CN104267082A - Method for rapidly measuring BOD (biochemical oxygen demand) by collagenous fiber immobilized escherichia coli serving as biosensor recognition element - Google Patents
Method for rapidly measuring BOD (biochemical oxygen demand) by collagenous fiber immobilized escherichia coli serving as biosensor recognition element Download PDFInfo
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- CN104267082A CN104267082A CN201410537863.2A CN201410537863A CN104267082A CN 104267082 A CN104267082 A CN 104267082A CN 201410537863 A CN201410537863 A CN 201410537863A CN 104267082 A CN104267082 A CN 104267082A
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Abstract
The invention discloses a method for rapidly measuring BOD (biochemical oxygen demand) by collagenous fiber immobilized escherichia coli serving as a biosensor recognition element, and belongs to the field of biosensors. The method comprises a method for preparing a collagenous fiber immobilized escherichia coli biological recognition element by selecting collagenous fiber immobilized escherichia coli as a biological recognition element, a method for rapidly measuring the BOD by collagenous fiber immobilized escherichia coli serving as the biological recognition element and a storage and activation method of the biological recognition element. According to the invention, one biological recognition element used for a microbial sensor is provided and has the advantages of good mass transfer performance, high stability, good microbial activity and the like, by means of the good mass transfer performance of the biological recognition element, the fixed quantity of microorganisms in the sensor can be properly increased, the biological membrane permeability is not influenced, and accordingly, the sensor has a wider linear response range and higher sensitivity; the linear response range of the sensor to a GGA standard liquid is 50-600 mg/L of BOD, the linearly correlation coefficient is larger than 0.999, the response time is 3-15 min, a microbial membrane can be continuously used for more than 200 times, the immobilized microorganisms which are stored for six months still maintain good performance after activation.
Description
Technical field
The invention belongs to field of biosensors, provide the preparation for the collagenous fibres immobilization Escherichia coli biological identification element of biological sensor of oxygen amount needed biochemically and using method thereof.
Background technology
Biochemical oxygen demand (Biochemical Oxygen Demand, BOD) is an important indicator weighing water pollution degree, represents the dissolved oxygen content that Organic substance in water consumes when microbial biochemical action breaks.The classic method of domestic and international mensuration BOD value is biochemical oxygen demand inoculation dilution method (BOD on the 5th
5method), namely sample cultivates 5d under 20 ± 1 DEG C of conditions, working sample cultivate before and after dissolved oxygen DO, both differences are biochemical oxygen demand on the 5th, but this method takes time and effort, poor reproducibility and unsuitable field monitoring.In recent years, microbiological sensor method because its minute is short, easy and simple to handle, be easy to the advantages such as field monitoring and be applied to the detection of BOD gradually.
BOD microbiological sensor is primarily of immobilized microorganism, chromacoder and signal output apparatus composition, and wherein the performance such as sensitivity, responding range of immobilized microorganism to sensor all has material impact.Common microbial fixed carrier has polyvinyl alcohol (PVA), active cross-linked resin, collosol and gel etc., but the most mass-transfer performance of these carriers and biocompatibility poor, make the problems such as less by its obtained sensor ubiquity response, measurement range is narrower, microorganism utilization factor is lower.
Collagenous fibres are a kind of natural macromolecular materials, and the modified collagenous fibres such as metallic ion zirconium (Zr), iron (Fe) have good mass-transfer performance, biocompatibility, thermal stability, resistance to biodegradability and the advantage such as soda acid accommodation widely.It can be used as microbial fixed carrier to prepare microbiological sensor, fixation of microbe consumption can be increased largely and keep comparatively high microorganism utilization factor.
Escherichia coli be a kind ofly be easy to cultivate, respiratory activity is strong, the bacterium of wide accommodation, the modified collagenous fibres of metallic ion zirconium (Zr) have larger fixed amount to Escherichia coli and it can be made to keep more stable activity.This patent is fixed Escherichia coli using metallic ion zirconium (Zr) modified collagen fiber (ZICF) for carrier and is prepared BOD microbiological sensor as biological identification element, the mass-transfer performance good based on ZICF and biocompatibility, the lifting of the present invention to the performance such as transducer sensitivity, measurement range has greater significance.
Summary of the invention
The object of this invention is to provide a kind of method using collagenous fibres immobilization Escherichia coli as biological identification element Fast Measurement BOD, it is characterized in that, the method comprises selects collagenous fibres immobilization Escherichia coli as biological identification element, prepare the method for collagenous fibres immobilization Escherichia coli biological identification element, utilize collagenous fibres immobilization Escherichia coli as the rapid assay methods of biological identification element to BOD, and the activation method of the colibacillary store method of this immobilization and this microbe-sensitive film.
Described collagenous fibres immobilization Escherichia coli are as follows as the reason of biological identification element:
1) collagenous fibres are as a kind of natural macromolecular material, have that good, the resistance to aeration intensity of permeability is high, good biocompatibility, with low cost, the advantage such as good mechanical property, soda acid wide accommodation;
2) collagenous fibres after metal ion-modified, on the basis keeping the original premium properties of collagenous fibres, have had more for thermotolerance well and resistance to biodegradability;
3) mass-transfer performance that ZICF is good makes fixation of microbe consumption in sensor suitably to increase, thus widens the linear response range of sensor, promotes the sensitivity of sensor;
4) collagenous fibres are fixed Escherichia coli and are had the advantages that fixed amount is large, microbial activity is high, and these features can make sensor obtain wider linear response range;
5) collagenous fibres immobilization Escherichia coli have long service life as biological identification element, and recognition component is preserved and the simple feature of activation method.
The fibre immobilized carrier of described metallic ion zirconium (Zr) modified collagen (ZICF) prepares (patent of invention: collagen fiber supported metal ion adsorption material and its production and use, the patent No.: 200410040145.0) by existing method.
The colibacillary preparation method of described immobilization: take dry weight 0.5g ZICF and be placed in sterilized water and soak 5-12h.Escherichia coli are activation culture 18-24h in nutrient broth medium, collecting cell after centrifugal, stroke-physiological saline solution are cleaned, and is added obtained bacteria suspension in stroke-physiological saline solution, and regulates bacteria suspension concentration to be 10
-7-10
10cfu/mL.Add in above-mentioned bacteria suspension after ZICF is filtered, detect to without free bacterium with stroke-physiological saline solution cleaning after 37 DEG C of concussion absorption 5h, obtain collagenous fibres immobilization Escherichia coli.
The preparation method of described biological identification element: each weighing weight in wet base is the collagenous fibres immobilization Escherichia coli of 0.10-0.30g, immobilization Escherichia coli are placed between two-layer 200 order nylon wires, make it to form one deck immobilized microorganism film, during use, this film is close on the semi-permeable diaphragm before dissolved oxygen electrode head, and fixes with electrode cap.
Describedly utilize collagenous fibres immobilization Escherichia coli as the rapid assay methods of biological identification element to BOD: during measurement, the measuring cup filling 0.07mol/L sodium hydrogen phosphate, potassium phosphate buffer (PBS damping fluid) will be placed in the colibacillary dissolved oxygen electrode head of immobilization, carry out aeration with the aeration intensity of 24L/h to damping fluid, measuring tempeature is 36-37 DEG C, pH is 6-7; Add after oxygen to be dissolved (DO) value stabilization containing organic sample, immobilized microorganism decomposing organic matter causes DO value in water to drop to another stationary value, tries to achieve the difference of the twice DO value in front and back, by the BOD value of typical curve calculation sample.
The colibacillary store method of described immobilization: collagenous fibres immobilization Escherichia coli are in the PBS damping fluid of 6-7 at pH, 4 DEG C of condition lower seals are preserved.Collagenous fibres immobilization Escherichia coli still have excellent activity after this law preserves 6 months.
The colibacillary activation method of described immobilization: the immobilization Escherichia coli be stored under 4 DEG C of conditions are placed 24h under 36-37 DEG C of condition in PBS damping fluid; To the repeated multiple times mensuration of certain density glucose glutamic acid (GGA) solution after composition BOD sensor, continuous 5 relative deviations of DO drop-out value are less than 5%, namely complete the colibacillary activation of immobilization.
Accompanying drawing explanation
Fig. 1 is BOD
5for the more typical response curve of GGA solution of 0-600mg/L scope.
Embodiment
Example 1: using the collagenous fibres immobilization Escherichia coli of the present invention's proposition as biological identification element, to the mensuration of the GGA solution of variable concentrations.
Configure BOD respectively
5concentration is 0,10,20,50,100,150,200,300,400,500,600, the GGA solution of 700mg/L, to be combined with dissolved oxygen electrode as biological identification element with collagenous fibres immobilization Escherichia coli and to form BOD microbiological sensor, measure the GGA solution of above-mentioned concentration.Condition determination is: temperature 36 DEG C, pH=6, aeration intensity 24h/L, and magnetic agitation rotor speed makes dissolved oxygen displayed value at 4-8mg/L.
Measurement result shows, has good linear relationship, at BOD between DO drop-out value and sample BOD concentration
5within the scope of concentration 0-600mg/L, linearly dependent coefficient is greater than 0.999, has fabulous measuring accuracy.
Example 2: the collagenous fibres immobilization Escherichia coli proposed using the present invention as biological identification element, 36 DEG C, pH is 6, to BOD under aeration intensity 24L/h condition
5value is 50mg/L, 100mg/L, the GGA solution of 200 mg/L measures, and the result reappearance of determination sensor, result is as shown in table 1.
As shown in table 1 is sensor reappearance measurement result
Known by data analysis in his-and-hers watches, sensor has good reappearance, maximum relative deviation 3.8%, meets the accuracy requirement of BOD Quick Measurement.
Above example is only be described embodiments of the present invention; and not scope of the present invention is limited; for a person skilled in the art; can improve above-mentioned explanation or be out of shape, but all these improve or are out of shape the protection domain that all should fall into claim of the present invention and determine.
Claims (8)
1. one kind using collagenous fibres immobilization Escherichia coli as the method for biological identification element Fast Measurement BOD, it is characterized in that, the method comprise select collagenous fibres immobilizations Escherichia coli as biological identification element, prepare collagenous fibres immobilization Escherichia coli biological identification element technique and utilize collagenous fibres immobilization Escherichia coli as the Fast Measurement of biological identification element to BOD, and the activation method of the colibacillary store method of this immobilization and this microorganism recognition component.
2. described in claim 1 using collagenous fibres immobilization Escherichia coli as the method for biological identification element Fast Measurement BOD, it is characterized in that, described microbial fixed carrier is the modified collagenous fibres (ZICF) of metallic ion zirconium (Zr).
3. described in claim 1 using collagenous fibres immobilization Escherichia coli as the method for biological identification element Fast Measurement BOD, it is characterized in that, described immobilized microorganism kind is Escherichia coli (colon bacillus;
escherichia coli).
4. the colibacillary preparation of collagenous fibres immobilization described in claim 1: take dry weight 0.5g ZICF and be placed in sterilized water and soak 5-12h; Escherichia coli are activation culture 18-24h in nutrient broth medium, collecting cell obtained bacteria suspension in stroke-physiological saline solution after centrifugal, stroke-physiological saline solution are cleaned, and regulates bacteria suspension concentration to be 10
7-10
10cfu/mL; Add in above-mentioned bacteria suspension after ZICF is filtered, detect to without free bacterium with stroke-physiological saline solution cleaning after 37 DEG C of concussion absorption 5h, obtain collagenous fibres immobilization Escherichia coli.
5. the preparation of collagenous fibres immobilization Escherichia coli biological identification element described in claim 1: each weighing weight in wet base is the collagenous fibres immobilization Escherichia coli of 0.10-0.30g, immobilization Escherichia coli are placed between two-layer 200 order nylon wires, make it to form one deck immobilized microorganism film, during use, this film is close on the semi-permeable diaphragm before dissolved oxygen electrode head.
6. utilize collagenous fibres immobilization Escherichia coli as the Fast Measurement of bio-sensitive film to BOD described in claim 1: during measurement, to be placed in the colibacillary dissolved oxygen electrode head of immobilization and fill 0.07mol/L sodium hydrogen phosphate, in the measuring cup of potassium phosphate buffer (PBS damping fluid), with the aeration intensity of 24L/h, aeration is carried out to damping fluid by limited aeration pipe, measuring tempeature is 37 DEG C, pH is 7, add after oxygen to be dissolved (DO) value stabilization containing organic sample solution, immobilized microorganism decomposing organic matter causes DO value in water to drop to another stationary value, try to achieve the difference of the twice DO value in front and back, by typical curve calculate survey the BOD value of water sample.
7. the colibacillary store method of collagenous fibres immobilization according to claim 1, is characterized in that, the colibacillary preservation condition of collagenous fibres immobilization is: 0.07mol/L PBS damping fluid, pH=6-7, temperature 4 DEG C.
8. the colibacillary activation method of collagenous fibres immobilization according to claim 1, it is characterized in that, the colibacillary activation method of collagenous fibres immobilization is: the immobilization Escherichia coli be stored under 4 DEG C of conditions are placed 24h under 36-37 DEG C of condition in original solution; Repeatedly measure secondary to glucose glutamic acid (GGA) solution that concentration is 50-200 mg/L after composition BOD sensor, continuous 5 relative deviations of DO drop-out value are less than 5%, namely complete the colibacillary activation of immobilization.
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| CN202083662U (en) * | 2011-05-06 | 2011-12-21 | 湖北博凡生物科技有限公司 | Online tester for biochemical oxygen demand BOD |
| CN103969303A (en) * | 2014-05-15 | 2014-08-06 | 四川农业大学 | Method for rapidly determining BOD (Biochemical Oxygen Demand) by taking collagenous fiber loaded saccharomyces cerevisiae as biosensor identification element |
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2014
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Patent Citations (6)
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| JP2010169598A (en) * | 2009-01-23 | 2010-08-05 | Nihon Suido Consultants Co Ltd | Water quality estimating apparatus and water quality estimating method |
| CN101639472A (en) * | 2009-08-31 | 2010-02-03 | 清华大学 | Reactor type BOD rapid measuring method and measuring instrument |
| KR20110099661A (en) * | 2010-03-02 | 2011-09-08 | 주식회사 과학기술분석센타 | Catalyst using immobilization of microorganism of working electrode for bod measuring sensor and manufacturing process thereof |
| CN102262123A (en) * | 2010-05-27 | 2011-11-30 | 江苏同和涂装机械有限公司 | Quick biochemical oxygen demand (BOD) measuring instrument for reactor type biosensor |
| CN202083662U (en) * | 2011-05-06 | 2011-12-21 | 湖北博凡生物科技有限公司 | Online tester for biochemical oxygen demand BOD |
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Application publication date: 20150107 |