WO2008133464A1 - Biochip for quantifying amino acid and method for analying amino acid using the same - Google Patents
Biochip for quantifying amino acid and method for analying amino acid using the same Download PDFInfo
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- WO2008133464A1 WO2008133464A1 PCT/KR2008/002395 KR2008002395W WO2008133464A1 WO 2008133464 A1 WO2008133464 A1 WO 2008133464A1 KR 2008002395 W KR2008002395 W KR 2008002395W WO 2008133464 A1 WO2008133464 A1 WO 2008133464A1
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- the present invention relates to a biochip for the quantitative analysis of amino acids and a method for analyzing amino acids using the same, and more particularly to a biochip for the quantitative analysis of amino acids, in which a plurality of E. coli mutants auxotrophic for different amino acids, the growth of which increases in proportion to the concentrations of specific amino acids, are immobilized on a flat substrate, as well as a method for analyzing the concentrations of amino acids using the biochip.
- Amino acids are the basic structural building units of proteins, and examples thereof include 20 kinds of amino acids, including glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, aspartate, asparagine, glutamate, tyrosine, lysine, arginine, histidine, phenylalanine, glutamine, tryptophan and proline. These amino acids are classified into two groups: essential amino acids, which are not synthesized in vivo and must be supplied in the food, and nonessential amino acids. Such amino acids are important in human metabolic processes, and when the supply thereof is insufficient, various disorders will occur, thus leading to various diseases.
- the concentration of amino acids in blood or urine indicates the nutritional status of the body, and thus the monitoring of the concentration of these amino acids in body fluids is important for verifying the therapeutic effects of diet or vitamin supplements.
- the concentrations of phenylalanine, leucine and methionine are used as important markers in diagnosing congenital metabolic diseases in newborns.
- Methods for analyzing amino acid concentration include thin-layer chromatography, electrophoresis, mass spectrometry, liquid chromatography and the like, and among them, ion-exchange liquid chromatography with post-column reactions, and with liquid column chromatography with pre-column reactions, are mainly used as commercial amino acid analyzers, because they are convenient to use and has high measurement accuracy (Slocum, R.H., Techniques in diagnostic human biochemical genetics: a laboratory manual, 87, 1991). Recently, tandem mass spectrometry (MS/MS), which performs high speed analysis and can handle a large amount of samples, has been recognized as important equipment for the diagnosis of congenital metabolic diseases in newborns.
- MS/MS tandem mass spectrometry
- microbiological assays which use microorganisms auxotrophic for specific amino acids, have been used for more than 60 years.
- these methods have not been widely used, because they have problems in that they require long time for analysis is long and that analysis samples must be aseptically treated ⁇ Appl. Microbiol. Biotechnol., 76:91, 2007).
- methods of analyzing amino acids using bioluminescent auxotrophic E. coli mutants have been reported (LB. Zabala et al., Appl. Microbiol. BiotechnoL, 62:268, 2003).
- amino acids must be analyzed in solution using a microtiter plate without immobilizing an auxotrophic mutant on a substrate. For this reason, when various kinds of amino acids are analyzed, an E. coli mutant, an analysis sample and a solution such as buffer must be sequentially added to each well. Accordingly, when there are plural amino acids to be analyzed are plural, a large amount of a sample and a plurality of pippeting procedures are required, and thus it is unsuitable for use as commercial analysis kits.
- the present inventors have made many efforts to develop a method for analyzing the concentrations of various kinds of amino acids in a sample in an accurate and simple manner.
- the present inventors have developed a biochip, in which a plurality of E.
- coli mutants auxotrophic for different amino acids are immobilized on a flat substrate, and have found that, when the proliferation of cells in a sample applied to the biochip is measured, the concentrations of various kinds of amino acids in the sample can be analyzed in an accurate and simple manner, thereby completing the present invention.
- Another object of the present invention is to provide a method for analyzing the concentrations of amino acids in a sample using the biochip.
- the present invention provides a biochip for the quantitative analysis of amino acids, in which a plurality of E. coli mutants auxotrophic for different amino acids are immobilized on a flat substrate.
- the growth of the E. coli mutants preferably increases in proportion to the concentrations of the specific amino acids.
- the E. coli mutants are preferably obtained using a method selected from the group consisting of mutagen treatment, homologous recombination and transposon insertion.
- the immobilization of the E. coli mutant on the flat substrate is preferably performed using an immobilizing substance selected from the group consisting of agar, agarose, sodium alginate, sol-gel, chitosan, collagen, carrageenan, polyvinyl alcohol, polyurethane, polyethylene glycol and polyacrylamide.
- an immobilizing substance selected from the group consisting of agar, agarose, sodium alginate, sol-gel, chitosan, collagen, carrageenan, polyvinyl alcohol, polyurethane, polyethylene glycol and polyacrylamide.
- the amino acids are preferably selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, aspartate, asparagine, glutamate, tyrosine, lysine, arginine, histidine, phenylalanine, glutamine, tryptophan, proline, taurine, sarcosine, homocysteine, betaine, citrulline, hydroxylproline and beta-alanine.
- the flat substrate is preferably made of a material selected from the group consisting of plasties, glass, silicon, hydrogels, ceramics, metals and porous membranes.
- the present invention provides a method for analyzing the concentrations of amino acids, the method comprises the steps of: (a) treating an amino acid-containing sample to said biochip and incubating the sample treated biochip; (b) adding a biomarker to the biochip treated with the amino acid- containing sample; and (c) analyzing the concentrations of amino acids in the sample by measuring the luminescence of the biomarker according to the proliferation of the mutant cells in the sample.
- the biomarker is preferably selected from the group consisting of fluorescent dyes, luminescent dyes and color-developing dyes. Also, the biomarker is preferably a substance which can penetrate the cell membrane of the mutants.
- the mutants preferably have a luminescent gene-containing recombinant vector introduced therein, and the luminescent gene is preferably a gene encoding luciferase.
- the present invention provides a method for analyzing the concentrations of amino acids, the method comprises the steps of: (a) applying an amino acid-containing sample and a luminescent gene transcription inducer to a biochip, in which mutants having a luminescent gene-containing recombinant vector introduced into a plurality of E. coli mutants auxotrophic for different amino acids, are immobilized on a flat substrate, and incubating the treated biochip; and
- the amino acid-containing sample is preferably selected from the group consisting of blood, food, injection solutions, nutrients and urine.
- the biochip preferably has at least two kinds of amino acid auxotrophic strains immobilized thereon.
- the biochip is preferably configured such that the amino acid auxotrophic strains immobilized at the respective locations grow at the same time.
- FIG. 1 shows a process of measuring the concentrations of amino acids using the inventive biochip for the quantitative analysis of amino acids.
- FIG. 2 illustrates photographs showing the results of luminescent analysis conducted to examine the selective specificities of the inventive biochip for 20 kinds of amino acids (glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, aspartate, asparagine, glutamate, tyrosine, lysine, arginine, histidine, phenylalanine, glutamine, tryptophan and proline).
- amino acids glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, aspartate, asparagine, glutamate, tyrosine, lysine, arginine, histidine, phenylalanine, glutamine, tryptophan and proline.
- FIG. 3 shows the growth of a methionine auxotrophic mutant according to the concentration of methionine, measured using a fluorescent dye syto9 and the inventive biochip for the quantitative analysis of six amino acids (methionine, isoleucine, leucine, threonine, arginine and lysine), which has a methionine auxotrophic mutant immobilized thereon.
- FIG. 4 illustrates photographs showing the results of luminescent analysis conducted using the inventive biochip for the quantitative analysis of six amino acids in order to examine the growth of amino acid auxotrophic mutants according to the concentration of each amino acid.
- FIG. 5 shows the change in luminescence according to the concentration of each amino acid, measured using the inventive biochip for the quantitative analysis of six amino acids.
- FIG. 6 shows the change in luminescence according to the concentration of each amino acid, measured using the inventive biochip for the quantitative analysis of 20 amino acids (glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, aspartate, asparagine, glutamate, tyrosine, lysine, arginine, histidine, phenylalanine, glutamine, tryptophan and proline).
- amino acids glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, aspartate, asparagine, glutamate, tyrosine, lysine, arginine, histidine, phenylalanine, glutamine, tryptophan and proline.
- FIG. 7 shows the change in luminescence according to the concentration of each of four amino acids (isoleucine, lysine, arginine and tyrosine), measured through ATP analysis using the inventive biochip for the quantitative analysis of 20 amino acids.
- FIG. 8 shows the change in luminescence according to the concentration of histidine in a human serum sample, measured using the inventive biochip for the quantitative analysis of 20 amino acids.
- the present invention relates to a a biochip for the quantitative analysis of amino acids, in which a plurality of E. coli mutants auxotrophic for different amino acids are immobilized on a flat substrate.
- Amino acid auxotrophic strains according to the present invention can be prepared by any one of the following three methods.
- the methods include a method of treating autotrophic microorganisms, having the ability to produce amino acids, with a chemical substance (e.g., NTG (N-methyl-N-nitro-N-nitrosoguanidine)), a method of obtaining amino acid auxotrophic mutants by inducing mutations using UV light, and a method of inactivating the ability of microorganisms to synthesize amino acids using DNA recombination technology or a transposon.
- a chemical substance e.g., NTG (N-methyl-N-nitro-N-nitrosoguanidine)
- the method that uses DNA recombination technology can be performed by introducing a nucleotide sequence or vector, having homology with a specific gene associated with the growth of microorganisms, into target microorganisms to induce homologous recombination.
- the introduced nucleotide sequence or vector contains a selection marker capable of selecting mutated microorganisms, which allows mutants to be easily selected.
- amino acid auxotrophic mutants were prepared by inducing mutations using a transposon.
- auxotrophic mutants for different amino acids were prepared by introducing a selection marker-containing transposon into an E. coli ATCC 11105 strain to randomly induce mutations in the chromosome, and then selecting strains, the growth of which was inhibited in an amino acid-deficient environment.
- the introduction of a transposon into the host cell may be performed by, but is not limited to, e.g., transformation or electroporation.
- a competent strain was made by a suitable method, such that external substances, such as DNA or transposons, would be easily introduced into the strain. Then, the microorganism was mixed with a transposon, the transposon was inserted into the microbial cell using a conventional technique such as an electric pulse method, and then the cell was cultured. Then, transformed strains were screened by an antibiotic-resistant gene contained in the transposon. Alternatively, the cell was cultured in an antibiotic-containing plate medium, and antibiotic-resistant strains, in which gene inactivation occurred due to the insertion of the transposon, were selected. Among the selected antibiotic-resistant strains, strains, which did not grow in minimal medium, but grew in a medium containing each of amino acids, were selected, and auxotrophic mutants for the corresponding amino acids were isolated.
- the present invention relates to a method for analyzing the concentrations of amino acids using the biochip.
- the method for analyzing the concentrations of amino acids according to the present invention comprises the steps of: (a) applying an amino acid-containing sample to said biochip and incubating the sample treated biochip; (b) adding a biomarker to the biochip treated with the amino acid-containing sample; and (c) analyzing the concentrations of amino acids in the sample by measuring the luminescence of the biomarker according to the proliferation of the mutant cells in the sample.
- the concentrations of amino acids in an unknown sample can be quantitatively measured by adding an amino acid- containing M9 minimal medium to each microplate of the biochip, incubating the biochip at 37 ° C for 4 hours, and then examining the growth of the amino acid auxotrophic mutants.
- any sample may be used, as long as it contains amino acids.
- the sample may include, but are not limited to, body fluids, such as blood, urine or serum, and food.
- the method of examining the growth of the amino acid auxotrophic mutants may be performed using various methods, such as fluorescence staining methods or enzyme immunoassays.
- the concentrations of amino acids in a sample may be measured by adding an amino acid-containing sample to the biochip for amino acid analysis, in which the above-prepared E. coli mutants auxotrophic for different amino acids are immobilized on a flat substrate, culturing the cells of the mutants, staining the cells with a fluorescent substance (e.g., syto9 (Invitrogen, USA)), which can penetrate the cell membrane of the mutants, at room temperature for 1 hour, and measuring the luminescence of the fluorescent substance according to the proliferation of the cells using a fluorimeter.
- a fluorescent substance e.g., syto9 (Invitrogen, USA)
- amino acid auxotrophic mutants in order to quantitatively analyze the growth of the amino acid auxotophic mutants according to the amino acid concentrations in proportion to the expression of a recombinant luciferase gene, amino acid auxotrophic mutants, into which a recombinant plasmid pTAC-luc containing a gene encoding luciferase is introduced, were prepared.
- the amino acid auxotrophic mutants prepared according to the present invention are strains derived from E. coli ATCC 11105 and are auxotrophic for arginine, methionine, histidine, valine, isoleucine, leucine, phenylalanine, alanine, threonine, cysteine, glutamate, glutamine, aspartate, asparagine, tryptophan, tyrosine, serine, proline, glycine and lysine.
- the above-prepared amino acid auxotrophic mutants were immobilized on a flat substrate. In a preferred embodiment, the mutants were mixed with agar and solidified.
- agar in order to immobilize the amino acid auxotrophic mutants, 0.7% agar was completely dissolved by heating at 100°C , and then was cooled slowly to about 50 ° C . Then, the cooled agar was mixed with each of the amino acid auxotrophic mutants, and 0.1 ml of each of the mixtures was dispensed into a microplate and left to stand at room temperature, such that the substrate could be sufficiently solidified.
- the substrate prepared by the immobilization of the auxotrophic mutants can be changed depending on temperature, and the number of cells immobilized on the substrate may vary depending on temperature.
- the concentrations of amino acids can be measured by measuring the proliferation of cells through the luminescence of the luminescent gene.
- the present invention relates to a method for analyzing the concentrations of amino acids, the method comprises the steps of: (a) applying an amino acid-containing sample and a luminescent gene transcription inducer to a biochip, in which mutants having a luminescent gene-containing recombinant vector introduced into a plurality of E. coli mutants auxotrophic for different amino acids, are immobilized on a flat substrate, and incubating the treated biochip; and (b) measuring the expression of the luminescent gene or the amount of ATP according to the proliferation of the mutant cells in the sample, thus analyzing the concentrations of amino acids in the sample.
- a recombinant vector containing a luminescent gene was introduced into a plurality of E. coli mutants auxotrophic for different amino acids, and the resulting mutants were immobilized on a flat substrate to prepare a biochip. Then, the quantitative analysis of amino acid concentrations was attempted by measuring the expression of the luminescent gene using the biochip or by detecting a substance showing luminescence by binding to ATP, the concentration of which increased with the growth of cells.
- E. coli strain (ATCC 11105) was inoculated into 3 ml of LB medium (10 g/L trypton, 5 g/L yeast extract and 10 g/L NaCl) and cultured overnight. Then, the strain cells were collected by centrifugation and washed twice with 10% glycerol aqueous solution. Then, the cells were centrifuged again and suspended in 10% glycerol aqueous solution, thus preparing a competent E. coli strain such that a substance, such as DNA or transposons, would be easily introduced there into.
- LB medium 10 g/L trypton, 5 g/L yeast extract and 10 g/L NaCl
- the resulting strain was cultured at 37 ° C for 1 hour. Then, the cultured strain was diluted with LB medium at a ratio of 1 : 10 and plated on a selection medium containing 50 mg/L kanamycin. Then, the strain was cultured overnight at 37 ° C , thus obtaining a transformed strain.
- the transformed strain was plated on a selection medium, containing 50 mg/L kanamycin, and M9 minimal medium (Table 1) containing 1 nM cyanocobalamin, and was cultured overnight at 37 " C .
- amino acid auxotrophic mutants were performed by selecting strains, which grew on an antibiotic-containing agar plate, but did not grow in minimal medium, and isolating an amino acid auxotrophic mutant, in which a specific gene associated with microbial growth was inactivated by an antibiotic- resistant gene.
- the primarily selected auxotrophic mutant was inoculated into 3 ml of a kanamycin-containing LB medium and was then cultured overnight. Then, the culture medium was centrifuged to collect a strain, and the remaining LB medium component was removed using minimal medium. Then, the strain was inoculated in a cell culture tube, containing 0.1 ml of M9 minimal medium and 50 ⁇ M of each amino acid, at a ratio of 1 :500, and then was cultured at 37 0 C overnight. Then, auxotrophic mutants for the corresponding amino acids, which grew in the medium containing each amino acid, were selected.
- amino acid auxotrophic mutants In order to quantitatively analyze the growth of the amino acid auxotrophic mutants according to the concentration of amino acids through the expression of the recombinant luminescent gene, amino acid auxotrophic mutants, into which a recombinant plasmid pTAC-luc, containing a luciferase-encoding gene is introduced, were prepared.
- a recombinant plasmid pTAC-luc the Tl lac of a pETDuet-1 plasmid (Novagen, San Diego, CA, USA) was substituted with a tac promoter.
- a Photinus pyralis (firefly)-derived luciferase-encoding gene was amplified by PCR using a pGL3 -Basic plasmid (Promega, WI, USA) as a template with oligoprimers of SEQ ID NO: 1 (JVcoI-luc-F) and SEQ ID NO: 2 (JECORI-IUC-R).
- SEQ ID NO: 1 5'-CATGCCATGGAAGACGCCAAAAACATAAAGAAAGGC-S'
- SEQ ID NO: 2 5'-CGGAATTCCTAGAATTACACGGCGATCTTTCCGC-S'
- the amplified gene was digested with restriction enzymes Nc ⁇ l and EcoRI, and then ligated into a Ptac plasmid treated with the same restriction enzymes, thus constructing a recombinant plasmid pTAC-luc.
- the constructed recombinant plasmid pTAC-luc was introduced into each of the amino acid auxotrophic mutants, prepared in Example I 5 using an electric pulse method, thus preparing amino acid auxotrophic mutants containing the recombinant luminescent gene.
- each culture broth was centrifuged to collect the mutants, which were then suspended in M9 minimal medium and centrifuged again. The process of suspension and centrifugation was repeated three times, .thus removing the remaining LB medium component.
- Each of the resulting amino acid auxotrophic mutants was suspended in M9 minimal medium, and about 1 x 10 6 cells of each of the mutants were taken and mixed with an agar solution.
- the agar solution was prepared by sterilizing 0.7% agar solution at high pressure and high temperature, and then cooling the sterilized solution to about 50 ° C .
- 0.1 ml of each of the mixtures was dispensed into two rows of a 96-well microplate arranged in 8 columns by 12 rows, and was then left to stand at room temperature for 30 minutes, such that the substrate could be solidified, thus preparing a biochip for the quantitative analysis of six amino acids.
- Each of the 20 amino acid auxotrophic mutants (auxotrophic for arginine, methionine, histidine, valine, isoleucine, leucine, phenylalanine, alanine, threonine, cysteine, glutamate, glutamine, aspartate, asparagine, tryptophan, tyrosine, serine, proline, glycine and lysine), prepared in Example 2, which contain the recombinant luciferase gene, was mixed with the agar solution according to the above-described method.
- the biochip having 20 spots was treated with 10 ⁇ M of each of amino acid solutions, and after 3 hours, the luminescence of the luciferase gene was measured. As a result, it could be observed that the luminescence of the luciderase gene was detected only at the spot corresponding to each of the supplied amino acids (FIG. 2).
- Example 3-1 To the biochip for the quantitative analysis of 6 amino acids, prepared in Example 3-1, which has a methionine auxotrophic mutant immobilized thereon, a minimal medium containing 3 ⁇ M methionine was added, and then the biochip was incubated at 37 °C for 4 hours. Then, a fluorescent dye syto9 (Invitrogen, USA) capable of penetrating the cell membrane of the mutants was added to sterilized water to each of final concentrations of 0.2 mM and 2 mM, and 0.1 ml of each of the solutions was added to the biochip having the methionine auxotrophic mutant immobilized thereon, and then was left to stand at room temperature for 1 hour.
- a fluorescent dye syto9 Invitrogen, USA
- the change in the luminescence of the luciferase gene was measured at 488/525 using a luminometer.
- the concentration of the amino acid was proportional to the luminescence which increased along with the proliferation of cells.
- Example 3-1 Into each well of the biochip for the quantitative analysis of 6 amino acids, prepared in Example 3-1, a solution, containing 0.1 ml of M9 minimal medium, 1 nM cyanocobalamin and each of amino acids having varying concentrations as shown in Table 3, was dispensed. As a control group, only 0.1 ml of M9 minimal medium was dispensed into the first column of the biochip. The biochip was cultured at 37 °C for 4 hours, and then 0.1 ml of a BacTiter-GloTM Microbial Cell Viability Assay Kit (Promega, USA) was added to each well of the biochip and allowed to react at room temperature for 10 minutes. Then, the amount of ATP generated from the mutants was measured using a laminometer.
- a solution containing 0.1 ml of M9 minimal medium, 1 nM cyanocobalamin and each of amino acids having varying concentrations as shown in Table 3, was dispensed.
- As a control group only
- the concentrations of the amino acids were proportional to the amount of ATP which increased along with the proliferation of cells.
- the recombinant plasmid pTAC-luc prepared in Example 2, shows luminescence, when the substrate D-luciferin and IPTG inducing transcription of a luciferase- encoding gene, are supplied.
- the concentration of each of the amino acids was proportional to the expression level of luciferase, which increased along with the proliferation of cells.
- Example 3-2 To the biochip for the quantitative analysis of 20 amino acids, prepared in Example 3-2, a BacTiter-GloTM Microbial Cell Viability Assay Kit (Promega, USA) was added, and the change in luminescence according to the amount of ATP was measured.
- a solution containing 0.1 ml of M9 minimal medium, 1 nM cyanocobalamin and four different amino acids (isoleucine, lysine, arginine and tyrosine) at varying concentrations as shown in Table 4, was dispensed into the biochip prepared in Example 3-2.
- the biochip was incubated at 37 °C for 3 hours, and then 0.1 ml of a BacTiter-GloTM Microbial Cell Viability Assay Kit (Promega, USA) was added to each well of the biochip and allowed to react at room temperature for 10 minutes. Then, the amount of ATP generated from each of the mutants was measured using a luminometer. Because the amount of ATP generated was proportional to the number of cells, the use of the measurement values allowed the concentrations of the amino acids in the sample to be analyzed.
- the concentrations of the amino acids were proportional to the amount of ATP, which increased along with the proliferation of cells.
- the concentrations of amino acids in human serum were measured through the change in luminescence.
- the concentrations of amino acids in the serum were measured using the analysis method described in Example 5-3.
- the luminescence of the recombinant luciferase gene was increased in proportion to the concentrations of amino acids in the serum.
- the concentration of histidine in human serum was measured through the change in luminescence.
- M9 minimal medium containing each of 0, 6, 12 and 18 ⁇ M histidine was added to the biochip.
- the biochip having the serum added thereto was incubated at 37 ° C for 3 hours, and then the concentration of the amino acid in the serum was measured using the analysis method described in Example 5-3.
- the measurement value was 15830 (RLU), and the concentration of histidine in human serum, calculated based on the standard curve, was about 10.5 ⁇ M (FIG. 8).
- the present invention provides a biochip for the quantitative analysis of amino acids, in which E. coli mutants auxotrophic for different amino acids, the growth of which increases in proportion to the concentrations of specific amino acids, are immobilized on a flat substrate, and a method for analyzing amino acids using the biochip.
- the concentrations of amino acids in a sample can be simply analyzed only by adding the sample and measuring an increase in the number of cells.
- the analysis method has an economical effect of greatly reducing the costs, required for amino acid analysis, because it does not require expensive equipment or reagents.
- the inventive biochip for the quantitative analysis of amino acids is used, various kinds of amino acids can be analyzed in an accurate and simple manner.
- the biochip according to the present invention allows miniaturization of analysis kits, and thus it can be applied in an analysis process requiring small samples or an analysis process requiring a large amount of analytes.
Abstract
Disclosed herein are a biochip for the quantitative analysis of amino acids and a method for analyzing amino acids using the same. More particularly, disclosed are a biochip for the quantitative analysis of amino acids, in which a plurality of E. coli mutants auxotrophic for different amino acids, the growth of which increases in proportion to the concentrations of specific amino acids, are immobilized on a flat substrate, and a method for analyzing the concentrations of amino acids using the biochip. The use of the disclosed biochip allows the concentrations of various kinds of amino acids to be analyzed in an accurate and simple manner and, in addition, provides an economical effect of greatly reducing the costs, required for amino acid analysis, because it does not require expensive analysis equipments or reagents.
Description
BIOCHIP FOR QUANTIFYING AMINO ACID AND METHOD FOR ANALYING AMINO ACID USING THE SAME
TECHNICAL FIELD
The present invention relates to a biochip for the quantitative analysis of amino acids and a method for analyzing amino acids using the same, and more particularly to a biochip for the quantitative analysis of amino acids, in which a plurality of E. coli mutants auxotrophic for different amino acids, the growth of which increases in proportion to the concentrations of specific amino acids, are immobilized on a flat substrate, as well as a method for analyzing the concentrations of amino acids using the biochip.
BACKGROUND ART
Amino acids are the basic structural building units of proteins, and examples thereof include 20 kinds of amino acids, including glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, aspartate, asparagine, glutamate, tyrosine, lysine, arginine, histidine, phenylalanine, glutamine, tryptophan and proline. These amino acids are classified into two groups: essential amino acids, which are not synthesized in vivo and must be supplied in the food, and nonessential amino acids. Such amino acids are important in human metabolic processes, and when the supply thereof is insufficient, various disorders will occur, thus leading to various diseases.
The concentration of amino acids in blood or urine indicates the nutritional status of the body, and thus the monitoring of the concentration of these amino acids in body fluids is important for verifying the therapeutic effects of diet or vitamin
supplements. In addition to this verification of therapeutic effects, the concentrations of phenylalanine, leucine and methionine are used as important markers in diagnosing congenital metabolic diseases in newborns.
Methods for analyzing amino acid concentration include thin-layer chromatography, electrophoresis, mass spectrometry, liquid chromatography and the like, and among them, ion-exchange liquid chromatography with post-column reactions, and with liquid column chromatography with pre-column reactions, are mainly used as commercial amino acid analyzers, because they are convenient to use and has high measurement accuracy (Slocum, R.H., Techniques in diagnostic human biochemical genetics: a laboratory manual, 87, 1991). Recently, tandem mass spectrometry (MS/MS), which performs high speed analysis and can handle a large amount of samples, has been recognized as important equipment for the diagnosis of congenital metabolic diseases in newborns.
Such prior methods for analyzing amino acids have problems in that, because they require complex extraction and derivatization procedures, they are unsuitable for handling a large amount of samples, and the derivatization reactions of amino acids differ depending on the kind of sample, thus making accurate quantitative analysis difficult. Also, the quantative analysis of amino acids has a shortcoming in that it uses expensive equipment and complex analysis methods. For this reason, there is a need to develop a method which can analyze the concentrations of amino acids in samples in a simple and accurate manner.
As other methods for analyzing amino acids, microbiological assays, which use microorganisms auxotrophic for specific amino acids, have been used for more than 60 years. However, these methods have not been widely used, because they have problems in that they require long time for analysis is long and that analysis samples must be aseptically treated {Appl. Microbiol. Biotechnol., 76:91, 2007).
In attempts to solve such problems, methods of analyzing amino acids using bioluminescent auxotrophic E. coli mutants have been reported (LB. Zabala et al., Appl. Microbiol. BiotechnoL, 62:268, 2003). However, in such methods, amino acids must be analyzed in solution using a microtiter plate without immobilizing an auxotrophic mutant on a substrate. For this reason, when various kinds of amino acids are analyzed, an E. coli mutant, an analysis sample and a solution such as buffer must be sequentially added to each well. Accordingly, when there are plural amino acids to be analyzed are plural, a large amount of a sample and a plurality of pippeting procedures are required, and thus it is unsuitable for use as commercial analysis kits.
In addition, an analysis method, comprising preparing a lysine auxotrophic E. coli mutant and measuring the growth of the mutant at various amino acids using a spectrophotometer, was reported (X. Li et al, Lett. Appl. Microbiol., 36:458, 2003). However, this method has a problem in that the concentrations of various kinds of amino acids cannot be measured at the same time in a simple manner.
Meanwhile, a method of analyzing tryptophan by immobilizing an amino acid auxotrophic microorganism on a silicon substrate and measuring the change in pH of medium, which occurs due to the change of metabolic action of E. coli according to the change in the amount of tryptophan in a sample, was reported (Atsushi Seki et al., Sensors and Actuators B, 94:253, 2003). This method has an advantage in that it shortens the analysis time, but has a problem in that it is difficult to perform accurate quantitative analysis, unlike the prior microbiological assay of measuring the growth of microorganisms according to the amount of amino acid.
Thus, in the art to which the present invention pertains, there is an urgent need to develop a method capable of accurately analyzing a plurality of samples at the same time in a simple and cost-effective manner.
Accordingly, the present inventors have made many efforts to develop a method for analyzing the concentrations of various kinds of amino acids in a sample in an accurate and simple manner. As a result, the present inventors have developed a biochip, in which a plurality of E. coli mutants auxotrophic for different amino acids, the cell growth of which increases in proportion to the concentrations of specific amino acids, are immobilized on a flat substrate, and have found that, when the proliferation of cells in a sample applied to the biochip is measured, the concentrations of various kinds of amino acids in the sample can be analyzed in an accurate and simple manner, thereby completing the present invention.
SUMMARY OF INVENTION
It is a main object of the present invention to provide a biochip for the quantitative analysis of amino acids, which can analyze various kinds of amino acids at the same time.
Another object of the present invention is to provide a method for analyzing the concentrations of amino acids in a sample using the biochip.
To achieve the above objects, in one aspect, the present invention provides a biochip for the quantitative analysis of amino acids, in which a plurality of E. coli mutants auxotrophic for different amino acids are immobilized on a flat substrate.
In the present invention, the growth of the E. coli mutants preferably increases in proportion to the concentrations of the specific amino acids. Also, the E. coli mutants are preferably obtained using a method selected from the group consisting of mutagen treatment, homologous recombination and transposon insertion.
In the present invention, the immobilization of the E. coli mutant on the flat substrate is preferably performed using an immobilizing substance selected from
the group consisting of agar, agarose, sodium alginate, sol-gel, chitosan, collagen, carrageenan, polyvinyl alcohol, polyurethane, polyethylene glycol and polyacrylamide.
In the present invention, the amino acids are preferably selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, aspartate, asparagine, glutamate, tyrosine, lysine, arginine, histidine, phenylalanine, glutamine, tryptophan, proline, taurine, sarcosine, homocysteine, betaine, citrulline, hydroxylproline and beta-alanine.
In the present invention, the flat substrate is preferably made of a material selected from the group consisting of plasties, glass, silicon, hydrogels, ceramics, metals and porous membranes.
In another aspect, the present invention provides a method for analyzing the concentrations of amino acids, the method comprises the steps of: (a) treating an amino acid-containing sample to said biochip and incubating the sample treated biochip; (b) adding a biomarker to the biochip treated with the amino acid- containing sample; and (c) analyzing the concentrations of amino acids in the sample by measuring the luminescence of the biomarker according to the proliferation of the mutant cells in the sample.
In the present invention, the biomarker is preferably selected from the group consisting of fluorescent dyes, luminescent dyes and color-developing dyes. Also, the biomarker is preferably a substance which can penetrate the cell membrane of the mutants.
In addition, in the present invention, the mutants preferably have a luminescent gene-containing recombinant vector introduced therein, and the luminescent gene is preferably a gene encoding luciferase.
In still another aspect, the present invention provides a method for analyzing the concentrations of amino acids, the method comprises the steps of: (a) applying an amino acid-containing sample and a luminescent gene transcription inducer to a biochip, in which mutants having a luminescent gene-containing recombinant vector introduced into a plurality of E. coli mutants auxotrophic for different amino acids, are immobilized on a flat substrate, and incubating the treated biochip; and
(b) measuring the expression of the luminescent gene or the amount of ATP according to the proliferation of the mutant cells in the sample, thus analyzing the concentrations of amino acids in the sample.
In the present invention, the amino acid-containing sample is preferably selected from the group consisting of blood, food, injection solutions, nutrients and urine.
In the present invention, the biochip preferably has at least two kinds of amino acid auxotrophic strains immobilized thereon. The biochip is preferably configured such that the amino acid auxotrophic strains immobilized at the respective locations grow at the same time.
Other features and aspects of the present invention will be apparent from the following detailed description and the appended claims.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows a process of measuring the concentrations of amino acids using the inventive biochip for the quantitative analysis of amino acids.
FIG. 2 illustrates photographs showing the results of luminescent analysis conducted to examine the selective specificities of the inventive biochip for 20 kinds of amino acids (glycine, alanine, valine, leucine, isoleucine, threonine, serine,
cysteine, methionine, aspartate, asparagine, glutamate, tyrosine, lysine, arginine, histidine, phenylalanine, glutamine, tryptophan and proline).
FIG. 3 shows the growth of a methionine auxotrophic mutant according to the concentration of methionine, measured using a fluorescent dye syto9 and the inventive biochip for the quantitative analysis of six amino acids (methionine, isoleucine, leucine, threonine, arginine and lysine), which has a methionine auxotrophic mutant immobilized thereon.
FIG. 4 illustrates photographs showing the results of luminescent analysis conducted using the inventive biochip for the quantitative analysis of six amino acids in order to examine the growth of amino acid auxotrophic mutants according to the concentration of each amino acid.
FIG. 5 shows the change in luminescence according to the concentration of each amino acid, measured using the inventive biochip for the quantitative analysis of six amino acids.
FIG. 6 shows the change in luminescence according to the concentration of each amino acid, measured using the inventive biochip for the quantitative analysis of 20 amino acids (glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, aspartate, asparagine, glutamate, tyrosine, lysine, arginine, histidine, phenylalanine, glutamine, tryptophan and proline).
FIG. 7 shows the change in luminescence according to the concentration of each of four amino acids (isoleucine, lysine, arginine and tyrosine), measured through ATP analysis using the inventive biochip for the quantitative analysis of 20 amino acids.
FIG. 8 shows the change in luminescence according to the concentration of histidine in a human serum sample, measured using the inventive biochip for the
quantitative analysis of 20 amino acids.
DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS
In one aspect, the present invention relates to a a biochip for the quantitative analysis of amino acids, in which a plurality of E. coli mutants auxotrophic for different amino acids are immobilized on a flat substrate.
Amino acid auxotrophic strains according to the present invention can be prepared by any one of the following three methods. The methods include a method of treating autotrophic microorganisms, having the ability to produce amino acids, with a chemical substance (e.g., NTG (N-methyl-N-nitro-N-nitrosoguanidine)), a method of obtaining amino acid auxotrophic mutants by inducing mutations using UV light, and a method of inactivating the ability of microorganisms to synthesize amino acids using DNA recombination technology or a transposon.
The method that uses DNA recombination technology can be performed by introducing a nucleotide sequence or vector, having homology with a specific gene associated with the growth of microorganisms, into target microorganisms to induce homologous recombination. Herein, the introduced nucleotide sequence or vector contains a selection marker capable of selecting mutated microorganisms, which allows mutants to be easily selected.
In one example of the present invention, amino acid auxotrophic mutants were prepared by inducing mutations using a transposon.
Specifically, auxotrophic mutants for different amino acids were prepared by introducing a selection marker-containing transposon into an E. coli ATCC 11105 strain to randomly induce mutations in the chromosome, and then selecting strains,
the growth of which was inhibited in an amino acid-deficient environment. The introduction of a transposon into the host cell may be performed by, but is not limited to, e.g., transformation or electroporation.
In the case where the transposon was introduced into the host cell by transformation, a competent strain was made by a suitable method, such that external substances, such as DNA or transposons, would be easily introduced into the strain. Then, the microorganism was mixed with a transposon, the transposon was inserted into the microbial cell using a conventional technique such as an electric pulse method, and then the cell was cultured. Then, transformed strains were screened by an antibiotic-resistant gene contained in the transposon. Alternatively, the cell was cultured in an antibiotic-containing plate medium, and antibiotic-resistant strains, in which gene inactivation occurred due to the insertion of the transposon, were selected. Among the selected antibiotic-resistant strains, strains, which did not grow in minimal medium, but grew in a medium containing each of amino acids, were selected, and auxotrophic mutants for the corresponding amino acids were isolated.
In another aspect, the present invention relates to a method for analyzing the concentrations of amino acids using the biochip. The method for analyzing the concentrations of amino acids according to the present invention comprises the steps of: (a) applying an amino acid-containing sample to said biochip and incubating the sample treated biochip; (b) adding a biomarker to the biochip treated with the amino acid-containing sample; and (c) analyzing the concentrations of amino acids in the sample by measuring the luminescence of the biomarker according to the proliferation of the mutant cells in the sample.
According to the present invention, the concentrations of amino acids in an unknown sample can be quantitatively measured by adding an amino acid- containing M9 minimal medium to each microplate of the biochip, incubating the
biochip at 37 °C for 4 hours, and then examining the growth of the amino acid auxotrophic mutants. As the sample, any sample may be used, as long as it contains amino acids. Examples of the sample may include, but are not limited to, body fluids, such as blood, urine or serum, and food.
The method of examining the growth of the amino acid auxotrophic mutants may be performed using various methods, such as fluorescence staining methods or enzyme immunoassays.
For example, the concentrations of amino acids in a sample may be measured by adding an amino acid-containing sample to the biochip for amino acid analysis, in which the above-prepared E. coli mutants auxotrophic for different amino acids are immobilized on a flat substrate, culturing the cells of the mutants, staining the cells with a fluorescent substance (e.g., syto9 (Invitrogen, USA)), which can penetrate the cell membrane of the mutants, at room temperature for 1 hour, and measuring the luminescence of the fluorescent substance according to the proliferation of the cells using a fluorimeter.
In another Example of the present invention, in order to quantitatively analyze the growth of the amino acid auxotophic mutants according to the amino acid concentrations in proportion to the expression of a recombinant luciferase gene, amino acid auxotrophic mutants, into which a recombinant plasmid pTAC-luc containing a gene encoding luciferase is introduced, were prepared.
The amino acid auxotrophic mutants prepared according to the present invention are strains derived from E. coli ATCC 11105 and are auxotrophic for arginine, methionine, histidine, valine, isoleucine, leucine, phenylalanine, alanine, threonine, cysteine, glutamate, glutamine, aspartate, asparagine, tryptophan, tyrosine, serine, proline, glycine and lysine.
In another Example of the present invention, the above-prepared amino acid auxotrophic mutants were immobilized on a flat substrate. In a preferred embodiment, the mutants were mixed with agar and solidified. That is, in order to immobilize the amino acid auxotrophic mutants, 0.7% agar was completely dissolved by heating at 100°C , and then was cooled slowly to about 50 °C . Then, the cooled agar was mixed with each of the amino acid auxotrophic mutants, and 0.1 ml of each of the mixtures was dispensed into a microplate and left to stand at room temperature, such that the substrate could be sufficiently solidified. The substrate prepared by the immobilization of the auxotrophic mutants can be changed depending on temperature, and the number of cells immobilized on the substrate may vary depending on temperature.
As a result, when a luminescent gene is introduced into the amino acid auxotrophic mutants, the concentrations of amino acids can be measured by measuring the proliferation of cells through the luminescence of the luminescent gene.
In still another aspect, the present invention relates to a method for analyzing the concentrations of amino acids, the method comprises the steps of: (a) applying an amino acid-containing sample and a luminescent gene transcription inducer to a biochip, in which mutants having a luminescent gene-containing recombinant vector introduced into a plurality of E. coli mutants auxotrophic for different amino acids, are immobilized on a flat substrate, and incubating the treated biochip; and (b) measuring the expression of the luminescent gene or the amount of ATP according to the proliferation of the mutant cells in the sample, thus analyzing the concentrations of amino acids in the sample.
In the present invention, a recombinant vector containing a luminescent gene was introduced into a plurality of E. coli mutants auxotrophic for different amino acids, and the resulting mutants were immobilized on a flat substrate to prepare a biochip. Then, the quantitative analysis of amino acid concentrations was attempted by
measuring the expression of the luminescent gene using the biochip or by detecting a substance showing luminescence by binding to ATP, the concentration of which increased with the growth of cells.
Examples
Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.
Example 1: Preparation of amino acid auxotrophic E. coli mutants using Tn5 transposon
An E. coli strain (ATCC 11105) was inoculated into 3 ml of LB medium (10 g/L trypton, 5 g/L yeast extract and 10 g/L NaCl) and cultured overnight. Then, the strain cells were collected by centrifugation and washed twice with 10% glycerol aqueous solution. Then, the cells were centrifuged again and suspended in 10% glycerol aqueous solution, thus preparing a competent E. coli strain such that a substance, such as DNA or transposons, would be easily introduced there into.
1 μl of an EZ-T5TM<KAN>Tn5 transposon (EPICENTRE Biotechnologies, USA) was introduced into the competent E. coli strain by an electric pulse method (Ausbuel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY(1987), Miller and Nickoloff, J.A., Escherichia coli electrotransformation, in Molecular Biology, 47, Nickoloff, J.A., ed., Humana Press, Totowa, NY, 105(1995)), and then an SOC medium (20 g bacto-tryptone, 5 g bacto-yeast extract, 0.5 g NaCl, 10 ml of 250 raM KCl, 20 mM MgCl2, 20 rnM glucose/L) was added to the strain. The resulting strain was cultured at 37 °C for 1 hour.
Then, the cultured strain was diluted with LB medium at a ratio of 1 : 10 and plated on a selection medium containing 50 mg/L kanamycin. Then, the strain was cultured overnight at 37 °C , thus obtaining a transformed strain. The transformed strain was plated on a selection medium, containing 50 mg/L kanamycin, and M9 minimal medium (Table 1) containing 1 nM cyanocobalamin, and was cultured overnight at 37 "C .
The selection of amino acid auxotrophic mutants was performed by selecting strains, which grew on an antibiotic-containing agar plate, but did not grow in minimal medium, and isolating an amino acid auxotrophic mutant, in which a specific gene associated with microbial growth was inactivated by an antibiotic- resistant gene.
Table 1: Composition of minimal medium
In order to obtain an auxotrophic mutant, showing specificity for each of amino acids, from the primarily selected amino acid auxotrophic mutant, the primarily selected auxotrophic mutant was inoculated into 3 ml of a kanamycin-containing LB medium and was then cultured overnight. Then, the culture medium was centrifuged to collect a strain, and the remaining LB medium component was removed using minimal medium.
Then, the strain was inoculated in a cell culture tube, containing 0.1 ml of M9 minimal medium and 50 μM of each amino acid, at a ratio of 1 :500, and then was cultured at 370C overnight. Then, auxotrophic mutants for the corresponding amino acids, which grew in the medium containing each amino acid, were selected.
The growth of the auxotrophic mutants showing specificity for each amino acid was measured using a spectrophotometer (Table 2).
Table 2: Measurement results (OD60O values) for growth of each auxotrophic mutant in M9 + amino acid medium
Example 2: Preparation of auxotrophic E. colt mutants containing recombinant
luminescent gene
In order to quantitatively analyze the growth of the amino acid auxotrophic mutants according to the concentration of amino acids through the expression of the recombinant luminescent gene, amino acid auxotrophic mutants, into which a recombinant plasmid pTAC-luc, containing a luciferase-encoding gene is introduced, were prepared.
Specifically, in order to construct a recombinant plasmid pTAC-luc, the Tl lac of a pETDuet-1 plasmid (Novagen, San Diego, CA, USA) was substituted with a tac promoter. Also, a Photinus pyralis (firefly)-derived luciferase-encoding gene was amplified by PCR using a pGL3 -Basic plasmid (Promega, WI, USA) as a template with oligoprimers of SEQ ID NO: 1 (JVcoI-luc-F) and SEQ ID NO: 2 (JECORI-IUC-R). SEQ ID NO: 1 : 5'-CATGCCATGGAAGACGCCAAAAACATAAAGAAAGGC-S'
SEQ ID NO: 2: 5'-CGGAATTCCTAGAATTACACGGCGATCTTTCCGC-S'
The amplified gene was digested with restriction enzymes Ncøl and EcoRI, and then ligated into a Ptac plasmid treated with the same restriction enzymes, thus constructing a recombinant plasmid pTAC-luc.
The constructed recombinant plasmid pTAC-luc was introduced into each of the amino acid auxotrophic mutants, prepared in Example I5 using an electric pulse method, thus preparing amino acid auxotrophic mutants containing the recombinant luminescent gene.
Example 3: Preparation of biochip for quantitative analysis of amino acids
3-1 : Preparation of biochip for quantitative analysis of 6 amino acids
To prepare a biochip for amino acid analysis, each of 6 mutants (methionine auxotrophic mutant, isoleucine auxotrophic mutant, leucine auxotrophic mutant, threonine auxotrophic mutant, arginine auxotrophic mutant and lysine auxotrophic mutant) among the amino acid auxotrophic mutants obtained in Example 1 was inoculated into 3 ml of kanamycin-containing LB medium, and was then cultured overnight.
Then, each culture broth was centrifuged to collect the mutants, which were then suspended in M9 minimal medium and centrifuged again. The process of suspension and centrifugation was repeated three times, .thus removing the remaining LB medium component. Each of the resulting amino acid auxotrophic mutants was suspended in M9 minimal medium, and about 1 x 106 cells of each of the mutants were taken and mixed with an agar solution. Herein, the agar solution was prepared by sterilizing 0.7% agar solution at high pressure and high temperature, and then cooling the sterilized solution to about 50 °C .
0.1 ml of each of the mixtures was dispensed into two rows of a 96-well microplate arranged in 8 columns by 12 rows, and was then left to stand at room temperature for 30 minutes, such that the substrate could be solidified, thus preparing a biochip for the quantitative analysis of six amino acids.
3-2: Preparation of biochip for quantitative analysis of 20 amino acids
Each of the 20 amino acid auxotrophic mutants (auxotrophic for arginine, methionine, histidine, valine, isoleucine, leucine, phenylalanine, alanine, threonine, cysteine, glutamate, glutamine, aspartate, asparagine, tryptophan, tyrosine, serine, proline, glycine and lysine), prepared in Example 2, which contain the recombinant luciferase gene, was mixed with the agar solution according to the above-described method. Then, 0.1 ml of each of the mixtures was dispensed onto a flat plastic substrate made of polystyrene material, such that one spot per mutant was formed
on the substrate. Then, the substrate was left to stand at room temperature for 30 minutes, such that it could be solidified, thus preparing a biochip for the quantitative analysis of 20 amino acids.
Example 4: Measurement of selective specificity of biochip for quantitative analysis of amino acids
In order to examine whether the biochip for the quantitative analysis of 20 amino acids, prepared in Example 3-2, has high specificities for amino acids, the biochip having 20 spots was treated with 10 μM of each of amino acid solutions, and after 3 hours, the luminescence of the luciferase gene was measured. As a result, it could be observed that the luminescence of the luciderase gene was detected only at the spot corresponding to each of the supplied amino acids (FIG. 2).
Example 5: Analysis of amino acids using biochip for quantitative analysis of amino acids
5-1 : Analysis with luminometer
To the biochip for the quantitative analysis of 6 amino acids, prepared in Example 3-1, which has a methionine auxotrophic mutant immobilized thereon, a minimal medium containing 3 μM methionine was added, and then the biochip was incubated at 37 °C for 4 hours. Then, a fluorescent dye syto9 (Invitrogen, USA) capable of penetrating the cell membrane of the mutants was added to sterilized water to each of final concentrations of 0.2 mM and 2 mM, and 0.1 ml of each of the solutions was added to the biochip having the methionine auxotrophic mutant immobilized thereon, and then was left to stand at room temperature for 1 hour. Then, the change in the luminescence of the luciferase gene was measured at 488/525 using a luminometer.
As a result, as shown in FIG. 3, the concentration of the amino acid was proportional to the luminescence which increased along with the proliferation of cells.
5-2: Analysis by intracellular ATP in biochip for quantitative analysis of 6 amino acids
Into each well of the biochip for the quantitative analysis of 6 amino acids, prepared in Example 3-1, a solution, containing 0.1 ml of M9 minimal medium, 1 nM cyanocobalamin and each of amino acids having varying concentrations as shown in Table 3, was dispensed. As a control group, only 0.1 ml of M9 minimal medium was dispensed into the first column of the biochip. The biochip was cultured at 37 °C for 4 hours, and then 0.1 ml of a BacTiter-GloTM Microbial Cell Viability Assay Kit (Promega, USA) was added to each well of the biochip and allowed to react at room temperature for 10 minutes. Then, the amount of ATP generated from the mutants was measured using a laminometer.
As a result, as shown in FIG. 4 and FIG. 5, the concentrations of the amino acids were proportional to the amount of ATP which increased along with the proliferation of cells.
Table 3: Auxotrophic mutants, used in biochip for quantitative analysis of amino acids, and amino acid concentrations
The recombinant plasmid pTAC-luc, prepared in Example 2, shows luminescence, when the substrate D-luciferin and IPTG inducing transcription of a luciferase- encoding gene, are supplied.
For this reason, to the biochip for the quantitative analysis of 20 amino acids, prepared in Example 3-2, 100 μl of a sample, containing each amino acid, 1 nM cyanocobalamin and 1 mM IPTG, was added, and then incubated at 37 °C for 3 hours. Then, 100 μl of a luciferin solution (1 mM D-luciferin in 0.1 M Na-citrate buffer (pH 5.0)) was added thereto. After about 10 minutes, the luminescence in the biochip was measured using a luminometer (Perkin Elmer).
As a result, as can be in FIG. 6, the concentration of each of the amino acids was proportional to the expression level of luciferase, which increased along with the proliferation of cells.
5-4: Analysis by intracellular ATP in biochip for quantitative analysis of 20 amino acids
To the biochip for the quantitative analysis of 20 amino acids, prepared in Example 3-2, a BacTiter-GloTM Microbial Cell Viability Assay Kit (Promega, USA) was added, and the change in luminescence according to the amount of ATP was measured.
Specifically, a solution, containing 0.1 ml of M9 minimal medium, 1 nM cyanocobalamin and four different amino acids (isoleucine, lysine, arginine and tyrosine) at varying concentrations as shown in Table 4, was dispensed into the biochip prepared in Example 3-2. The biochip was incubated at 37 °C for 3 hours, and then 0.1 ml of a BacTiter-GloTM Microbial Cell Viability Assay Kit (Promega,
USA) was added to each well of the biochip and allowed to react at room temperature for 10 minutes. Then, the amount of ATP generated from each of the mutants was measured using a luminometer. Because the amount of ATP generated was proportional to the number of cells, the use of the measurement values allowed the concentrations of the amino acids in the sample to be analyzed.
As a result, as shown in FIG. 7, the concentrations of the amino acids were proportional to the amount of ATP, which increased along with the proliferation of cells.
Table 4: Amino acid auxotrophic mutants, used in biochip for amino acid analysis, and amino acid concentrations
Example 6: Analysis of amino acids in serum sample using biochip for quantitative analysis of amino acids
Using the inventive biochip for the quantitative analysis of amino acids, the concentrations of amino acids in human serum were measured through the change in luminescence. Specifically, to the biochip for the quantitative analysis of 20 amino acids, prepared in Example 3-2, human serum, which is the supernatant obtained by centrifugation, was added at a concentration of M9 minimal medium: serum = 20: 1, and then incubated at 37 °C for 3 hours. Then, the concentrations of amino acids in the serum were measured using the analysis method described in Example 5-3.
As a result, the luminescence of the recombinant luciferase gene was increased in proportion to the concentrations of amino acids in the serum.
Also, using the inventive biochip for the quantitative analysis of amino acids, the concentration of histidine in human serum was measured through the change in luminescence. Specifically, to the inventive biochip for the quantitative analysis of 20 amino acids, prepared in Example 3-2, human serum, which was the supernatant obtained by centrifugation, was added at a concentration of M9 minimal medium: serum = 20:1. Moreover, in order to obtain a standard curve of histidine concentration, 0.1 ml of M9 minimal medium containing each of 0, 6, 12 and 18 μM histidine was added to the biochip. Then, the biochip having the serum added thereto was incubated at 37°C for 3 hours, and then the concentration of the amino acid in the serum was measured using the analysis method described in Example 5-3.
As a result, the measurement value was 15830 (RLU), and the concentration of histidine in human serum, calculated based on the standard curve, was about 10.5 μM (FIG. 8).
INDUSTRIALAPPLICABILITY
As described in detail above, the present invention provides a biochip for the quantitative analysis of amino acids, in which E. coli mutants auxotrophic for different amino acids, the growth of which increases in proportion to the concentrations of specific amino acids, are immobilized on a flat substrate, and a method for analyzing amino acids using the biochip. According to the analysis method of the present invention, the concentrations of amino acids in a sample can be simply analyzed only by adding the sample and measuring an increase in the number of cells. In addition, the analysis method has an economical effect of greatly reducing the costs, required for amino acid analysis, because it does not
require expensive equipment or reagents. Moreover, when the inventive biochip for the quantitative analysis of amino acids is used, various kinds of amino acids can be analyzed in an accurate and simple manner. In addition, the biochip according to the present invention allows miniaturization of analysis kits, and thus it can be applied in an analysis process requiring small samples or an analysis process requiring a large amount of analytes.
Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.
Claims
THE CLAIMS
What is Claimed is:
L A biochip for the quantitative analysis of amino acids, in which a plurality of E. coli mutants auxotrophic for different amino acids are immobilized on a flat substrate.
2. The biochip for the quantitative analysis of amino acids according to claim I5 wherein the growth of said E. coli mutants increases in proportion to the concentrations of the specific amino acids.
3. The biochip for the quantitative analysis of amino acids according to claim 1, wherein said E. coli mutants are obtained using a method selected from the group consisting of mutagen treatment, homologous recombination and transposon insertion.
4. The biochip for the quantitative analysis of amino acids according to claim 1, wherein the immobilization of the E. coli mutant on the flat substrate is performed using an immobilizing substance selected from the group consisting of agar, agarose, sodium alginate, sol-gel, chitosan, collagen, carrageenan, polyvinyl alcohol, polyurethane, polyethylene glycol and polyacrylamide.
5. The biochip for the quantitative analysis of amino acids according to claim 1, wherein the amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, aspartate, asparagine, glutamate, tyrosine, lysine, arginine, histidine, phenylalanine, glutamine, tryptophan, proline, taurine, sarcosine, homocysteine, betaine, citrulline, hydroxylproline and beta-alanine.
6. The biochip for the quantitative analysis of amino acids according to claim 1, wherein the flat substrate is made of a material selected from the group consisting of plastics, glass, silicon, hydrogel, ceramics, metals and porous membranes.
7. The biochip for the quantitative analysis of amino acids according to claim 1, wherein said mutants have a luminescent gene-containing recombinant vector introduced therein.
8. The biochip for the quantitative analysis of amino acids according to claim 7, wherein the luminescent gene is a gene encoding luciferase
9. A method for analyzing the concentrations of amino acids, the method comprising the steps of:
(a) applying an amino acid-containing sample to said biochip of claim 1 and incubating the sample treated biochip;
(b) adding a biomarker to the biochip treated with the amino acid-containing sample; and
(c) analyzing the concentrations of amino acids in the sample by measuring the luminescence of the biomarker according to the proliferation of the mutant cells in the sample.
10. The method for analyzing the concentrations of amino acids according to claim 9, wherein the biomarker is selected from the group consisting of fluorescent dyes, luminescent dyes and color-developing dyes.
11. The method for analyzing the concentrations of amino acids according to claim 9, wherein the biomarker is a substance which can penetrate the cell membrane of the mutants.
12. A method for analyzing the concentrations of amino acids, the method comprising the steps of:
(a) applying an amino acid-containing sample and a luminescent gene transcription inducer to the biochip of claim 7, and incubating the sample treated biochip; and
(b) analyzing the concentrations of amino acids in the sample by measuring the expression of the luminescent gene or the amount of ATP according to the proliferation of the mutant cells in the sample.
13. The method for analyzing the concentrations of amino acids according to claim 9 or 12, wherein the amino acid-containing sample is selected from the group consisting of blood, food, injection solutions, nutrients and urine.
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| KR101211095B1 (en) * | 2010-05-20 | 2012-12-11 | 주식회사 랩 지노믹스 | Microorganism for quantifying homocysteine and use thereof |
| US20130095497A1 (en) * | 2010-05-20 | 2013-04-18 | Labgenomics Co., Ltd. | Microorganism for quantifying homocysteine, and use thereof |
| US8759021B2 (en) * | 2010-05-20 | 2014-06-24 | Labgenomics Co., Ltd. | Microorganism for quantifying homocysteine, and use thereof |
| KR102047827B1 (en) * | 2019-05-28 | 2019-11-22 | 한국생산기술연구원 | Biochip for amino acid quantitative analysis, kit comprising the biochip for amino acid quantitative analysis, and method of quantitative analysis using the same |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101688232A (en) | 2010-03-31 |
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