CN101614687B - Integrated calorimetric biosensor for enrichment and detection - Google Patents
Integrated calorimetric biosensor for enrichment and detection Download PDFInfo
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- CN101614687B CN101614687B CN2009101266779A CN200910126677A CN101614687B CN 101614687 B CN101614687 B CN 101614687B CN 2009101266779 A CN2009101266779 A CN 2009101266779A CN 200910126677 A CN200910126677 A CN 200910126677A CN 101614687 B CN101614687 B CN 101614687B
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Abstract
The invention relates to an integrated calorimetric biosensor for enrichment and detection. The biosensor is characterized in that a system is a flow injection analysis system consisting of a constant flow pump, a filling valve, a thermostat, a micro-reactor, a reference micro-reactor, a detector and flow path. The detection flow is characterized in that the detection is based on flow injection analysis method, and enrichment and detection reactions occur on an enrichment carrier. The integrated thermal biosensor for enrichment and detection has the advantages of integrating separation, enrichment, sensitization and detection, omitting conventional steps such as elution, enhancing detection sensibility and selectivity, and analytical automaticity and precision, realizing high-sensibility and high-selectivity detection and analysis, and greatly enhancing inherent advantages and applications of the calorimetric biosensor.
Description
Technical field
Patent of the present invention relates to a kind of calorimetric biosensor that collects enrichment and be detected on one, can be applied in scene, on-line monitoring and the trace analysis etc. of processes such as food, medicine, biochemistry, environment and chemical industry.
Background technology
The reach of science require can the be enough lower consumption of analysis field, easier method and apparatus, faster analysis speed provide in real time, accurately, comprehensive information, the accuracy that its obvious development trend is analytical equipment, integrated and robotization.
Calorimetric biosensor is the biology sensor of analyzing by the thermal change of measuring the biochemical reaction process generation.With respect to method for biosensor such as galvanochemistry and optics, calorimetric biosensor is not subjected to the interference of build-in attributes such as color, muddiness and galvanochemistry, has advantage, is particularly useful for the untreated actual samples of scene, on-line monitoring and analysis.In addition, the detection principle of calorimetric method is simple, reliable, helps the exploitation of microsystem.
Conventional amounts hot type biosensor application is in trace analysis, its sensitivity and selectivity are not near as people's will, its specificity can be by improving in conjunction with specific biosensor, but its sensitivity can not improve by the sensitivity that improves the calorimetric transducer simply, because amplify when this tends to cause undesired signal, and cause the instability of output signal.Be only basic road by suitable enrichment to sample to be tested.Enrichment is to get rid of the important means of disturbing, reducing detection limit, improve analysis precision, can improve the intensity of useful signal greatly, and improve its specificity relatively.At present, calorimetric biosensor in conjunction with enrichment function does not appear in the newspapers and production marketing, and the flow process that traditional on-line preconcentration method adopts is after finishing component separation to be measured, enrichment on the solid phase carrier, with eluent with the sample fraction wash-out, then eluent is sent into detecting device and detect, complex operation, length consuming time have increased the complicacy and the uncertainty that detect simultaneously, also increase the propagation of error process, restricted their application.
At above-mentioned background, the present invention relates to a kind of enrichment, detect integrated calorimetric biosensor, collect separation, enrichment, enhanced sensitivity, be detected on one, and omit traditional steps such as wash-out, the combined high precision thermal sensor can be realized the detection and the analysis of high sensitivity and high selectivity.
Summary of the invention
The biology sensor that the purpose of this invention is to provide a kind of high-adaptability, specificity and sensitivity, it adopts simply, reliable calorimetric method, and utilize enrichment to strengthen sensitivity and the specificity that detects, the integrated further intermediate link that reduced of enrichment, detection has improved automaticity and the precision analyzed.
Technical scheme of the present invention is to realize like this, a kind of enrichment, detection integrated calorimetric biosensor system that adopts flowing injecting analysis technology as robotization Continuous Flow Analysis method, comprise constant flow pump, fillup valve, thermostat, reactor, detecting device and stream etc., be characterized in the power that constant flow pump provides the constant flow of sample liquid, current-carrying and reagent to carry; Fillup valve is realized sample liquid, current-carrying and combination of agents and control; Thermostat makes sample liquid, current-carrying and the combination of agents constant temperature of reaction that reaches setting before entering reactor; Reactor is made up of the detection microreactor and the reference microreactor of twin symmetrical structure, the two inner enrichment carrier (resin, activated charcoal etc.) of filling; Obtain detecting the reaction heat signal difference of microreactor and reference microreactor as the thermal sensor of detecting device, after signal Processing and display unit or Computer Processing, show; Reactor and thermal sensor all are placed in the adiabatic environment.Enrichment, detection integrated calorimetric biosensor adopt following testing process: the first step, prepare and stabilization process, naturally pile up in detecting microreactor and reference microreactor and fill the enrichment carrier, total system feeds current-carrying and to system stability, obtains steady baseline; Second step, enrichment process, sample liquid and current-carrying potpourri constant flow flow through the detection microreactor, and current-carrying flows through the reference microreactor with flow simultaneously, and process continues setting-up time as enrichment time, and at this moment, tested component is enriched on the enrichment carrier; The 3rd step, reaction and testing process, agent combination and the same flow of current-carrying potpourri, same time, flow through and detect microreactor and reference microreactor simultaneously, and react on the enrichment carrier therein, various reaction liberated heats change in the omnidistance monitoring of thermal sensor two reactors, and its signal changes the amount that has reflected tested component; In the 4th step, the raffinate in eliminating detection microreactor and the reference microreactor in enrichment carrier and the stream carries out next sample 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 this calorimetric biosensor collection separation, enrichment, enhanced sensitivity, be detected on one, enrichment has effectively strengthened sensitivity, enrichment, the traditional steps such as wash-out of the integrated omission of detection, reduce the propagation of error process simultaneously, further improved the sensitivity and the selectivity that detect.And its separation, enrichment, mensuration are once finished, and have reduced manual intervention, improve automaticity and the precision analyzed, and this will strengthen the Inherent advantage and the application of calorimetric biosensor greatly.
Description of drawings
Fig. 1 is enrichment, detection integrated calorimetric biosensor system schematic
Fig. 2 is the process flow diagram of heavy metal embodiment in the testing environment water sample
Fig. 3 is for detecting the process flow diagram of organophosphorus pesticide embodiment in the fruits and vegetables
Embodiment
The present invention does an explanation in conjunction with the accompanying drawings, enrichment as shown in Figure 1, detection integrated calorimetric biosensor system.Wherein C is a current-carrying; S is a sample; R is a reagent; P is a constant flow pump; V is a fillup valve; T is a thermostat; M is for detecting microreactor; MC is the reference 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, sample liquid S and reagent R, 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, sample liquid S, reagent R, finish regularly, quantitatively inject, realize operations such as programmed control and auto injection; Thermostat T detects microreactor M, the current-carrying C of reference microreactor MC to entering, constant temperature is carried out in the combination of sample liquid S, reagent R, can guarantee to be reflected at the reappearance under the optimum temperature condition; Detect microreactor M and reference microreactor MC and adopt twin symmetrical structure, non-specific heat interference can be effectively eliminated in being provided with of reference microreactor MC, detect the inner enrichment carrier (resin, activated charcoal etc.) of filling of microreactor M and reference microreactor MC, carry out enrichment and course of reaction on it, simultaneously the also surface area by augmenting response and mix intensified response efficient effectively; Obtain detecting the reaction heat signal difference of microreactor M and reference microreactor MC as the thermal sensor of detecting device D, and through signal Processing and display unit C﹠amp; D, or be connected with computer PC, carry out signal Processing, obtain the amount of tested component; Detect the adiabatic environment of microreactor M, reference microreactor MC and thermostat T outside, can effectively eliminate thermal loss, improve precision.Use the concrete enforcement testing process of above-mentioned enrichment, detection integral biological sensing system to be: the first step, prepare and stabilization process, in detecting microreactor M and reference microreactor MC, pile up naturally and fill the enrichment 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 of sample liquid S and current-carrying C and current-carrying C detect microreactor M and reference microreactor MC to flow through respectively with flow, and at this moment, tested component is enriched on the carrier, is enrichment process, continues setting-up time as enrichment time; The 3rd step, reaction and testing process, switch fillup valve V, to reagent R and current-carrying C combination, the same flow of its potpourri, same time, flow through and detect microreactor M and reference microreactor MC simultaneously, and react on the enrichment carrier therein, various reaction liberated heats change in the omnidistance monitoring of thermal sensor two reactors, and its signal changes the amount that has reflected tested component; The 4th step, switch fillup valve V, current-carrying C washes away system's stream, and the raffinate among eliminating detection reaction device M and the reference microreactor MC in enrichment carrier and the stream carries out next sample test.
Different application field according to enrichment, detection integrated calorimetric biosensor provides two test cases.
Test case 1:
Use described enrichment, detect the heavy metal ion in the integrated calorimetric biosensor testing environment water sample, testing process as shown in Figure 2.Adopt enzyme inhibition method to carry out, heavy metal ion can suppress the activity of urase, during environmental sample heavy metal free ion, and urea hydrolysis under the effect of urase, when heavy metal ion, the activity of urase is suppressed, and the enzymatic reaction degree descends.What of heavy metal ion amount can be reflected the fierce degree of enzyme reaction in the environmental sample, and then can know in the variation by reaction heat.Detect microreactor and reference microreactor size and be Φ 5mm * 30mm, the enrichment carrier is particle diameter 30～35 purpose D401 chelating resins, use the urase of 2mM, 0.5M urea as substrate, PH7.0, the phosphate buffer of 0.1M, enrichment time is 5min, the thermostat set temperature is 35 ℃, and flow velocity is 1ml/min.Obtain, the detectability of Cu2+ ion and Hg2+ ion can reach 0.005mg/L in the environmental water sample.
Test case 2:
Use described enrichment, detect the organophosphorus pesticide in the integrated calorimetric biosensor detection fruits and vegetables, testing process as shown in Figure 3.Adopt enzyme inhibition method to carry out, residues of pesticides can suppress the activity of cholinesterase, during the environmental sample non agricultural chemical residuum, and BuCh hydrolysis under the effect of cholinesterase, when residues of pesticides, the activity of cholinesterase is suppressed, and the enzymatic reaction degree descends.What of organophosphorus pesticide residual quantity can be reflected in the inhibition degree of agricultural chemicals to cholinesterase activity in the fruits and vegetables, and then can know in the variation by reaction heat.Detect microreactor and reference microreactor size and be Φ 5mm * 30mm, the enrichment carrier uses particle diameter 30～35 purpose activated charcoals, and the BuCh that uses the cholinesterase of 2mM and 20mM is as substrate, PH7.0, the phosphate buffer of 0.1M.The thermostat set temperature is 30 ℃, and enrichment time is 5min, and flow velocity is 2ml/min.Detection obtains, and the detectability of organophosphorus pesticide reaches 0.01mg/L in the fruits and vegetables sample.
Claims (4)
1. an enrichment, detect integrated calorimetric biosensor, the Flow Injection Analysis system that it is made up of constant flow pump, fillup valve, thermostat, reactor, detecting device, signal Processing and display unit, computing machine and stream, it is characterized in that the power that constant flow pump (P) provides the constant flow of sample liquid (S), current-carrying (C) and reagent to carry; Fillup valve (V) is realized sample liquid (S), current-carrying (C) and combination of agents and control; Thermostat (T) makes sample liquid (S), current-carrying (C) and the combination of agents constant temperature of reaction that reaches setting before entering reactor; Reactor is made up of the detection microreactor (M) and the reference microreactor (MC) of twin symmetrical structure, the two inner enrichment carrier of filling; Obtain detecting the reaction heat signal difference of microreactor (M) and reference microreactor (MC) as the thermal sensor of detecting device (D), through signal Processing and display unit (C﹠amp; D) or after computing machine (PC) processing show.
2. enrichment according to claim 1, detection integrated calorimetric biosensor is characterized in that described enrichment carrier is resin or activated charcoal.
3. enrichment according to claim 1, detection integrated calorimetric biosensor is characterized in that described reactor and thermal sensor all are placed in the adiabatic environment.
4. application rights requires the testing process of 1 described enrichment, detection integrated calorimetric biosensor, it is characterized in that:
A. pile up naturally in detecting microreactor (M) and reference microreactor (MC) and fill the enrichment carrier, total system feeds current-carrying (C) and to system stability, obtains steady baseline;
B. sample liquid (S) flows through with current-carrying (C) potpourri constant flow and detects microreactor (M), and current-carrying (C) flows through reference microreactor (MC) with flow simultaneously, and process continues setting-up time as enrichment time, and at this moment, tested component is enriched on the enrichment carrier;
C. the same flow of reagent and current-carrying (C) potpourri, same time, flow through and detect microreactor (M) and reference microreactor (MC) simultaneously, and react on the enrichment carrier therein, various reaction liberated heats change in the omnidistance monitoring of thermal sensor two reactors, and its signal changes the amount that has reflected tested component;
D. get rid of the raffinate that detects in microreactor (M) and middle enrichment carrier of reference microreactor (MC) and the stream, carry out next sample test.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102946988B (en) * | 2010-06-09 | 2015-04-15 | 英派尔科技开发有限公司 | Adjustable pressure microreactor |
| CN102661970B (en) * | 2012-05-02 | 2014-03-26 | 青岛大学 | Neural-network-based enrichment process thermal monitoring method |
| CN106198296A (en) * | 2015-06-01 | 2016-12-07 | 中国人民解放军63971部队 | A kind of DMMP concentration determination method |
| CN107064208B (en) * | 2017-02-04 | 2019-04-16 | 青岛大学 | A kind of universal calorimetric biosensor |
| CN107421982B (en) * | 2017-02-04 | 2019-04-16 | 青岛大学 | A kind of universal calorimetric biosensor |
| CN107064216B (en) * | 2017-04-19 | 2019-07-02 | 哈尔滨工业大学 | A kind of portable COD detection device based on microfluidic chip technology |
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| EP0532480A1 (en) * | 1991-09-13 | 1993-03-17 | Consiglio Nazionale Delle Ricerche | Calorimeter for time/temperature measurements of thermosetting resins (thermosets) |
| 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 |
| CN101526490A (en) * | 2009-03-06 | 2009-09-09 | 青岛大学 | Thermal biosensor based on reversible immobilized enzyme |
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Patent Citations (5)
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|---|---|---|---|---|
| EP0532480A1 (en) * | 1991-09-13 | 1993-03-17 | Consiglio Nazionale Delle Ricerche | Calorimeter for time/temperature measurements of thermosetting resins (thermosets) |
| 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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