CN1584579A - Method and apparatus for detecting microbe by piezoelectric quartz crystal sensor - Google Patents
Method and apparatus for detecting microbe by piezoelectric quartz crystal sensor Download PDFInfo
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- CN1584579A CN1584579A CN 200410023232 CN200410023232A CN1584579A CN 1584579 A CN1584579 A CN 1584579A CN 200410023232 CN200410023232 CN 200410023232 CN 200410023232 A CN200410023232 A CN 200410023232A CN 1584579 A CN1584579 A CN 1584579A
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- microorganism
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Abstract
A method and device for detecting microbe with piezoelectric quarts crystal transducer includes leading microbial metablin CO2 from culture cell to basic solution detection cell, measuring reaction variation with transducer and counting it out with frequency counter, obtaining linear relation between FDT and microbial concentration for carrying out microbial quantitative and qualitative detection by measuring response curve of microbe in different concentration.
Description
Technical field
The invention belongs to the microbiological sensor field, particularly a kind of piezoelectric quartz crystal sensor that utilizes detects method of microorganism and device.
Background technology
Traditional microbioassay method has cell number scale, turbidimetry, dry weight method and mycelia length measurment method etc., and there are shortcomings such as operation is loaded down with trivial details, the mensuration cycle is long in these methods, can not satisfy in the practical application needs to the microorganism fast measuring.Occurred many calorifics, optics, galvanochemistry and biochemical properties of microorganism of utilizing in recent years and carried out method for measuring, as abiotic electrochemical method, bio-electrochemical process and cell component determination method etc.
The microbioassay method all has important effect in many fields such as Food Inspection, clinical medicine and environmental monitorings.Nowadays, mortality ratio lungy reaches a record high, and tuberculosis has become the leading killer and the maximum cause of death in the infectious disease.The appearance of particularly anti-multiple medicines bacterial strain brings new challenge for prevention lungy.Therefore, the method that detects tubercle bacillus is fast and efficiently demanded urgently coming out.Chang Yong certain methods all exists some drawbacks now.It is the most traditional method of diagnosis of tuberculosis that bacteriology is cultivated, and also is to think reliable analytical method at present.The accuracy rate height is its a great advantage, but this kind method is very consuming time, does not satisfy the requirement of quick diagnosis.Also be used for the detection of tubercle bacillus based on the biochemical method of Enzyme Linked Immunoadsorbent Assay, this method selectivity height, highly sensitive, low cost, but its complex operation, it is big to produce false-positive possibility.DNA and rna probe and the application of reacting in conjunction with the polymerase chain amplification also can cause a large amount of false positive results, and experimental provision cost height, are difficult in developing country and promote.Now larger hospital's maximum fast detecting large-scale instrument lungy of utilization comprises Bactec 460 systems and Bactec MGIT 960 systems, and there are shortcomings such as radiocontamination, instrumentation and difficult in maintenance, instrument costliness in Bactec 460 systems.Although Bactec MGIT 960 systems have realized robotization substantially, but all reagent dependence on import, and the cost of instrument own is just high, therefore its popularization is subjected to the restriction of economic condition, and be unable to reach in tuberculosis incidence high impoverished nation or developing country, and its detection time is longer relatively, and the general positive goes out the fast person of result and takes about 8-10 days, slow person took about one month, and feminine gender also took about 40 days.In our research group, designed piezoelectric immunosensor based on the mass-basis response of PQC, it is simple, easy to operate, but sensitive inadequately, and high difficult acquisition of antibody price; Another FDT piezoelectric sound wave impedance transducer also is used to detect tubercle bacillus, response is fast, highly sensitive, but need the electricity of control nutrient culture media to lead it is worked in the sensitivity interval of instrument, and often cause because nutritional deficiency and bacteria growing inhibiting or do not have a growth phenomenon at all.
Summary of the invention
The object of the present invention is to provide a kind of piezoelectric quartz crystal sensor that utilizes simple in structure, that cost is low, highly sensitive to detect method of microorganism and device.
For achieving the above object, a kind of piezoelectric quartz crystal sensor that utilizes of the present invention detects method of microorganism, may further comprise the steps:
1) place culture pond to cultivate microorganism to be detected;
2) aqueous slkali is injected the detection cell that is embedded with pair of electrodes;
3) with the CO of microorganisms in the culture pond
2Introduce detection cell;
4) electricity of the online detection detection cell of piezoelectric quartz crystal sensor is led variation, and exports with the frequency oscillator signal form;
5) frequency of frequency counter survey frequency oscillator signal, and the frequency counting result delivered to computing machine, after machine is handled as calculated, the output testing result.
Above-mentioned steps 2) aqueous slkali described in is Ba (OH)
2Solution or Ca (OH)
2Solution.
The microorganism detection device of realizing said method comprises detection cell, culture pond, calibration cell, pair of electrodes, quartz oscillator, frequency counter, computing machine etc., described detection cell is the sealing pond that fills aqueous slkali, and place on the calibration cell, be provided with culture pond in the detection cell, pair of electrodes is inserted in the detection cell, the other end of electrode connects with quartz oscillator, and the output of quartz oscillator connects the input end of frequency counter, and the output terminal of frequency counter connects with computing machine.
Advantage of the present invention: 1) culture pond and the detection cell with microorganism is provided with separately, and this structure has overcome microbial nutrition and required to require inconsistent contradiction with the detection sensitivity.2) with the CO of culture pond microorganisms
2Be incorporated in the detection cell that fills alkali lye CO
2Generate carbonate deposition, excessive CO with alkaline reaction
2React with carbonate deposition again, make the electric artificial delivery of detection cell give birth to obvious variation, piezoelectric quartz crystal sensor is led the electricity of detection cell the variation that becomes the piezoelectric quartz crystal sensor frequency, by carrying out qualitative or detection by quantitative to microorganism to the piezoelectric quartz crystal sensor frequency counting, this detection method is highly sensitive, reliably, and can shorten detection time of microorganism greatly, measure tubercle bacillus with method of the present invention, measure once and only need about 82 hours, shorten the minute of tubercle bacillus greatly, can be widely used in clinical diagnosis lungy.
The present invention is further illustrated below in conjunction with the drawings and specific embodiments.
Description of drawings
Fig. 1 is a structural drawing of the present invention.
Fig. 2 is for detecting the typical response curve of tubercle bacillus with the present invention.
Real-time frequency displacement response curve when Fig. 3 exists for the variable concentrations tubercle bacillus.
Fig. 4 is the logarithm of the concentration of tubercle bacillus in the sample and the relation curve between the FDT.
Embodiment
Referring to Fig. 1, pick-up unit of the present invention comprises culture pond 4, detection cell 2, calibration cell 3, quartz oscillator 7, frequency counter 8, computing machine 9, and culture pond 4 is nested together with detection cell 2 and places on the calibration cell 3.Wherein 1 for rubber plug be the sealing-plug of detection cell 2,5 is bacterium, calibration cell 3 is used for controlling the temperature of culture pond 4 and detection cell 2, a pair of stainless steel electrode or gold-plated electrode 6 through Passivation Treatment inserts in the detection cell 2, the other end of pair of electrodes 6 connects with quartz oscillator 7, the output of quartz oscillator 7 connects the input of frequency counter 8, and the output of frequency counter 8 connects computing machine 9.Wherein culture pond 4, detection cell 2, calibration cell 3, electrode 6, quartz oscillator 7 are formed piezoelectric quartz crystal sensor.
When carrying out microorganism detection, place the nutrient culture media in the culture pond 4 to cultivate microorganism, because culture pond 4 is in the airtight detection cell 2 CO of microbial metabolism with apparatus of the present invention
2Introducing fills Ba (OH)
2In the detection cell 2 of solution.CO
2With Ba (OH)
2Reaction generates BaCO
3And Ba (HCO
3)
2Thereby the rapid variation that electricity is led before and after inducing reaction, thereby the electricity between the electrode 6 is led also along with variation, quartz oscillator 7 responds this variation delicately, and with the output of the form of frequency, count by frequency counter 8, deliver to again in the computing machine 9 and handle, thereby microorganism is carried out quantitative detecting analysis.
For accelerating CO in the culture pond
2Effusion, improve sensitivity, can in nutrient culture media, add minor N aHCO
3Solution.
Frequency-time curve of microorganism by measuring variable concentrations can obtain the relation of microorganism concn and frequency detecting time (FDT), thereby carry out quantitative test.The foundation that qualitative analysis is judged is: occur flex point on the frequency response curve, then be considered as bacterium.Ba in the detection cell (OH)
2Absorb CO
2, electricity is led and is changed greatly, and characteristic is arranged, and causes tangible electroresponse signal, than other method sensitivity, reliable.
Measure tubercle bacillus with method of the present invention, measure once and only need about 82 hours, can be widely used in clinical diagnosis lungy.
Fig. 2 is for detecting the typical response curve bacillus of tubercle bacillus with the present invention.Before the numeration, placed 12 hours earlier, eliminate the CO that is dissolved with in the nutrient culture media
2Influence.Because experimental implementation is to carry out in room temperature, and cultivate at 37 ℃.Thus, along with variation of temperature, a part of CO
2Overflow, by Ba (OH)
2Absorb.Along with the growth of tubercle bacillus, reaction is proceeded.Ba (OH)
2Change into BaCO earlier
3After become Ba (HCO again
3)
2, electricity is led also corresponding changing, and reduces afterwards earlier to raise.Utilize constructed sensor, the electricity that causes in the growth of bacillus tubercle process is led variation, just can be detected online, and show with frequency displacement-time response curve.Therefore,, do the graph of a relation of frequency displacement and FDT, just the tubercle bacillus that exists in the judgement sample quantitatively according to the frequency displacement response curve.
Among Fig. 2, the slight a bit drift of frequency about about 180Hz, belongs to noise during beginning.After a period of time, the B dot frequency begins obvious decline, and the pairing time of this point is called FDT, and frequency descended about tens hours continuously, reached a platform then.In the AB section, there is not tangible change in electric, this is that their physiologically active is suppressed because tubercle bacillus is kept in the refrigerator, and its recovery needs a period of time, the nutrient environment that simultaneous adaptation is new also needs the time, the CO that discharges
2Amount be very little, thereby SOLUTION PROPERTIES does not have much variations.In the BC stage, frequency change is very fast, is referred to as the activation stage.Tubercle bacillus is in exponential phase, constantly absorbs the nutritional labeling in the nutrient culture media, and well-grown discharges a large amount of CO
2, CO
2With BaCO
3React and change into Ba (HCO
3)
2, the electricity of solution is led and is increased sharply, and correspondingly frequency also descends comparatively fast.And the CD stage is in relative static conditions, owing to constantly drain some noxious materials in metabolic process, as free fatty acid, superoxide etc., in addition a little less than the surge capability of nutrient culture media, the pH value of solution 7.2 becomes about 5.0 when beginning.Because these unfavorable factors, the physiologically active of tubercle bacillus is suppressed once more, thereby is difficult to absorb nutriment and excretion metabolism product again.Because no CO
2Release, reaction terminating, the electricity of solution are led and are kept constant, and platform occurs.
Fig. 3 is the frequency displacement-time response curve of the tubercle bacillus of variable concentrations.1-0；2-3×10
2cells/ml；3-10
4cells/ml；4-10
5cells/ml；5-10
6cells/ml；6-10
7cells/ml。The concentration of tubercle bacillus is 3 * 10
2Cells/ml to 10
7Change between cells/ml.As can be seen from the figure, the concentration difference of tubercle bacillus, its FDT difference, concentration is big more, and its FDT is more little.Thereby according to the FDT value, we can obtain the concentration of tubercle bacillus.Fig. 4 has illustrated between the logarithm of the concentration of tubercle bacillus in the sample and the FDT and has had the better linearity relation.Regression curve can be used equation (1) expression.
LogC＝7.42742-3.8853×10
-5FDT (1)
In equation (1), C is the initial concentration of the tubercle bacillus that exists in the sample, and FDT is frequency pairing detection time when beginning to descend fast, and related coefficient is 0.9763 (n=5).3 * 10
2Cells/ml to 10
7In the cells/ml scope, in case obtain the FDT value, the initial concentration of tubercle bacillus can calculate according to equation (1) in the sample.Therefore, this equation can be used as the basis that tubercle bacillus carries out quantitative test.
Microorganism can discharge CO in growth course
2By measuring the CO that discharges in the metabolic process
2And being carried out detection by quantitative, microorganism reaches the series electrical utmost point piezoelectric quartz crystal sensor that its incubation is detected.
Claims (5)
1, a kind of piezoelectric quartz crystal sensor that utilizes detects method of microorganism, may further comprise the steps:
1) place culture pond to cultivate microorganism to be detected;
2) aqueous slkali is injected the detection cell that is embedded with pair of electrodes;
3) with the CO of microorganisms in the culture pond
2Introduce detection cell;
4) electricity of the online detection detection cell of piezoelectric quartz crystal sensor is led variation, and exports with the frequency oscillator signal form;
5) frequency of frequency counter survey frequency oscillator signal, and the frequency counting result delivered to computing machine, after machine is handled as calculated, the output testing result.
2, a kind of piezoelectric quartz crystal sensor that utilizes according to claim 1 detects method of microorganism, and it is characterized in that: described aqueous slkali is Ba (OH)
2Perhaps Ca (OH)
2Solution.
3, a kind of piezoelectric quartz crystal sensor that utilizes according to claim 1 detects method of microorganism, it is characterized in that: add minor N aHCO in the described culture pond
3Solution.
4, a kind of device that utilizes piezoelectric quartz crystal sensor to detect microorganism, comprise detection cell, culture pond, calibration cell, pair of electrodes, quartz oscillator, frequency counter, computing machine, described detection cell is the sealing pond that fills aqueous slkali, and place on the calibration cell, be provided with culture pond in the detection cell, pair of electrodes is inserted in the detection cell, the other end of electrode connects with quartz oscillator, the output of quartz oscillator connects the input end of frequency counter, and the output terminal of frequency counter connects with computing machine.
5, a kind of microorganism detection device that utilizes the series voltage quartz crystal sensor according to claim 4, it is characterized in that: described electrode is stainless steel electrode or gold-plated electrode.
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| CNB2004100232325A CN100338458C (en) | 2004-05-24 | 2004-05-24 | Method and apparatus for detecting microbe by piezoelectric quartz crystal sensor |
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| US5155019A (en) * | 1982-08-31 | 1992-10-13 | Becton, Dickinson And Company | Detection of the presence of biological activity in a sealed container utilizing infrared analysis of carbon dioxide and apparatus therefor |
| US5688660A (en) * | 1995-03-15 | 1997-11-18 | Colgate-Palmolive Company | Method for determining product biodegradability |
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| GB2319837A (en) * | 1996-11-28 | 1998-06-03 | Donald Wood | Measuring carbon dioxide emissions from soil |
| US6589761B1 (en) * | 1999-06-19 | 2003-07-08 | Marv Freadman | Method and apparatus for detecting bacteria |
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| US20040067592A1 (en) * | 2002-09-13 | 2004-04-08 | Rabasco John Joseph | Process for controlling microbial contamination of polymeric emulsions using carbon dioxide detection |
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