CN101974606A - High-throughput biosensor array chip, construction method thereof and application - Google Patents
High-throughput biosensor array chip, construction method thereof and application Download PDFInfo
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Abstract
The invention discloses a high-throughput biosensor array chip, the chip adopts an ELISA plate as a base, and the ELISA plate contains at least two biosensors which are constructed on the basis of the same host bacteria and can respond to the detection of different target substrates. The invention provides a construction method of the chip, which comprises the following steps: taking single colonies of the at least two biosensors which are constructed on the basis of the same host bacteria and can respond to the detection of the different target substrates, inoculating into a culture medium for culture, centrifugating, diluting, taking diluted cell suspension, arranging according to the design and adding into holes of the ELISA plate of the base. The invention further discloses a method for simultaneously detecting at least two target substrates in a sample, which comprises the following steps: taking the sample to be detected, preparing into water solution, adding into the chip of the invention, carrying out co-culture, and detecting the target substrates through an ELISA reader. The chip can detect at least two target substrates in the sample once with high throughput and has the advantages of convenience and rapidness.
Description
Technical field
The present invention relates to a kind of high-throughput biosensor array chip, and construction process, the present invention also relates to a kind of method that detects at least two kinds of target substrates in the sample simultaneously.
Background technology
Society's every profession and trade in production, transportation, use and disposal process, often be accompanied by the pollutent that has environmental risk in a large number residual, reveal and discharging.In recent years, the fast development along with China's industry, agricultural and urban construction is emerged in an endless stream by the environmental pollution accident that above-mentioned pollutent caused, and the number and the area in contaminated place enlarge year by year, and people's lives health has been caused significant threat.In these pollutents, be that the complicated persistence organic pollutant of representative has trace, extensively exists and characteristics such as of a great variety with agricultural chemicals and petrochemicals, proposed great challenge for environmental monitoring and risk assessment.Existing traditional chemical analytical procedure is in the face of complicated persistence organic pollutant, have complex operation (complicated pretreatment process), (several hours to several days time), apparatus expensive and the more high shortcoming of cost grown consuming time, and bio-toxicity and environmental ecology risk under can't effective evaluation combined pollution condition.These problems have seriously restricted the environmental assessment and the risk assessment of relevant contaminated site, limited the rapid evaluation of sudden environmental emergency accident, being novel detection technique, is that the development of the biological Fast Detection Technique of representative provides excellent opportunity with biosensor (biosensor) particularly.
Biosensor has obtained the degree of depth and has paid attention to and widespread use since 1962 are proposed by Clark and Lyons at aspects such as zymotechnique, environmental monitoring, food engineering, clinical medicine, military affairs and military medicines.The biosensor at initial stage mainly is the biosensor of making based on the development enzyme electrodes, but owing to zymin costs an arm and a leg, and less stable, therefore the application with enzyme biologic sensor is subjected to certain limitation.In the last few years, along with the continuous development of molecular biology and microbial immobilized technology, DNA transmitter (gene chip) and microorganism cells transmitter became the main flow of biosensor development gradually.Microbiological sensor can be estimated the biological effect (bioavailability) of target substance in the environment really, and the correlative study result shows that it has stability and reliability, compares the accuracy with height with the traditional analysis method.The microbiological sensor detection method is simple, can directly apply to water sample, or be used for complex sample (as soil or medical sample) through pre-treatment, and its characteristics low-cost and that respond fast, can satisfy the needs in this market, field, have both simple to operate and can realize advantage such as quantitative assay.In the environmental engineering Application Areas, biosensor can be used for the environmental evaluation of specific pollutants contaminated site and water body, and the pollution level that also can be used for specific pollutants contaminated site and water body detects.In the medical and health field, biosensor can be used for medical diagnosis on disease and medicine controlled releasing (for example, regulate Regular Insulin by the monitoring blood sugar concentration and inject level, in the hope of realizing the intelligent control to diabetes drug treatment).
Meanwhile, existing microbiological sensor research mainly is confined to make up the individual biosensor that produces response at the different chemical material, the property of there are differences, culture condition vary between different host bacterium, the construction process owing to adopt, and these biosensors are applied to the detection of a certain principal character substrate in the clean water body mostly.And in actual environment or human sample, often the number of chemical material exists simultaneously, and this has not only proposed high requirement to the specificity of biosensor, has also brought individual biosensor can't obtain the comprehensive semiochemical difficult problem of sample.
Do not have multiple biosensor at present under identical cultivation and inductive condition, can carry out the relevant report of the high-throughput biosensor array chip of specificly-response simultaneously to the plurality of target substrate.
Summary of the invention
The purpose of this invention is to provide a kind of high-throughput biosensor array chip, it can carry out the specific detection response at least two kinds of target substrates in the sample simultaneously under identical cultivation and inductive condition.
In order to achieve the above object, the invention provides a kind of high-throughput biosensor array chip, it is characterized in that: it is substrate that this chip adopts enzyme plate, comprises at least two kinds of biosensors to different target substrate detection response that make up based on same host bacterium on this enzyme plate.
High-throughput biosensor array chip of the present invention, it is further characterized in that: described same host bacterium is acinetobacter calcoaceticus (Acinetobacter sp.) ADP1.
High-throughput biosensor array chip of the present invention, it is further characterized in that: described biosensor is selected from: acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk, acinetobacter calcoaceticus (Acinetobacter sp..) ADPWH_BenM and acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA.
High-throughput biosensor array chip of the present invention, it is further characterized in that: described enzyme plate is 96 hole enzyme plates.
The invention provides a kind of construction process of high-throughput biosensor array chip, this method comprises: be taken to few two kinds of single colony inoculations based on the biosensor that detection responds to the different target substrate of same host bacterium structure and cultivate in substratum, centrifugal, dilution, get the cell suspending liquid of dilution, distribute according to design, join in the substrate enzyme plate hole.
The invention provides a kind of construction process of high-throughput biosensor array chip, it is further characterized in that: described same host bacterium is acinetobacter calcoaceticus (Acinetobacter sp.) ADP1.
The invention provides a kind of construction process of high-throughput biosensor array chip, it is further characterized in that: described biosensor is selected from: acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM and acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA.
The invention provides a kind of construction process of high-throughput biosensor array chip, it is further characterized in that: described substratum is the LB substratum; Described centrifugal carrying out 2 times, for the first time centrifugal after, abandoning supernatant, precipitation adds isopyknic sterilized water, it is centrifugal to carry out second time, abandoning supernatant, precipitation adding liquid mineral medium dilutes.
The invention provides a kind of construction process of high-throughput biosensor array chip, it is further characterized in that: described enzyme plate is 96 hole enzyme plates.
Further, the invention provides a kind of method that detects at least two kinds of target substrates in the sample simultaneously, this method comprises: get sample to be tested, make the aqueous solution, join in the high-throughput biosensor array chip of the present invention, cultivate altogether, detect target substrates by the microplate reader measurement.
The invention provides a kind of method that detects at least two kinds of target substrates in the sample simultaneously, it is further characterized in that: described same host bacterium is acinetobacter calcoaceticus (Acinetobacter sp.) ADP1.
The invention provides a kind of method that detects at least two kinds of target substrates in the sample simultaneously, it is further characterized in that: described biosensor is selected from: acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM and acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA.
The invention provides a kind of method that detects at least two kinds of target substrates in the sample simultaneously, it is further characterized in that: described enzyme plate is 96 hole enzyme plates.
The invention provides a kind of method that detects at least two kinds of target substrates in the sample simultaneously, it is further characterized in that: the temperature of cultivating is 20 ℃ to 37 ℃ altogether, and the frequency that microplate reader is measured is 2 to 15 minutes.
The invention provides a kind of method that detects at least two kinds of target substrates in the sample simultaneously, it is further characterized in that: the temperature of cultivating is 30 ℃ altogether, and the frequency that microplate reader is measured is 5 minutes.
Based on what same host bacterium made up the different target substrate is detected the biosensor that responds by integrated on chip, more help the same culture conditions of finding that this difference biosensor is had, realize that disposable high-throughput carries out the purpose of specific detection response simultaneously to different target substrate in the sample.
In order to understand the present invention better, reach specific embodiment now in conjunction with the accompanying drawings the present invention is described in detail.
Need to prove that at this used biomaterial of the present invention, reagent, instrument all belong to biomaterial, reagent, the instrument of this area routine if no special instructions, all can buy by commercial sources.Employed substratum and method are the substratum and the method for this area routine if no special instructions among the present invention.
Description of drawings
Fig. 1 is the design pattern of high-throughput biosensor array chip of the present invention.
Fig. 2 is the diagrams of the different culture condition of five kinds of biosensors of the present invention to the response of target contaminant.
Fig. 3 is the specific diagram of biosensor acinetobacter calcoaceticus of the present invention (Acinetobacter sp.) ADPWH_alk to the alkane response.
Fig. 4 is the specific diagram of biosensor acinetobacter calcoaceticus of the present invention (Acinetobacter sp.) ADPWH_RecA to the genetoxic response.
Fig. 5 is the specific diagram of high-throughput biosensor array chip of the present invention to the target contaminant response.
Embodiment
Biosensor used in the present invention, with acinetobacter calcoaceticus (Acinetobacter sp.) ADP1 (BD413) is the host bacterium, comprises detecting salicylic biosensor (acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux), detect the biosensor (acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol) of benzene series thing, detect the biosensor (acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk) of petroleum-type alkane, detect benzoic biosensor (acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM) and detect genotoxic biosensor (acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA).This biosensor is acinetobacter calcoaceticus (Acinetobacter sp.) ADP1 that contains reporter gene in the karyomit(e) in the target gene, wherein the target gene of biosensor acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux is the SalA gene, the target gene of biosensor acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol is the pu gene, the target gene of biosensor acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk is the alkM gene, the target gene of biosensor acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM is the BenM gene, and the target gene of biosensor acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA is the RecA gene.Described target gene and reporter gene have the common promotor, and it is transcribed and is subjected to the target gene promoter regulation, and the target gene expression intensity is identical with the reporter gene expression intensity.Described reporter gene is a bioluminescence gene, is specially luxCDABE (for known gene order).(only fragrant rain Bioisystech Co., Ltd) all can be bought and be obtained to above-mentioned five kinds of biosensors by commercial sources or according to the disclosed document of prior art (patent publication No.: .WO2008/056144; Huang W.E.et al.Chromosomally located gene fusions constructed in Acinetobacter sp.ADP1 for the environmental detection of salicylate.Environ.Microbiol.7,1339-1348,2005; Huang W.E.et al.Characterizing the regulation of the Pu promoter in Acinetobacter baylyi ADP1.Environ.Microbiol.10,1668-1680,2008; Song Y.et al.Optimization of bacterial whole cell bioreporters for toxicity assay of environmental samples.Environ.Sci.﹠amp; Technol.43,7931-7938,2009) make up.
Structure and the use of embodiment 1. biosensor array chips under LB culture medium culturing condition.
1) the biosensor array chip makes up
Five kinds of biosensors, comprise acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux, acinetobacter calcoaceticus (Acinetobacter sp) ADPWH_Tol, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk and acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA, respectively get single colony inoculation to LB liquid nutrient medium (LB), after 30 ℃ of overnight incubation, centrifugal 10 minutes of 3000rpm, abandoning supernatant.Precipitation adds the equal-volume sterilized water, shakes up centrifugal 10 minutes of 3000rpm, abandoning supernatant.Precipitation adds 10 times of volume of liquid mineral mediums, shakes up, and obtains the cell suspending liquid of 10 times of dilutions.Get the above-mentioned cell suspending liquid of 180 μ L, distribute (as shown in Figure 1), join in 96 hole enzyme plates (the U.S. Corning Costar company) hole saturating at the bottom of the black according to design.This biosensor array chip can be preserved 45 days under 4 ℃ of conditions.
2) sample is prepared
Take by weighing certain mass sodium salicylate (160.1mg), Sodium Benzoate (144.1mg), octadecane (254.5mg) and mitomycin (33.4mg) respectively, join in the 100mL pure water, obtain the stock solution of sodium salicylate (10mM), phenylformic acid (10mM), octadecane (10mM) and mitomycin (1mM).Other gets toluene 20 μ L and joins in the 100mL pure water, and 40K-Hz ultrasonic wave 30 seconds obtains the saturated stock solution of toluene, and concentration is 600 μ M.
Take by weighing 27.02g six hydration Soduxins, join in the 100mL pure water, use 0.22 μ m filter membrane (Millipore) to filter, obtain aseptic Soduxin stock solution, this solution Soduxin concentration is 1M.
3) sample test
Get the biosensor array chip that obtains in the step 1), add 4 μ L Soduxin stock solutions in every hole respectively.Distribute according to biosensor design, every group of biosensor (6 hole) adds 20 μ L pure water respectively as negative control (the parallel sample in 3 holes), and the parallel sample in 3 holes adds corresponding substrate solution (sodium salicylate, Sodium Benzoate, octadecane, toluene and mitomycin stock solution) and the 18 μ L pure water of inducing of 2 μ L in addition.
Use multi-functional microplate reader (model Synergy 2, U.S. BioTek company) that noclilucence intensity in the 96 hole enzyme plates is measured.Culture temperature is 30 ℃, carries out one-shot measurement in per 10 minutes, vibrates 1 minute before each the measurement to guarantee that cytomixis is even.
4) data processing
Calculation procedure 3) three groups of parallel sample averages of every kind of biosensor that test process is obtained in obtain every kind of biosensor negative control and induce the different moment noclilucence intensity of group.Every kind of biosensor induces the different noclilucence constantly of group intensity divided by correspondence moment negative control group noclilucence intensity, obtains every kind of biosensor and induces the relative multiple of the different noclilucences constantly of group.Get every kind of biosensor and induce the different relative multiple peak values of noclilucence constantly of group and each 2 relative multiple of time point noclilucence of front and back thereof, calculating mean value obtains this biosensor noclilucence response multiple.
The biosensor array chip under LB culture medium culturing condition to inducing substrate response as shown in Figure 2 accordingly.Test result shows that five kinds of biosensors all can effectively be discerned and induce substrate in the biosensor array chip, produces significantly response.
Structure and the use of embodiment 2. biosensor array chips under the mineral medium culture condition.
1) the biosensor array chip makes up
Five kinds of biosensors, comprise acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk and acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA, respectively get single colony inoculation to the liquid mineral medium (MMS) that contains the 20mM Soduxin, after 30 ℃ of overnight incubation, centrifugal 10 minutes of 3000rpm, abandoning supernatant.Precipitation adds the equal-volume sterilized water, shakes up centrifugal 10 minutes of 3000rpm, abandoning supernatant.Precipitation adds 10 times of volume of liquid mineral mediums, shakes up, and obtains the cell suspending liquid of 10 times of dilutions.Get the above-mentioned cell suspending liquid of 180 μ L, distribute (as shown in Figure 1), join in 96 hole enzyme plates (the U.S. Corning Costar company) hole saturating at the bottom of the black according to design.This biosensor array chip can be preserved 45 days under 4 ℃ of conditions.
2) sample is prepared
Take by weighing certain mass sodium salicylate (160.1mg), Sodium Benzoate (144.1mg), octadecane (254.5mg) and mitomycin (33.4mg) respectively, join in the 100mL pure water, obtain the stock solution of sodium salicylate (10mM), phenylformic acid (10mM), octadecane (10mM) and mitomycin (1mM).Other gets toluene 20 μ L and joins in the 100mL pure water, and 40K-Hz ultrasonic wave 30 seconds obtains the saturated stock solution of toluene, and concentration is 600 μ M.
Take by weighing 27.02g six hydration Soduxins, join in the 100mL pure water, use 0.22 μ m filter membrane (Millipore) to filter, obtain aseptic Soduxin stock solution, this solution Soduxin concentration is 1M.
3) sample test
Get the biosensor array chip that obtains in the step 1), add 4 μ L Soduxin stock solutions in every hole respectively.Distribute according to biosensor design, every group of biosensor (6 hole) adds 20 μ L pure water respectively as negative control (the parallel sample in 3 holes), and the parallel sample in 3 holes adds corresponding substrate solution (sodium salicylate, Sodium Benzoate, octadecane, toluene and mitomycin stock solution) and the 18 μ L pure water of inducing of 2 μ L in addition.
Use multi-functional microplate reader (model Synergy 2, U.S. BioTek company) that noclilucence intensity in the 96 hole enzyme plates is measured.Culture temperature is 30 ℃, carries out one-shot measurement in per 10 minutes, vibrates 1 minute before each the measurement to guarantee that cytomixis is even.
4) data processing
Calculation procedure 3) three groups of parallel sample averages of every kind of biosensor that test process is obtained in obtain every kind of biosensor negative control and induce the different moment noclilucence intensity of group.Every kind of biosensor induces the different noclilucence constantly of group intensity divided by correspondence moment negative control group noclilucence intensity, obtains every kind of biosensor and induces the relative multiple of the different noclilucences constantly of group.Get every kind of biosensor and induce the different relative multiple peak values of noclilucence constantly of group and each 2 relative multiple of time point noclilucence of front and back thereof, calculating mean value obtains this biosensor noclilucence response multiple.
The biosensor array chip under the mineral medium culture condition to inducing substrate response as shown in Figure 2 accordingly.Test result shows, in the biosensor array chip in five kinds of biosensors except that acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol, all can effectively discern and induce substrate, produce significantly response.Wherein biosensor acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux and the response multiple of acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM under the mineral medium culture condition are higher than LB culture medium culturing condition.Biosensor acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk and the response multiple of acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA under response multiple under the mineral medium culture condition and LB culture medium culturing condition do not have significant difference.Biosensor acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol does not have remarkable response to toluene under the mineral medium culture condition, and under LB culture medium culturing condition toluene is had remarkable response.The above analysis result determines that LB culture medium culturing condition is that best biosensor array chip makes up condition.
Embodiment 3. biosensor acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk is to the specificity of alkane response.
1) the biosensor array chip makes up
Five kinds of biosensors, comprise acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk and acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA, respectively get single colony inoculation to LB liquid nutrient medium (LB), after 30 ℃ of overnight incubation, centrifugal 10 minutes of 3000rpm, abandoning supernatant.Precipitation adds the equal-volume sterilized water, shakes up centrifugal 10 minutes of 3000rpm, abandoning supernatant.Precipitation adds 10 times of volume of liquid mineral mediums, shakes up, and obtains the cell suspending liquid of 10 times of dilutions.Get the above-mentioned cell suspending liquid of 180 μ L, distribute (as shown in Figure 1), join in 96 hole enzyme plates (the U.S. Corning Costar company) hole saturating at the bottom of the black according to design.This biosensor array chip can be preserved 45 days under 4 ℃ of conditions.
2) sample is prepared
Take by weighing certain mass sodium salicylate (160.1mg), Sodium Benzoate (144.1mg), octadecane (254.5mg) and mitomycin (33.4mg) respectively, join in the 100mL pure water, obtain the stock solution of sodium salicylate (10mM), phenylformic acid (10mM), octadecane (10mM) and mitomycin (1mM).Other gets toluene 20 μ L and joins in the 100mL pure water, and 40K-Hz ultrasonic wave 30 seconds obtains the saturated stock solution of toluene, and concentration is 600 μ M.
Take by weighing 27.02g six hydration Soduxins, join in the 100mL pure water, use 0.22 μ m filter membrane (Millipore) to filter, obtain aseptic Soduxin stock solution, this solution Soduxin concentration is 1M.
Get the above-mentioned substrate stock solution of inducing of certain volume, the configuration testing sample, as shown in table 1.
The different samples of table 1. are induced substrate content
3) sample test
Get the biosensor array chip that obtains in the step 1), add 4 μ L Soduxin stock solutions in every hole respectively.Biosensor acinetobacter calcoaceticus in the biosensor array chip (Acinetobacter sp.) ADPWH_alk (6 hole) adds the sample 6 of acquisition in the step 2 respectively as negative control (the parallel sample in 3 holes) and 20 μ L sample 1-5 solution (the parallel sample in 3 holes).
Use multi-functional microplate reader (model Synergy 2, U.S. BioTek company) that noclilucence intensity in the 96 hole enzyme plates is measured.Culture temperature is 30 ℃, carries out one-shot measurement in per 10 minutes, vibrates 1 minute before each the measurement to guarantee that cytomixis is even.
4) data processing
Calculation procedure 3) three groups of parallel sample averages of every kind of biosensor that test process is obtained in obtain every kind of biosensor negative control and induce the different moment noclilucence intensity of group.Every kind of biosensor induces the different noclilucence constantly of group intensity divided by correspondence moment negative control group noclilucence intensity, obtains every kind of biosensor and induces the relative multiple of the different noclilucences constantly of group.Get every kind of biosensor and induce the different relative multiple peak values of noclilucence constantly of group and each 2 relative multiple of time point noclilucence of front and back thereof, calculating mean value obtains this biosensor noclilucence response multiple.
Biosensor acinetobacter calcoaceticus in the biosensor array chip (Acinetobacter sp.) ADPWH_alk to the response of 6 kinds of samples as shown in Figure 3.Test result shows, biosensor acinetobacter calcoaceticus in the biosensor array chip (Acinetobacter sp.) ADPWH_alk only produces significantly response to octadecane, response to all the other sodium salicylates, phenylformic acid, toluene and mitomycin is all identical with negative control, and response has specificity to alkane to show biosensor in the biosensor array chip (Acinetobacter sp.) ADPWH_alk.
1) the biosensor array chip makes up
Five kinds of biosensors, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk and acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA, respectively get single colony inoculation to LB liquid nutrient medium (LB), after 30 ℃ of overnight incubation, centrifugal 10 minutes of 3000rpm, abandoning supernatant.Precipitation adds the equal-volume sterilized water, shakes up centrifugal 10 minutes of 3000rpm, abandoning supernatant.Precipitation adds 10 times of volume of liquid mineral mediums, shakes up, and obtains the cell suspending liquid of 10 times of dilutions.Get the above-mentioned cell suspending liquid of 180 μ L, distribute (as shown in Figure 1), join in 96 hole enzyme plates (the U.S. Corning Costar company) hole saturating at the bottom of the black according to design.This biosensor array chip can be preserved 45 days under 4 ℃ of conditions.
2) sample is prepared
Take by weighing certain mass sodium salicylate (160.1mg), Sodium Benzoate (144.1mg), octadecane (254.5mg) and mitomycin (33.4mg) respectively, join in the 100mL pure water, obtain the stock solution of sodium salicylate (10mM), phenylformic acid (10mM), octadecane (10mM) and mitomycin (1mM).Other gets toluene 20 μ L and joins in the 100mL pure water, and 40K-Hz ultrasonic wave 30 seconds obtains the saturated stock solution of toluene, and concentration is 600 μ M.
Take by weighing 27.02g six hydration Soduxins, join in the 100mL pure water, use 0.22 μ m filter membrane (Millipore) to filter, obtain aseptic Soduxin stock solution, this solution Soduxin concentration is 1M.
Get the above-mentioned substrate stock solution of inducing of certain volume, the configuration testing sample, as shown in table 2.
The different samples of table 2. are induced substrate content
3) sample test
Get the biosensor array chip that obtains in the step 1), add 4 μ L Soduxin stock solutions in every hole respectively.Biosensor acinetobacter calcoaceticus in the biosensor array chip (Acinetobacter sp.) ADPWH_RecA (6 hole) adds the sample 6 of acquisition in the step 2 respectively as negative control (the parallel sample in 3 holes) and 20 μ L sample 1-5 solution (the parallel sample in 3 holes).
Use multi-functional microplate reader (model Synergy 2, U.S. BioTek company) that noclilucence intensity in the 96 hole enzyme plates is measured.Culture temperature is 30 ℃, carries out one-shot measurement in per 10 minutes, vibrates 1 minute before each the measurement to guarantee that cytomixis is even.
4) data processing
Calculation procedure 3) three groups of parallel sample averages of every kind of biosensor that test process is obtained in obtain every kind of biosensor negative control and induce the different moment noclilucence intensity of group.Every kind of biosensor induces the different noclilucence constantly of group intensity divided by correspondence moment negative control group noclilucence intensity, obtains every kind of biosensor and induces the relative multiple of the different noclilucences constantly of group.Get every kind of biosensor and induce the different relative multiple peak values of noclilucence constantly of group and each 2 relative multiple of time point noclilucence of front and back thereof, calculating mean value obtains this biosensor noclilucence response multiple.
Biosensor acinetobacter calcoaceticus in the biosensor array chip (Acinetobacter sp.) ADPWH_RecA to the response of 6 kinds of samples as shown in Figure 4.Test result shows, biosensor acinetobacter calcoaceticus in the biosensor array chip (Acinetobacter sp.) ADPWH_alk only produces significantly response to mitomycin, all identical with negative control to all the other sodium salicylates, phenylformic acid, toluene and benzoic response, response has specificity to the mitomycin genetoxic to show biosensor acinetobacter calcoaceticus in the biosensor array chip (Acinetobacter sp.) ADPWH_RecA.
Embodiment 5. biosensor array chips are to the specificity of target contaminant response
1) the biosensor array chip makes up
Five kinds of biosensors, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk and acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA, respectively get single colony inoculation to LB liquid nutrient medium (LB), after 30 ℃ of overnight incubation, centrifugal 10 minutes of 3000rpm, abandoning supernatant.Precipitation adds the equal-volume sterilized water, shakes up centrifugal 10 minutes of 3000rpm, abandoning supernatant.Precipitation adds 10 times of volume of liquid mineral mediums, shakes up, and obtains the cell suspending liquid of 10 times of dilutions.Get the above-mentioned cell suspending liquid of 180 μ L, distribute (as shown in Figure 1), join in 96 hole enzyme plates (the U.S. Corning Costar company) hole saturating at the bottom of the black according to design.This biosensor array chip can be preserved 45 days under 4 ℃ of conditions.
2) sample is prepared
Take by weighing certain mass sodium salicylate (160.1mg), Sodium Benzoate (144.1mg), octadecane (254.5mg) and mitomycin (33.4mg) respectively, join in the 100mL pure water, obtain the stock solution of sodium salicylate (10mM), phenylformic acid (10mM), octadecane (10mM) and mitomycin (1mM).Other gets toluene 20 μ L and joins in the 100mL pure water, and 40K-Hz ultrasonic wave 30 seconds obtains the saturated stock solution of toluene, and concentration is 600 μ M.
Take by weighing 27.02g six hydration Soduxins, join in the 100mL pure water, use 0.22 μ m filter membrane (Millipore) to filter, obtain aseptic Soduxin stock solution, this solution Soduxin concentration is 1M.
Get the above-mentioned substrate stock solution of inducing of certain volume, the configuration testing sample, as shown in table 3.
The different samples of table 3. are induced substrate content
3) sample test
Get the biosensor array chip that obtains in the step 1), add 4 μ L Soduxin stock solutions in every hole respectively.Biosensor (6 hole) adds the sample 32 of acquisition in the step 2 respectively as negative control (the parallel sample in 3 holes) and 20 μ L sample 1-31 solution (the parallel sample in 3 holes) in every group of biosensor array chip.
Use multi-functional microplate reader (model Synergy 2, U.S. BioTek company) that noclilucence intensity in the 96 hole enzyme plates is measured.Culture temperature is 30 ℃, carries out one-shot measurement in per 10 minutes, vibrates 1 minute before each the measurement to guarantee that cytomixis is even.
4) data processing
Calculation procedure 3) three groups of parallel sample averages of every kind of biosensor that test process is obtained in obtain every kind of biosensor negative control and induce the different moment noclilucence intensity of group.Every kind of biosensor induces the different noclilucence constantly of group intensity divided by correspondence moment negative control group noclilucence intensity, obtains every kind of biosensor and induces the relative multiple of the different noclilucences constantly of group.Get every kind of biosensor and induce the different relative multiple peak values of noclilucence constantly of group and each 2 relative multiple of time point noclilucence of front and back thereof, calculating mean value obtains this biosensor noclilucence response multiple.
The biosensor array chip to the response of 32 kinds of target stains matter samples as shown in Figure 5.Test result shows, biosensor acinetobacter calcoaceticus in the biosensor array chip (Acinetobacter sp.) ADPWH_lux, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk and acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA all only produce significantly response to each self-induction substrate, response to all the other materials is all identical with negative control, shows that the biosensor array chip has specificity to the detection of pollutent in the sample.
Embodiment 6. biosensor array chips are to the specificity of target contaminant response
Substantially the same manner as Example 5, difference is
3) sample test
Get the biosensor array chip that obtains in the step 1), add 4 μ L Soduxin stock solutions in every hole respectively.Biosensor (6 hole) adds the sample 32 of acquisition in the step 2 respectively as negative control (the parallel sample in 3 holes) and 20 μ L sample 1-31 solution (the parallel sample in 3 holes) in every group of biosensor array chip.
Use multi-functional microplate reader (model Synergy 2, U.S. BioTek company) that noclilucence intensity in the 96 hole enzyme plates is measured.Culture temperature is 20 ℃, carries out one-shot measurement in per 2 minutes, vibrates 1 minute before each the measurement to guarantee that cytomixis is even.
The result is identical with embodiment 5.
Embodiment 7. biosensor array chips are to the specificity of target contaminant response
Substantially the same manner as Example 5, difference is
3) sample test
Get the biosensor array chip that obtains in the step 1), add 4 μ L Soduxin stock solutions in every hole respectively.Biosensor (6 hole) adds the sample 32 of acquisition in the step 2 respectively as negative control (the parallel sample in 3 holes) and 20 μ L sample 1-31 solution (the parallel sample in 3 holes) in every group of biosensor array chip.
Use multi-functional microplate reader (model Synergy 2, U.S. BioTek company) that noclilucence intensity in the 96 hole enzyme plates is measured.Culture temperature is 37 ℃, carries out one-shot measurement in per 15 minutes, vibrates 1 minute before each the measurement to guarantee that cytomixis is even.
The result is identical with embodiment 5.
Claims (15)
1. high-throughput biosensor array chip is characterized in that: it is substrate that this chip adopts enzyme plate, comprises at least two kinds on this enzyme plate and based on what same host bacterium made up the different target substrate is detected the biosensors of response.
2. high-throughput biosensor array chip according to claim 1 is characterized in that: described same host bacterium is acinetobacter calcoaceticus (Acinetobacter sp.) ADP1.
3. high-throughput biosensor array chip according to claim 2, it is characterized in that: described biosensor is selected from: acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM and acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA.
4. high-throughput biosensor array chip according to claim 3 is characterized in that: described enzyme plate is 96 hole enzyme plates.
5. the construction process of a high-throughput biosensor array chip, this method comprises: be taken to few two kinds of single colony inoculations based on the biosensor that detection responds to the different target substrate of same host bacterium structure and cultivate in substratum, centrifugal, dilution, get the cell suspending liquid of dilution, distribute according to design, join in the substrate enzyme plate hole.
6. the construction process of a kind of high-throughput biosensor array chip according to claim 5 is characterized in that: described same host bacterium is acinetobacter calcoaceticus (Acinetobacter sp.) ADP1.
7. the construction process of a kind of high-throughput biosensor array chip according to claim 6 is characterized in that: described biosensor is selected from: acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM and acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA.
8. the construction process of a kind of high-throughput biosensor array chip according to claim 7 is characterized in that: described substratum is the LB substratum; Described centrifugal carrying out 2 times, for the first time centrifugal after, abandoning supernatant, precipitation adds isopyknic sterilized water, it is centrifugal to carry out second time, abandoning supernatant, precipitation adding liquid mineral medium dilutes.
9. the construction process of a kind of high-throughput biosensor array chip according to claim 8 is characterized in that: described enzyme plate is 96 hole enzyme plates.
10. method that detects at least two kinds of target substrates in the sample simultaneously, this method comprises: get sample to be tested, make the aqueous solution, join in the described high-throughput biosensor array of claim 1 chip, cultivate altogether, detect target substrates by the microplate reader measurement.
11. a kind of method that detects at least two kinds of target substrates in the sample simultaneously according to claim 10 is characterized in that: described same host bacterium is acinetobacter calcoaceticus (Acinetobacter sp.) ADP1.
12. a kind of method that detects at least two kinds of target substrates in the sample simultaneously according to claim 11, it is characterized in that: described biosensor is selected from: acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_lux, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_Tol, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_alk, acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_BenM and acinetobacter calcoaceticus (Acinetobacter sp.) ADPWH_RecA.
13. a kind of method that detects at least two kinds of target substrates in the sample simultaneously according to claim 12 is characterized in that: described enzyme plate is 96 hole enzyme plates.
14. a kind of method that detects at least two kinds of target substrates in the sample simultaneously according to claim 13 is characterized in that: the temperature of cultivating is 20 ℃ to 37 ℃ altogether, and the frequency that microplate reader is measured is 2 to 15 minutes.
15. a kind of method that detects at least two kinds of target substrates in the sample simultaneously according to claim 14 is characterized in that: the temperature of cultivating is 30 ℃ altogether, and the frequency that microplate reader is measured is 5 minutes.
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| CN107217017A (en) * | 2017-05-27 | 2017-09-29 | 青岛农业大学 | One plant of acinetobacter calcoaceticus and its application in oil degradation |
| CN107217017B (en) * | 2017-05-27 | 2020-12-08 | 青岛农业大学 | Acinetobacter and application thereof in petroleum degradation |
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