstall process, promptly contrary immobilization process, the controlled condition of change enzyme immobilization, enzyme breaks away from from function carrier, and the effluent of microreactor M does not recycle, and can be used as waste liquid, can be used for the recovery of enzyme yet; The 5th step, switch fillup valve V, current-carrying C washes away system's stream, gets rid of the raffinate in microreactor M and the stream, and steady baseline, carries out next test.
Fig. 2 has provided the synoptic diagram that above-mentioned calorimetric biosensor based on reversible immobilised enzymes is implemented the combinatory analysis of many samples.The implication of arrow mark is respectively a, begins to control contrary fixing condition (introduce contrary immobilized reagent, regulate temperature etc.), carries out the uninstall process of enzyme; B, introducing current-carrying, the systems stabilisation baseline; The immobilization of enzyme is carried out in c, introducing enzyme liquid, reagent and current-carrying combination; The substrate S1 of S1, introducing enzyme E1 correspondence reacts; The substrate S2 of S2, introducing enzyme E2 correspondence reacts.Dependence test with two kinds of enzyme E1 and E2 is the example explanation, at first introduce the stabilization process that current-carrying C carries out system, after obtaining steady baseline, carry out the test of enzyme E1, by introducing the potpourri of enzyme liquid E1, reagent R and current-carrying C, carry out the immobilization of enzyme, during test, introduce substrate S1, carry out enzyme reaction, repeat to introduce substrate S1, can continuous many sample testings.System fed current-carrying C after test finished, steady baseline, the condition of the contrary immobilised enzymes of conversion and control, by introducing contrary immobilized reagent, regulating pH and temperature etc., carry out the contrary immobilization process of enzyme E1, introduce raffinate in the current-carrying C scavenge system then, and steady baseline, what carry out subsequently is the test of enzyme E2, introduces the potpourri of enzyme liquid E2, reagent R and current-carrying C, carry out the immobilization of enzyme, introduce substrate S2, carry out enzyme reaction, introduce current-carrying C after test finishes, steady baseline enters next test loop.
According to the difference of control mode, provide two test cases that detect hydrogen peroxide based on the calorimetric biosensor of reversible immobilised enzymes.Hydrogen peroxide carries out following reaction under catalatic effect, utilize the reaction enthalpy based on the calorimetric biosensor test process of reversible immobilised enzymes, reflects the amount of hydrogen peroxide.
2H
2O
2→2H
2O+O
2+ΔH
Test case 1:
Rely on the Ag-Ab cross reaction to control reversible immobilization process.Fixation support is an Ago-Gel, and functional group is a rabbit AHS albumin (anti-HSA), and substrate is a hydrogen peroxide.Current-carrying is the damping fluid of 0.1M, pH7.0 potassium phosphate.Flow velocity is set at 1ml/min, and temperature of reaction is 30 ℃.Use hydrogen peroxidase and human serum albumins (HSA) mixed liquor of 20U/ml to fix, the set time is 2min.0.2M, glycocoll-hydrochloric acid and the damping fluid mixed liquor of pH2.2 wash away immobilised enzymes, unloads, the time is 2min.
Test case 2:
Rely on pH to control reversible immobilization process.Fixation support is ceramic hydroxyapatite, and substrate is a hydrogen peroxide.Flow velocity is set at 0.1ml/min, and temperature of reaction is 30 ℃.5mM, pH6.5 phosphate buffer, the hydrogen peroxidase liquid mixture of pH6.5,20U/ml is fixed, 20 minutes set times.Introduce damping fluid, change the pH and the ionic strength of damping fluid, unload, the time is 10min.
Claims (5)
1. calorimetric biosensor based on reversible immobilised enzymes, the Flow Injection Analysis system that forms by constant flow pump, fillup valve, thermostat, microreactor, detecting device, signal and display unit, computing machine and circulation stream, it is characterized in that the power that constant flow pump (P) provides the constant flow of current-carrying (C), enzyme liquid (E), substrate (S) and reagent (R) to carry; Fillup valve (V) is realized the combination and the control of current-carrying (C), enzyme liquid (E), substrate (S) and reagent (R), and changeable mode of operation, i.e. open circuit mode and circulation pattern; Thermostat (T) makes being combined in of current-carrying (C), enzyme liquid (E), substrate (S) and reagent (R) enter the preceding constant design temperature that reaches of microreactor (M), and can change temperature of reaction by regulating design temperature; The inner reversible immobilized function carrier of enzyme of filling of microreactor (M); The thermal sensor that is used for detecting device (D) detects the variation of microreactor heat, its output terminal respectively with signal Processing and display unit (C﹠amp; D) be connected with computing machine (PC), carry out signal Processing, and show.
2. the calorimetric biosensor based on reversible immobilised enzymes according to claim 1 is characterized in that described open circuit mode is that the effluent of microreactor (M) is discharged system.
3. the calorimetric biosensor based on reversible immobilised enzymes according to claim 1 is characterized in that described circulation pattern is that the effluent of microreactor (M) is entered microreactor (M) by thermostat (T) circulation again.
4. the calorimetric biosensor based on reversible immobilised enzymes according to claim 1 is characterized in that described thermostat (T), microreactor (M) and connecting line and thermal sensor all are placed in the adiabatic environment.
5. application rights requires the testing process of 1 described calorimetric biosensor based on reversible immobilised enzymes, it is characterized in that:
A. pile up naturally in microreactor (M) and fill function carrier, system feeds current-carrying (C) and to system stability, obtains steady baseline;
B. enzyme loads (immobilization) process, switch fillup valve (V), be operated in circulation pattern, the potpourri constant flow circular flow of current-carrying (C), enzyme liquid (E) and reagent (R) is crossed microreactor (M) at this moment, process continues setting-up time as the set time, and enzyme is fixed on the function carrier;
C. reaction and testing process are switched fillup valve (V), are operated in open circuit mode, and substrate (S) is introduced into system, carries out enzyme reaction on the function carrier in microreactor (M), and the enzyme reaction liberated heat changes in the thermal sensor omnidistance monitoring microreactor (M);
D. uninstall process is contrary immobilization process, changes the controlled condition of enzyme immobilization, and enzyme breaks away from from function carrier;
E. feed current-carrying (C), get rid of the raffinate in microreactor (M) and the stream, carry out next test.
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| CN101614687B (en) * | 2009-03-06 | 2011-05-25 | 青岛大学 | Integrated calorimetric biosensor for enrichment and detection |
| CN103235006B (en) * | 2013-04-07 | 2015-02-25 | 青岛大学 | Calorimetric detection apparatus for acetone in breathing gas |
| CN110045764B (en) * | 2019-04-10 | 2021-04-20 | 万华化学集团股份有限公司 | Reactor temperature automatic control method based on feedforward-reaction mechanism model |
| JP2022067884A (en) * | 2020-10-21 | 2022-05-09 | 株式会社アドバンテスト | Biosensor and reactor for biosensor, and method for manufacturing reactor |
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| CN2519909Y (en) * | 2001-09-26 | 2002-11-06 | 上海理工大学 | Heat-measuring type biosensor for detecting residul pesticide |
| WO2007010379A2 (en) * | 2005-07-22 | 2007-01-25 | University Of Basel | Calorimetric assessment of microorganisms and use thereof |
| CN101071113A (en) * | 2007-06-12 | 2007-11-14 | 上海理工大学 | Reaction chamber and detecting chamber device for calorimetric biosensor |
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|---|---|---|---|---|
| CN2519909Y (en) * | 2001-09-26 | 2002-11-06 | 上海理工大学 | Heat-measuring type biosensor for detecting residul pesticide |
| WO2007010379A2 (en) * | 2005-07-22 | 2007-01-25 | University Of Basel | Calorimetric assessment of microorganisms and use thereof |
| CN101071113A (en) * | 2007-06-12 | 2007-11-14 | 上海理工大学 | Reaction chamber and detecting chamber device for calorimetric biosensor |
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