KR20140059609A - A dip-stick toxicity biosensor using smart functional microbeads - Google Patents
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Abstract
The present invention relates to a biosensor in the form of a dip-stick in which a functional microbead carrying a recombinant light emitting bacteria and a fluorescent substance is immobilized, and a method for detecting toxicity using the biosensor.
The biosensor in the form of a dip-stick in which the recombinant light-emitting bacteria for toxicity detection and the functionalized microbeads carrying the fluorescent substance are immobilized according to the present invention can be easily carried, It is expected to contribute to the improvement of public health ultimately by applying toxicological detection in new medicine development, environment, food as well as space aviation which is one of future science.
Description
The present invention relates to a biosensor in the form of a dip-stick in which a functional microbead carrying a recombinant light-emitting bacteria and a fluorescent substance is immobilized, and a method for detecting toxicity using the biosensor.
A biochip is a hybrid device made by integrating a living organism such as an enzyme, a protein, an antibody, a DNA, a living organism, an animal or plant cell, an organ, or a nerve cell derived from a living organism on a substrate made of glass, silicon and nylon As a hybrid device, it can analyze gene expression patterns, genetic defects, protein distribution, and reaction patterns, and can play a role in disease diagnosis and development of new drugs.
One of the components of the biochip is biochip, which is mostly disposable, so the annual consumption will be astronomical at the time when the biochip is generalized. Currently, domestic and overseas companies involved in biotechnology and medical care are concentrating their efforts on manufacturing biochips using substrates for biochips in order to preoccupy the market in these potentially huge markets.
Luminescent bacteria have been pre-screening for environmental toxicity and have played an important role in monitoring their toxicity classification, namely genetic toxicity, oxidative damage, membrane damage and protein damage. Cell chip development studies are under way to develop a high throughput based system that seeks to detect various toxicities of various luminescent bacteria at once (Korean Patent No. 473350).
The most serious problem in existing cell chip research is that a program for identifying, arranging and recognizing which cell is immobilized in which position among many cells is essential. In particular, existing researches focus on immobilization methods of cells rather than studies on self-identification, and in fact, they are based on electrical network-based chips, nylon membrane arrays, optical detection arrays, and agar- (Ahn, JM et al. , Lab. On a Chip , 10: 2695, 2010).
This method is a cell array chip that arranges cells. Still, recognition of each cell is indispensable and it is not suitable for rapid detection of actual toxicity because it takes a long analysis time. In the existing toxicity assessment and diagnosis of biosensors, these techniques are not cost and time efficient, their application range is not wide, and there are limited problems in application to biosensors.
Accordingly, the present inventors have found that when a biosensor in which a recombinant light emitting bacteria for toxicity detection and a functional microbead carrying a fluorescent substance are immobilized in a dip-stick form is prepared, if the biosenses are immersed in a sample containing a toxic substance, The presence or absence of toxic substances can be easily detected, and the present invention has been completed.
An object of the present invention is to provide a biosensor capable of easily detecting a toxic substance in a sample containing a toxic substance.
It is another object of the present invention to provide a method of manufacturing the biosensor.
It is still another object of the present invention to provide a method for detecting toxic substances using the biosensor.
In order to achieve the above object, the present invention provides a method for producing a microbead, comprising the steps of: (a) stirring a recombinant light-emitting bacteria and a fluorescent substance for toxicity detection together with a bead substrate and then preparing a microbead by electrospinning; And (b) disposing the prepared microbeads on a substrate on which the wells are formed, and fixing the mesh having a mesh size in which the microbeads can not escape, onto the substrate, the recombinant light-emitting bacteria and the fluorescent material for toxicity detection And a method of manufacturing a biosensor in the form of a dip-stick in which the supported functional microbeads are immobilized.
The present invention also provides a dip-stick type biosensor manufactured by the above-described method, wherein functional microbeads carrying the recombinant light-emitting bacteria for detecting toxicity and the fluorescent material are immobilized.
The present invention also provides a toxicity detection method using the biosensor.
The biosensor in the form of a dip-stick in which the recombinant light-emitting bacteria for toxicity detection and the functionalized microbeads carrying the fluorescent substance are immobilized according to the present invention can be easily carried, It is expected to contribute to the improvement of public health ultimately by applying toxicological detection in new medicine development, environment, food as well as space aviation which is one of future science.
1 shows a method for preparing a functional microbead carrying a recombinant light-emitting bacteria and a fluorescent substance for toxicity detection by electrospinning.
FIG. 2 shows a structure of a biosensor in the form of a dip-stick according to the present invention.
FIG. 3 shows a structure and a manufacturing method of a dip-stick type biosensor according to the present invention.
FIG. 4 is a photograph showing a result of detecting a toxic substance using a dip-stick type biosensor according to the present invention with a cooled CCD camera.
The present invention, in a continuity, comprises the steps of: (a) stirring a recombinant light-emitting bacteria and a fluorescent substance for toxicity detection together with a bead substrate, and then preparing a microbead by electrospinning; And (b) disposing the prepared microbeads on a substrate on which the wells are formed, and fixing the mesh having a mesh size in which the microbeads can not escape, onto the substrate, the recombinant light-emitting bacteria and the fluorescent material for toxicity detection Stick-type biosensor in which the supported functional microbeads are immobilized.
The dip-stick type biosensor of the present invention requires auxiliary devices such as a pipette to transmit a compound to a chip when bio-toxicity is measured using a bio-chip-based self-identifying code functional microbead, The biochip of the dip-stick type according to the present invention is produced by immersing a chip in a sample containing a compound to be measured, So that the cost and time required for the experiment can be reduced.
In one embodiment of the present invention, the wells of the chip carrying the functional micro-beads are shaved with an oblique surface so that the beads do not fall under the chip and the oxygen supply necessary for light emission of the light-emitting bacteria can be smoothly received. The material was made of glass, and the adsorption of the chemical was prevented to the utmost.
Since the dip-stick type biosensor of the present invention is easy to carry and can be used anywhere, it is widely applied not only to aerospace, which is one of future science, but also toxic detection in new drug development, environment, food, Of the total population.
In the present invention, the recombinant light-emitting bacteria for toxicity detection may be a bacteria transformed with a recombinant plasmid in which a promoter sensitive to toxicity is combined with a luminescent gene, and the toxicity may be genotoxicity, oxidative damage, Toxicity, and protein damage.
The fluorescent material used in the present invention can identify the kind of the recombinant light emitting bacteria carried on each well of the biosensor and the kind of the detectable toxicity by using different fluorescent materials depending on the kind of the recombinant light emitting bacteria for toxicity detection, The fluorescent material used in the present invention may be selected from the group consisting of Cy3 (green), Cy5 (red), FITC (green), Alexa, BODIPY, Rhodamine and Quantum- But the present invention is not limited thereto.
In the present invention, the bead substrate is preferably a natural polysaccharide and is preferably selected from the group consisting of alginic acid, sodium alginate, Glucomannan, kappa-carrageenin or a derivative thereof and agar. However, the present invention is not limited thereto, and any material capable of forming a microbead by supporting a recombinant light emitting bacteria and a fluorescent substance can be used without limitation.
In the present invention, the well of the biosensor substrate may be characterized in that the bottom surface of the well has a narrow bottom portion and a wide top portion. In this case, the bead does not fall under the chip and the contact area of the compound and the bead is increased And it is advantageous that the light emitting bacteria can smoothly receive the essential oxygen supply for light.
In the present invention, the recombinant light-emitting bacteria for toxicity detection may be selected from among the bacteria listed in Table 1, but the present invention is not limited thereto. The recombinant light-emitting bacteria may be transformed with a plasmid containing a promoter of a gene susceptible to toxicity and a luminescent gene Can be used without limitation.
In the present invention, the recombinant light emitting bacteria for toxicity detection may be selected from the group consisting of DPD2794, DPD2511, DPD2540, EBAlkA, EBSoxS, TV1061, KAN3 and GC2.
In one embodiment of the present invention, a biosensor in the form of a dip-stick in which a functional microbead carrying a recombinant light-emitting bacteria for detecting toxicity and a fluorescent substance is immobilized was prepared in the following manner:
The luminescent bacterial culture was centrifuged and concentrated. The concentrated cells were resuspended in 2% alginic acid solution, fluorescent material was added, mixed together, and placed in a sterilized syringe. To prepare the beads by electrospinning, insert a (+) electrode into a beaker containing 2% calcium chloride solution, connect the needle of the syringe containing the mixture to the (-) electrode, The mixture was continuously stirred to be beads in a 2% calcium chloride solution (Fig. 1). After stirring for 1 hour in a 2% calcium chloride solution to cure the beads, the functional microbeads were washed three times with LB medium to remove calcium chloride, and functional microbeads were prepared. The functional microbeads were arranged in four to five microbeads of two microbeads on an 85 mm x 8 mm chip having 10 holes of 1 mm in size. Thereafter, the mesh having a mesh size that can pass through the compound but can not escape is fixed on the chip, and a biosensor in the form of a dip-stick having fixed functional microbeads carrying the recombinant light- Respectively.
In another aspect, the present invention relates to a biosensor in the form of a dip-stick, which is manufactured by the above-described method and has functional immobilizing bacterial cells for detecting toxicity and fluorescent substance-bearing functional microbeads immobilized thereon.
In the present invention, the recombinant light-emitting bacteria for toxicity detection may be a bacteria transformed with a recombinant plasmid in which a promoter sensitive to toxicity is combined with a luminescent gene, and the toxicity may be genotoxicity, oxidative damage, Toxicity, and protein damage.
The fluorescent material used in the present invention can identify the kind of the recombinant light emitting bacteria carried on each well of the biosensor and the kind of the detectable toxicity by using different fluorescent materials depending on the kind of the recombinant light emitting bacteria for toxicity detection, The fluorescent material used in the present invention may be selected from the group consisting of Cy3 (green), Cy5 (red), FITC (green), Alexa, BODIPY, Rhodamine and Quantum- But the present invention is not limited thereto.
In the present invention, the well of the biosensor substrate may be characterized in that the bottom surface of the well has a narrow bottom portion and a wide top portion. In this case, the bead does not fall under the chip and the contact area of the compound and the bead is increased And it is advantageous that the light emitting bacteria can smoothly receive the essential oxygen supply for light.
In the present invention, the recombinant light-emitting bacteria for toxicity detection may be selected from among the bacteria listed in Table 1, but the present invention is not limited thereto, and the plasmids containing the promoter of the gene susceptible to toxicity and the luminescent gene Any transformed strain can be used without limitation.
In the present invention, the recombinant light emitting bacteria for toxicity detection may be selected from the group consisting of DPD2794, DPD2511, DPD2540, EBAlkA, EBSoxS, TV1061, KAN3 and GC2.
In another aspect, the present invention provides a toxicity detection method using the biosensor.
In the present invention, the toxicity detection may be performed by confirming whether or not the light emitting bacteria emit light.
In the present invention, the toxicity detection may be performed by immersing the biosensor in a sample containing a toxic substance.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.
Example 1: Preparation of recombinant luminescent bacteria for toxicity detection
The recombinant luminescent bacteria for toxicity detection used in the present invention was prepared by the following method.
The method for producing the recombinant light-emitting bacteria of the present invention is described in detail in Korean Patent Registration No. 10-0473350, which is a patent of the inventor of the present invention. to summarize,
1) The gene promoter is prepared from the genomic DNA of Escherichia coli RFM443 by preparing a primer suitable for the promoter portion in each gene and amplifying it using PCR. The primer set used to amplify the promoter of each gene is as follows:
DPD2794
SEQ ID NO: 1: 5'-ACTTAA GGATCC AGAGAAGCCTGTCGGCAC- 3 '
SEQ ID NO: 2: 5'-AGCTTT GAATTC CGCTTTCTGTTTGTTTT-3 '
EBAlkA
SEQ ID NO: 3: 5'-ACTTAA GGATCC GCAAGGCATTGAAGGCAG- 3 '
SEQ ID NO: 4: 5'-AGCAGC GAATTC ATCCCAACATCCACGACC-3 '
EBSoxS
SEQ ID NO: 5: 5'-AGCAGC GAATTC GCGGCTGGTCAATATGCTC-3 '
SEQ ID NO: 6: 5'-ACTTAA GGATCC GCTGCGTTTCGCCACTT- 3 '
DPD2511
SEQ ID NO: 7: 5'-ACTTA GGATCC CGAAATGAGGGCGGGAAA- 3 '
SEQ ID NO: 8: 5'-AGCAGC GAATTC GAACGTTGCTGACCACGA-3 '
The PCR primer product is treated with the restriction enzymes BamHI and EcoRI (underlined in the top sequence) and the plasmid vector pUCD615 (a vector carrying the luxCDABE operon without the promoter) is also cut with the same restriction enzyme. The recombinant plasmid pRecALux was prepared using T4 ligase at 14 ° C for 8 hours. DPD2540, TV1061, KAN3 and GC2 can also be manufactured by the same method using the information shown in Table 2. [
This PCR product is treated with restriction enzymes such as BamHI and EcoR1 and the plasmid vector pUCD615 (a vector carrying the luxCDABE operon without the promoter) is also cut with the same restriction enzyme. The recombinant plasmid shown in Table 2 was prepared using T4 ligase at 14 DEG C for 8 hours.
Promoter
This was transformed into E. coli RFM443, and RFM443 / pXXX was grown on agar plates containing kanamycin. The selected colonies were cultured in LB medium containing kanamycin to prepare recombinant light-emitting bacteria for toxic substance detection.
Example 2: Preparation of a biosensor in the form of a dip-stick having a functional microbead-immobilized recombinant light-emitting bacteria and a fluorescent substance
Using the recombinant light-emitting bacteria prepared in Example 1, functional microbeads were prepared and the detection ability of 7 different toxic substances was confirmed.
First, mitomycin C (MMC) causing genotoxicity, 1-methyl-1-nitroso-Nmethylguanidine (MNNG) causing DNA accu- racy during genotoxicity, H 2 O 2 , O 2 · causing oxidative damage by OH · · Paraquat, which causes oxidative damage, salicylic acid (SA), which causes cell membrane toxicity, ethanol that causes protein damage, and finally phenol, which can detect the phenolic system, were performed as follows.
Eight kinds of recombinant light-emitting bacteria, DPD2794, DPD2511, DPD2540, EBAlkA, EBSoxS, TV1061, KAN3 and GC2 prepared in Example 1 were cultured and functional microbeads were prepared by the following method.
Eight kinds of luminescent bacteria (100 ml) were cultured to OD600 = 0.7, and the cells were concentrated using a centrifuge at 3,000 rpm for 10 minutes. The concentrated cells were resuspended in 2% alginic acid solution, the fluorescent material was added, mixed together, and placed in a 12 ml sterile syringe (Table 3). To prepare beads by electrospinning, a (+) electrode was placed in a beaker containing 2% calcium chloride solution, and the needle of the syringe containing the mixture was connected to the (-) electrode. The voltage was allowed to flow through a high voltage DC unit and the dropping mixture was continuously stirred to be beads in a 2% calcium chloride solution (FIG. 1).
After stirring for 1 hour in 2% calcium chloride solution to cure the beads, the functional microbeads were washed three times with LB medium to remove calcium chloride.
To investigate the detection ability of functional microbeads using different luminescent bacteria, two or three microbeads were arrayed on each 85 mm x 8 mm chip with 10 holes of 1 mm size. Thereafter, a mesh having a mesh size that can pass through the compound but can not escape is fixed on the chip, and a biosensor in the form of a dip-stick having fixed functional microbeads carrying the recombinant light- Respectively. The overall structure of the dip-stick type biosensor is shown in FIG. 2, and includes the steps of exposing the above-mentioned seven chemical substances in a Dip-Stick manner, including an LB chip for testing the basic emission level of the chip, And measured for 3 hours using a cooled CCD camera every 10 minutes (Fig. 3).
As a result, it was confirmed that MMC reacted specifically with DPD2794 detecting genotoxicity, and MNNG reacted with DPD2974 detecting genotoxicity and EBAlkA detecting Alkylation.
H 2 O 2 exposure indirectly caused genotoxicity and responded to oxidative damage by DPD2794 and OH. Paraquat, which causes oxidative damage by O2 ·, specifically reacted with EBSoxS. Salicylic acid, which causes cell membrane damage by oxidative damage, was weakly reactive with DPD2511 and DPD2540.
The kinds of the recombinant luminescent bacteria carried on each bead were confirmed by fluorescence of the fluorescent material carried thereon (Fig. 4). Figure 4 shows the actual photographs taken with DPD2511, EBSoxS and a digital camera in response to paraquat and H 2 O 2. It can be seen that this reaction can be quantitatively analyzed by pixel density as well as the degree of luminescence identified by the eye there was.
Example 3: Detection of toxic substances using the biosensor of the present invention manufactured in a random arrangement
Using the functional microbeads prepared by the method of Example 2, a randomly arrayed Dip-Stick cell chip was produced to confirm the present invention. Eight functional microbeads were filled into a 1 mm sized Dip-Stick cell chip using a Pasteur pipette. The thus prepared randomly arranged Dip-Stick cell chip was tested for its detection ability using the toxic substance described in Example 2. First, the fluorescence image map was measured to confirm the self-identifying code of the randomly arranged functional microbeads, and the toxic substance was exposed to measure the emission for 2 hours using a cooled CCD camera every 10 minutes as in Example 2. [ As a result of the measurement, a chemical substance exhibiting a specific stressor in a functional microbead having specific fluorescence was detected and confirmed to exhibit luminescence.
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
<110> Korea University Research and Business Foundation <120> A Dip-stick Toxicity Biosensor Using Smart Functional Microbeads ≪ 130 > P12-B247 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 acttaaggat ccagagaagc ctgtcggcac 30 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agctttgaat tccgctttct gtttgtttt 29 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 acttaaggat ccgcaaggca ttgaaggcag 30 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 agcagcgaat tcatcccaac atccacgacc 30 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 agcagcgaat tcgcggctgg tcaatatgct c 31 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 acttaaggat ccgctgcgtt tcgccactt 29 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 acttaggatc ccgaaatgag ggcgggaaa 29 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 agcagcgaat tcgaacgttg ctgaccacga 30
Claims (18)
(a) preparing a microbead by electrospinning after stirring the recombinant light-emitting bacteria and the fluorescent substance for toxicity detection together with the bead substrate; And
(b) arranging the prepared microbeads on a substrate on which the wells are formed, and fixing the mesh having a mesh size on which the microbeads can not escape, onto the substrate.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106526172A (en) * | 2016-10-08 | 2017-03-22 | 北京国科华仪科技有限公司 | Immunochromatographic test strip based on electrostatic-spinning nitrocellulose nanofiber membrane and preparation method thereof |
| CN108613957A (en) * | 2018-05-28 | 2018-10-02 | 中国人民解放军火箭军疾病预防控制中心 | A kind of detection method for micro- plastics in aquatic food |
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| KR20010003154A (en) * | 1999-06-21 | 2001-01-15 | 김효근 | Immobilization and Detecting Method of toxicity in Gas Phase Using Bioluminescent Bacteria |
| KR20010069072A (en) * | 2000-01-12 | 2001-07-23 | 김효근 | Device for monitoring toxicity in contaminated soils and method for monitoring toxicity in contaminated soils using same |
| KR20110137200A (en) * | 2010-06-16 | 2011-12-22 | 고려대학교 산학협력단 | Functional microbead for fluorescent coding with recombinant bioluminescent bacteria and biochip for toxicity analysis |
| KR20120022690A (en) * | 2011-12-28 | 2012-03-12 | 고려대학교 산학협력단 | Functional microbead for fluorescent coding with recombinant bioluminescent bacteria and biochip for toxicity analysis |
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| KR20010003154A (en) * | 1999-06-21 | 2001-01-15 | 김효근 | Immobilization and Detecting Method of toxicity in Gas Phase Using Bioluminescent Bacteria |
| KR20010069072A (en) * | 2000-01-12 | 2001-07-23 | 김효근 | Device for monitoring toxicity in contaminated soils and method for monitoring toxicity in contaminated soils using same |
| KR20110137200A (en) * | 2010-06-16 | 2011-12-22 | 고려대학교 산학협력단 | Functional microbead for fluorescent coding with recombinant bioluminescent bacteria and biochip for toxicity analysis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106526172A (en) * | 2016-10-08 | 2017-03-22 | 北京国科华仪科技有限公司 | Immunochromatographic test strip based on electrostatic-spinning nitrocellulose nanofiber membrane and preparation method thereof |
| CN108613957A (en) * | 2018-05-28 | 2018-10-02 | 中国人民解放军火箭军疾病预防控制中心 | A kind of detection method for micro- plastics in aquatic food |
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