KR102047827B1 - Biochip for amino acid quantitative analysis, kit comprising the biochip for amino acid quantitative analysis, and method of quantitative analysis using the same - Google Patents

Abstract

Provided are a biochip for amino acid quantitative analysis capable of maintaining activity even after long-term storage for 90 days or more, a kit for amino acid quantitative analysis and a method for amino acid quantitative analysis. The biochip for amino acid quantitative analysis includes auxotrophic microorganisms, trehalose and agarose.

Description

BIOCHIP FOR AMINO ACID QUANTITATIVE ANALYSIS, KIT COMPRISING THE BIOCHIP FOR AMINO ACID QUANTITATIVE ANALYSIS, AND METHOD OF QUANTITATIVE ANALYSIS USING THE SAME

The present invention relates to amino acid quantification biochips, amino acid quantification kits and amino acid quantification methods. More particularly, the present invention relates to an amino acid quantitative biochip, an amino acid quantitative analysis kit, and an amino acid quantitative analysis method including trehalose, which can maintain activity even at a relatively high temperature for longer than 90 days.

Amino acids are substances of the basic units that make up proteins. Examples of major amino acids are glycine, alanine, valine, leucine, isoleucine, threonine, serine, cysteine, methionine, Aspartic acid, asparagine, glutamate, tyrosine, lysine, arginine, histidine, phenylalanine, glutamine, glutamine, Tryptophan and proline.

Such amino acids may be classified into essential and non-essential amino acids, depending on whether they can be synthesized in the body. Non-essential amino acids can be synthesized in the body, while essential amino acids are not synthesized in the body and can be consumed only through food. Among the major amino acids, valine, leucine, isoleucine, threonine, methionine, lysine, phenylalanine and tryptophan are examples of essential amino acids.

These amino acids are important for metabolic processes in the human body, and since the concentration of amino acids in the blood or urine reflects the nutritional status of the body, it is very important to detect the amino acid concentrations in these body fluids. For example, by monitoring the concentration of amino acids, it is possible to verify the therapeutic effect of diet or vitamin preparation, nutritional status analysis, diagnosis of congenital metabolic diseases, and the like.

Korea Patent Registration No. 10-1211095 (2012.12.11.) Chinese public patent CN 101688232 A (2010.03.31.) US published patent US 2007-0031913 A1 (2007.02.08.) US published patent US 2010-0017147 A1 (2010.01.21.) Korea Patent Registration No. 10-1541756 (2015.08.05.)

 Analytical Chemistry, 82 (10), 4072-4077, 2010  Analytical Chemistry, 73, 5972, 2001  Food Chemistry, 51, 3629, 2003

Conventional analytical techniques, such as high-performance liquid chromatography (HPLC), mass spectrometry (MS) and immunoassay, are currently used to quantify the concentration of amino acids. Such methods are widely used for the clinical monitoring of biological fluids, but have disadvantages such as complexity and inefficiency of labeling, low stability and low detection sensitivity. In addition, in order to perform these methods requires expensive precision analysis equipment and skilled workers, there is a limit that takes a lot of time and cost.

Patent Literature 1 (Korean Patent No. 10-1211095) and Non-Patent Literature 1 (Analytical Chemistry, 82 (10), 4072-4077, 2010) describe specific amino acids that increase cell growth in proportion to specific amino acid concentrations. Microbial biochips have been disclosed in which nutrient-required E. coli mutant strains are fixed on 96-well plate substrates to quantitatively analyze amino acids through luminescence proteins expressed according to cell growth.

By using the microbial biochip according to the disclosure of the patent document 1 and the non-patent document 1, not only can accurately and easily analyze the concentration of various types of amino acids at the same time, but also does not require expensive analysis equipment or reagents, amino acid analysis There is an economic effect that can significantly lower costs.

However, in order to utilize the biochip for amino acid analysis at the actual diagnosis site, the activity of the developed biochip must be preserved for a long time. However, the biochip according to the disclosure of Patent Literature 1 and Non-Patent Literature 1 has a problem in that activity decreases rapidly over time. The activity of such a biochip is a very important problem for applying to the actual diagnosis site because it causes a decrease in sensitivity of the sensor.

Patent document 2 (CN 101688232 A) discloses a biochip for quantitative analysis of amino acids similarly to patent document 1. Patent document 2 discloses the invention which is considerably similar to patent document 1 in that it uses the nutrient-conjugated E. coli mutant strain immobilized on the board | substrate, and the limitation which cannot be utilized in a real diagnosis site is also the same as patent document 1.

In addition, Patent Document 3 (US 2007-0031913 A1) discloses an invention using an electrode containing a medium and an enzyme reacting with a specific amino acid in order to measure the total concentration of the specific amino acid. Such an attempt is also revealed in Patent Document 4 (US 2010-0017147 A1). Patent Literature 3 and Patent Literature 4 also attempt to easily quantitatively analyze amino acids, but there is a problem that requires expensive precise analysis equipment.

Patent Document 5 (Korean Registered Patent No. 10-1541756), Non-Patent Document 2 (Analytical Chemistry, 73, 5972, 2001) and Non-Patent Document 3 (Food Chemistry, 51, 3629, 2003) detect a amino acid There is disclosed a conjugated compound for improving the sensitivity of the color and the fluorescence change. However, they are very limited to be used as biosensors.

The problem to be solved by the present invention is to provide a biochip that has high stability and detection sensitivity, and can be easily quantitative analysis of amino acids without expensive precision analysis equipment and skilled workers.

Furthermore, the present invention provides an amino acid quantitative biochip capable of long-term storage by maintaining activity despite time and having high detection sensitivity. For example, the present invention provides an amino acid quantitative analysis biochip that maintains linearity of amino acid diagnosis even if stored at -20 ° C for 90 days or more.

Another problem to be solved by the present invention is to provide an amino acid quantitative analysis kit that retains activity even for long-term storage.

Another problem to be solved by the present invention is to provide an amino acid quantitative analysis method that maintains activity even for long-term storage.

The objects of the present invention are not limited to the above-mentioned technical problem, and other technical problems not mentioned will be clearly understood by those skilled in the art from the following description.

Amino acid quantitative analysis biochip according to an embodiment of the present invention for solving the above problems includes nutrient-containing microorganisms, trehalose and agarose.

The trophic microorganism may include arginine demanding Escherichia coli.

Alternatively, the trophic microorganism may include isoleucine-required Escherichia coli.

Alternatively, the trophic microorganism may include lysine-required E. coli.

Alternatively, the trophic microorganism may include leucine-required E. coli.

Alternatively, the trophic microorganism may include methionine-required Escherichia coli.

Here, the amount of trehalose may be 4.9% (v / v) to 5.1% (v / v).

The arginine-required Escherichia coli may be prepared by selecting an arginine-required strain induced by inserting a transfer factor into E. coli ATCC1105 strain and inserting a plasmid expressing luciferase or fluorescent protein into the selected strain. .

The E. coli can express luciferase or fluorescent protein.

The transition factor may include a KAN-2 transition factor (Tn5).

In addition, the plasmid may comprise pTAC-luc.

In addition, the quantitative analysis biochip may include dimethyl sulfoxide, glycerol, ethylene glycol, polyethylene glycol and sucrose as active ingredients.

Further, the amino acid quantitative analysis biochip immobilized with the arginine-required Escherichia coli on agarose may have a diagnostic linearity (R ² ) of 0.980 or more when stored at −20 ° C. for 90 days or less.

Amino acid quantitative analysis kit according to an embodiment of the present invention for solving the other problem is a substrate; And any of the aforementioned amino acid quantitative analysis biochips disposed on the substrate.

A quantitative analysis method of amino acids according to an embodiment of the present invention for solving the another problem comprises the steps of preparing an amino acid quantitative analysis biochip comprising a nutrient-containing microorganism, trehalose and agarose; Contacting the amino acid quantitative analysis biochip with a sample; And measuring the luminescence of the biochip to quantify the amino acid content in the sample.

At this time, the diagnostic linearity (R ² ) of the amino acid quantitative analysis biochip may be 0.980 or more.

Preparing the amino acid quantitative analysis biochip, the step of culturing and cultivating the ureotrophic Escherichia coli and antibiotics in LB medium, after culturing, centrifugation, removing the supernatant and washing with M9 minimal medium, and After washing, it may comprise the step of solidifying by mixing with agarose and trehalose.

The method may further include storing the amino acid quantitative analysis biochip at -20 ° C between preparing the amino acid quantitative analysis biochip and contacting the biochip with a sample.

Specific details of other embodiments are included in the detailed description.

The inventors have invented a (R ² ≒ 1) preservative component that maintains the linearity (luminescence signal vs. amino acid concentration) of amino acid diagnosis for more than 90 days at -20 ° C, and the conditions for its use, thereby remarkably preserving the amino acid diagnostic biochip. Improved.

That is, according to the present invention, the amount of amino acid in the sample can be precisely determined through a microorganism expressing the growth in proportion to the amount of amino acids in the sample, for example, arginine, isoleucine, leucine, lysine and methionine, and thus luminescence or fluorescence. Not only can it be quickly quantified but also preserved for longer than 90 days and its activity is not lost, so it can be usefully used at actual diagnosis sites.

Effects according to embodiments of the present invention are not limited by the contents exemplified above, and more various effects are included in the present specification.

1 is a view showing a method for producing an amino acid quantitative analysis biochip according to an embodiment of the present invention and a method for quantifying amino acids using the same.
Figure 2 is a view showing the results of the 16 days diagnostic straightness confirmation experiment (Example 5-1) according to the type of preservatives performed in the immobilized state.
Figure 3 is a view showing the results of the 16-day diagnostic straightness confirmation experiment (Example 5-2) according to the kind of preservatives performed in the immobilized state.
4 is a diagram showing the results of a 90-day diagnostic straightness confirmation experiment (Example 6) according to the primary screened preservative type.
FIG. 5 shows the results of a 90-day arginine diagnostic bioluminescence intensity confirmation experiment (Example 7) according to the primary screened preservative type.
FIG. 6 is an actual bioluminescence image of chemi-doc measured on the 90-day biochip.
FIG. 7 shows the results of a 90-day arginine diagnostic slope confirmation experiment (Example 8) according to the primary screened preservative type.
8 shows the results of a diagnostic evaluation confirmation experiment (Example 9) for other amino acids.

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various forms, and only the embodiments are provided to make the disclosure of the present invention complete, and those of ordinary skill in the art to which the present invention pertains. It is provided to fully inform the scope of the invention, and the invention is defined only by the scope of the claims.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In this specification, 'and / or' includes each and all combinations of one or more of the items mentioned. In addition, the singular also includes the plural unless specifically stated in the text. As used herein, 'comprises' and / or 'comprising' does not exclude the presence or addition of one or more other components in addition to the mentioned components. Numerical ranges shown using 'to' indicate numerical ranges including the values described before and after the lower limit and the upper limit, respectively. "About" or "approximately" means a value or numerical range within 20% of the value or numerical range described thereafter.

As used herein, the term 'vector' is a means for efficiently inducing the expression of a gene of interest by introducing DNA into a host cell, and refers to a gene construct comprising essential regulatory elements operably linked to express a particular sequence. do. In addition, the term "transformer" as used herein refers to an organism in which the genetic trait has changed due to the introduction of foreign genetic material. In addition, the term 'operably linked' as used herein refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest and the like are functionally linked to perform a general function. do. For example, a promoter and a nucleic acid sequence encoding a protein or peptide can be operably linked to affect expression of the coding sequence.

Unless otherwise defined, all terms (including technical and scientific terms) used in the present specification may be used in a sense that can be commonly understood by those skilled in the art. In addition, the terms defined in the commonly used dictionaries are not ideally or excessively interpreted unless they are specifically defined clearly.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

One embodiment of the present invention provides a biochip for quantitative analysis of amino acids. The biochip for quantitative analysis of amino acids according to the present embodiment includes auxotrophs and trehalose, and may further include agarose for immobilizing them.

The microtrophic microorganism may be an amino acid-required Escherichia coli (auxotrophic E. coli). For example, the amino acid demanding Escherichia coli may grow in proportion to the concentration of any one, or more than one, of the amino acids in minimal medium. For example, the amino acid requirement Escherichia coli can be prepared through a transposon mutagenesis method. The transfer factor refers to a DNA sequence capable of causing mutation by a method such as transferring its position in a gene or inverting the sequence. When the transfer factor is inserted into the gene for synthesizing the amino acid, it can be induced to become an auxotroph of the amino acid by inactivating the function of the amino acid synthesis gene. For example, the transition factor may be a KAN-2 transition factor (Tn5).

In addition, the amino acid requirement Escherichia coli can express a luciferase or fluorescent protein. That is, the amino acid-required Escherichia coli may include a gene encoding luciferase or a fluorescent protein, and the expression of the gene may be proportional to the growth of Escherichia coli. Luciferase may be expressed in proportion to the growth of E. coli. For example, amino acid-required E. coli expressing the luciferase or fluorescent protein can be prepared through the insertion of pTAC-luc. In the amino acid requirement Escherichia coli according to the present embodiment, the amount of light emitted by luciferase according to the growth of Escherichia coli may have a high linearity proportionality to the concentration of amino acids.

In an exemplary embodiment, the amino acid requirement Escherichia coli, which can express the luciferase or fluorescent protein, is a mutation produced by inserting a transposon into the gene of the strain E. coli ATCC1105 by electroporation. Screening for mutations having a requirement for a particular amino acid from the sieves, and (ii) inserting pTAC-luc into the selected amino acid demanding mutant strain by electroporation. This will be described later in detail with examples.

In addition, the E. coli can grow quantitatively after a short time, for example, 3 to 4 hours of incubation because of its rapid growth rate. Therefore, the amino acid-required Escherichia coli according to the present embodiment having a gene encoding luciferase or fluorescent protein and the amount of light emitted by the luciferase or the like is linear with respect to the amino acid concentration can be effectively used to quantify the amino acid concentration in the sample. have. In an exemplary embodiment, the amino acid may be any of arginine, isoleucine, lysine, leucine and methionine.

The present invention can provide an amino acid quantitative assay kit. The amino acid quantitative analysis kit may include a substrate and an amino acid quantitative biochip disposed on the substrate. The substrate may include one or more of glass, silicon, polymer resin, ceramic, metal, or porous body.

The present invention may also provide a method for quantitative analysis of amino acids. Method for quantitative analysis of amino acids (a) preparing the amino acid quantitative analysis biochip or amino acid quantitative analysis kit comprising the same, (b) storing the amino acid quantitative analysis biochip, etc. at -20 ℃, (c) amino acid quantification Contacting the analytical biochip with a sample at room temperature, and (d) measuring the luminescence of the biochip to quantify the amino acid content in the sample.

That is, the amino acid-required Escherichia coli, which has a nutrient composition for a specific amino acid and a gene expressing luciferase, is contacted with the sample to be cultured and immobilized, and the amount of luciferase expression is measured to measure the amino acid in the sample. It can be converted into content.

In some embodiments, the step of preparing the (a) amino acid quantitative analysis biochip, (a-1) administering and culturing the trophic Escherichia coli and antibiotics in the LB medium, (a-2) centrifugation thereof, Removing the supernatant and washing with M9 minimal medium, and (a-3) solidifying by mixing the residue with agarose and trehalose.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

<Preparation of biochips using nutritional microorganisms>

1 is a view showing a method for producing an amino acid quantitative analysis biochip according to an embodiment of the present invention and a method for quantifying amino acids using the same.

The manufacturing process of the biochip for quantitative analysis of amino acids and a method of quantifying amino acids using the same have been described above, and a detailed description thereof may be more clearly understood through the following examples.

Example 1 Preparation of Amino Acid Auxotroph Escherichia Coli

Random insertion of transposons into the gene of E. coli W strain (ATCC1105) strain by electroporation followed by arginine (Arg) and isoleucine (Lrg) on LB (Luria-Bertani) medium plate and M9 medium plate Ile), lysine (Lys), leucine (Leu) or methionine (Met) strains showing auxotrophic (auxotrophic), respectively.

Example 1 is explained in more detail.

First, E. coli W (Accession No. ATCC1105) was prepared. Strains generated by randomly inserting 1 μl of KAN-2 transfer factor (Tn5) were cultured in LB medium plates containing kanamycin (kanamycin, Km; 50 μg / ml), and mutants were selected.

The selected mutants were selected from a M9 medium plate containing kanamycin and each amino acid (arginine, isoleucine, lysine, leucine and methionine), and a replica medium plate screening method was selected from an M9 medium plate containing kanamycin only. After use, the nutritional requirements for the amino acids of the candidate auxotrophs (ie arginine, isoleucine, lysine, leucine and methionine) in 3 ml of M9 liquid medium with or without the corresponding amino acid were confirmed.

Through this, arginine-required E. coli, isoleucine-required E. coli, lysine-required E. coli, leucine-required E. coli and methionine-required E. coli were prepared.

Example 2 Preparation of Bioluminescent Escherichia Coli

Example 2-1: Preparation of pTAC

pTAC was prepared by replacing the T7 lac promoter of the pETDuet-1 vector (Novagen, San Diego, CA) with the tac promoter.

Example 2-2 Preparation of pTAC-luc

First, luc (gene) from pGL3-Basic vector (Promega, WI) was amplified by PCR using the following pair of primers. The luc (gene) is a gene encoding luciferase. Since luciferase is an enzyme that exhibits bioluminescence, a strain into which luc (gene) is introduced may fluoresce as it grows.

Nco I-luc-F (5′-CATGCCATGGAAGACGCCAAAAACATAAAGAAAGGC-3 ′)

Eco RI-luc-R (5′-CGGAATTCCTAGAATTACACGGC GATCTTTCCGC-3 ′)

The Nco I-luc-F and Eco RI-luc-R are primers that recognize Nco I and Eco RI restriction enzymes, respectively.

The amplified luc fragment was digested with Nco I and Eco RI. And pTAC-luc was prepared by cleaving pTAC prepared in Example 2-1 with the same enzymes ( Nco I and Eco RI) and introducing the cleaved luc fragment thereto.

The primers used were synthesized in Bioneer (company) (Daejeon, Korea).

Example 2-3 Preparation of Bioluminescent Escherichia Coli

PTAC-luc prepared in Example 2-2 by electroporation using a Gene Pulser system (Bio-Rad, CA) to the arginine-required E. coli, isoleucine-required E. coli, lysine requirements prepared in Example 1 E. coli, leucine-required E. coli and methionine-required E. coli were introduced respectively.

Through the above-described process, amino acid quantitative strains that can be used for quantitative analysis of any of arginine, isoleucine, lysine, leucine and methionine were prepared.

Example 3: Preparation of Microbial Biochips

A microbial biochip was prepared using the arginine-required Escherichia coli, the isoleucine-required Escherichia coli, the lysine-required Escherichia coli, the leucine-required Escherichia coli, and the methionine-required Escherichia coli prepared in Example 2 prepared with pTAC-luc.

Example 3 is explained in more detail.

Example 3-1 Culture of Amino Acid Requirement Escherichia Coli

Each amino acid-required Escherichia coli and 50 µg / ml of antibiotics (ampicillin, kanamycin) were administered to the LB medium and cultured overnight in a shaker incubator. After incubation, centrifugation was performed at 8000 rpm and 4 ° C for 5 minutes. The supernatant was then discarded and the cultured cells washed twice with M9 minimal medium.

Example 3-2 Preservative Addition and Immobilization

The cell number of 2 × 10 ⁶ of the cells cultured in Example 3-1 was mixed with any one of 3% (low gelling) agarose and a preservative candidate group. At this time, the mixture of agarose and cultured cells was prepared by mixing at a volume ratio of 1: 1. 100 μl was then distributed to each well of a bioluminescent 96-well plate. Then waited for about 30 minutes until the agarose hardened. When the agarose completely hardened, it was packaged and stored at -20 ° C and -80 ° C.

Candidates for preservatives here are dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, polyethylene glycol, sucrose, trehalose, Six were used.

Example 3-3 Preservative Addition and Unfixing

The cell number of 2 × 10 ⁶ of the cells cultured in Example 3-1 was mixed with any one of the preservative candidate groups. 100 μl was then distributed to each well of a bioluminescent 96-well plate. And it stored at -20 degreeC conditions and -80 degreeC conditions. That is, a preservative was added and stored in the same manner as in Example 3-2, except that the agarose was not immobilized.

Example 4 Preliminary Diagnostic Evaluation of Storage of Biochips

Arginine-required E. coli biochips (including DMSO, glycerol, ethylene glycol, polyethylene glycol, sucrose, or trehalose) prepared in Example 3-2 and stored for 90 days at -20 ° C and -80 ° C conditions were subjected to 25 ° C. Thaw for 1 hour at.

And a solution consisting of 4 x M9 medium, vitamin B12 (Vit.B12), IPTG (isopropyl-1-thio-β-D-galactopyranoside), and arginine were prepared. The solution was prepared for each concentration gradient so that the concentration of arginine was from 0 to 25 μM and distributed on 100 μl of the thawed biochip.

Then, in this state and incubated for 4 hours at 37 ℃ conditions and the diagnostic evaluation using the chemi-doc, multi microplate reader all confirmed that the luminescent properties to some extent.

Example 5 Diagnosis of Diagnostic Straightness According to Preservative Types

Example 5-1: Diagnostic Straightness Measurement According to Preservative Type in Immobilized State

The preservation of Arginine-required Escherichia coli prepared in Example 3-2 and stored at -20 ° C and -80 ° C was confirmed according to the storage period. Specifically, diagnostic straightness (R ² value, luminescence signal vs. amino acid concentration) for arginine at a concentration of 0 to 25 μM of the biochip according to the retention period for up to 16 days was measured and the results are shown in FIG. 2.

Figure 2 is a result of evaluating the arginine diagnostic straightness of the range of 0 ~ 25μM concentration in agarose gel fixation conditions for six preservative candidate group for 16 days.

Specifically, FIG. 2A is a result showing diagnostic straightness when 5% of six preservative candidate groups (DMSO, glycerol, ethylene glycol, polyethylene glycol, sucrose, or trehalose) were added and stored at −20 ° C. (with agarose gel). Figure 2b is a result showing the diagnostic straightness when 5% of six preservative candidate groups, respectively, and stored at -80 ℃ (with agarose gel). Figure 2c is a result showing the diagnostic straightness when 10% of six preservative candidate groups, respectively, and stored at -20 ℃ (with agarose gel). Figure 2d is a result showing the diagnostic straightness when 10% of six preservative candidate groups, respectively, and stored at -80 ℃ (with agarose gel).

Referring to Figures 2a to 2d, it can be seen that the agarose gel-based kit without the preservative (control) significantly loses diagnostic straightness when stored at -20 ℃. In addition, when using DMSO, ethylene glycol and polyethylene glycol it was confirmed that the diagnostic straightness is somewhat unstable.

In addition, when the 5% (v / v) is added compared to the 10% (v / v) preservatives, it can be confirmed that the diagnostic straightness is significantly stable. This suggests that the concentration of the preservative is preferably about 4.9% (v / v) to 5.1% (v / v), or about 5.0% (v / v).

Example 5-2: Diagnostic Straightness Measurement According to Preservative Type in the Unimmobilized State

The preservation of Arginine-required Escherichia coli prepared in Example 3-3 and stored at −20 ° C. and −80 ° C. was confirmed according to the storage period. Specifically, diagnostic straightness (R ² value) for arginine at a concentration of 0 to 25 μM of the biochip according to the retention period for up to 16 days was measured, and the results are shown in FIG. 3.

Figure 3 is a result of evaluating the arginine diagnostic straightness in the range of 0 ~ 25μM concentration in a condition that is not fixed by agarose gel for six preservative candidate group for 16 days.

Specifically, Figure 3a is a result showing the diagnostic straightness when stored in the storage agent at -20 ℃ 5% each of the six preservative candidate groups (DMSO, glycerol, ethylene glycol, polyethylene glycol, sucrose, or trehalose) 5% agarose gel). Figure 3b is a result showing the diagnostic straightness when 5% of each of the six preservative candidate groups, and stored at -80 ℃ (without agarose gel). Figure 3c is a result showing the diagnostic straight-line when 10% of six preservative candidate groups, respectively, and stored at -20 ℃ (without agarose gel). Figure 3d is a result showing the diagnostic straightness when 10% of six preservative candidate groups, respectively, and stored at -80 ℃ (without agarose gel).

3A to 3D, it was confirmed that unstable diagnostic straightness was observed in the case of using the control agent, DMSO, ethylene glycol and polyethylene glycol, without using a preservative, even under conditions without immobilization with an agarose gel. Could.

Example 5-1 and Example 5-2 allowed the primary screening of preservative candidates, and further experiments were conducted using glycerol, sucrose and trehalose 5% (v / v) in the following examples. .

Example 6 Diagnosis of Diagnostic Straightness According to Preservative Type 2

Example 5 confirmed the shelf life of the primary screened preservative candidates (glycerol, sucrose, trehalose) according to the storage period of the arginine-required E. coli stored at -20 ℃ and -80 ℃ conditions. Specifically, diagnostic straightness (R ² value) for arginine at a concentration of 0 to 25 μM of the biochip according to the retention period for up to 90 days was measured and the results are shown in FIG. 4.

FIG. 4 shows the results of evaluating arginine diagnostic straightness in the range of 0-25 μM concentration over 90 days for three primary screened preservative candidate groups (5% (v / v)).

Specifically, FIG. 4A is a result showing diagnostic straightness when three preservative candidate groups (glycerol, sucrose, trehalose) were added and stored at -20 ° C (with agarose). 4B is a result showing the diagnostic straightness when three preservative candidate groups were added and stored at -80 ° C (with agarose).

4C is a result showing diagnostic straightness when three preservative candidate groups were added and stored at -20 ° C (without agarose). 4D is a result showing diagnostic straightness when three preservative candidate groups were added and stored at −80 ° C. (without agarose).

4A to 4D, it can be seen that the control straightness without treatment of the preservative completely loses diagnostic straightness at -20 ° C.

Moreover, 90 days after storage, when trehalose was used, it was confirmed that it has remarkably excellent diagnostic straightness. That is, when stored at -20 ° C under immobilized conditions, it can be seen that the biochip added with trehalose has a diagnostic straightness of about 0.980 or more, while glycerol has a diagnostic straightness of about 0.890 and sucrose about 0.780. In other words, it can be seen that the use of trehalose has about 10% p and 20% p improved diagnostic straightness compared to the case of using glycerol and sucrose.

Example 7 Arginine Diagnostic Bioluminescence Intensity Confirmation According to Preservative Type>

Example 5 evaluated the diagnostic bioluminescent intensity of arginine-required E. coli stored at −20 ° C. and −80 ° C. conditions for three primary screened preservative candidate groups (glycerol, sucrose, trehalose), and 5 is shown.

FIG. 5 shows the arginine diagnostic bioluminescence intensity at a concentration of 25 μM over 90 days for three primary screened preservative candidates (5% (v / v)).

Specifically, FIG. 5A shows the results of arginine diagnostic luminescence intensity when three preservative candidate groups (glycerol, sucrose, trehalose) were added and stored at -20 ° C (with agarose). FIG. 5B shows the results of arginine diagnostic luminescence intensity when three preservative candidate groups were added and stored at −80 ° C. (with agarose). FIG.

5C shows the results of arginine diagnostic luminescence intensity when three preservative candidate groups were added and stored at -20 ° C (without agarose). Fig. 5D shows the results of arginine diagnostic luminescence intensity when three preservative candidate groups were added and stored at -80 ° C (without agarose).

6 is an image measured by chemi-doc for arginine diagnostic luminescence of a biochip stored at −20 ° C. for 90 days.

5 and 6, it was confirmed that in most cases, the use of trehalose as a preservative showed superior emission intensity as compared with the other cases.

Example 8 Arginine Diagnostic Slope Confirmation According to Preservative Type

Example 5 evaluated the diagnostic slope of arginine-required E. coli stored at −20 ° C. and −80 ° C. conditions for three primary screened preservative candidate groups (glycerol, sucrose, trehalose) 7 is shown.

FIG. 7 shows arginine diagnostic slopes ranging from 0 to 25 μM concentration range over 90 days for three primary screened preservative candidates (5% (v / v)).

Specifically, FIG. 7A is a result showing an arginine diagnostic slope when three preservative candidate groups (glycerol, sucrose, trehalose) were added and stored at -20 ° C (with agarose). Fig. 7B shows the results of the arginine diagnostic slope when three preservative candidate groups were added and stored at -80 ° C (with agarose).

7C is the result which showed the arginine diagnostic slope at the time of adding three preservative candidate groups and storing at -20 degreeC (without agarose). FIG. 7D shows the results of an arginine diagnostic slope when three preservative candidate groups were added and stored at −80 ° C. (without agarose). FIG.

7A to 7D, it can be seen that in most cases, the use of trehalose as a preservative shows an excellent diagnostic slope compared to the other cases.

Example 9 Confirmation of Diagnostic Evaluation of Other Amino Acids

The isoleucine-required E. coli, lysine-required E. coli, leucine-required E. coli and methionine-required E. coli prepared in Example 3 were immobilized with agarose in the same manner as in Example 3-2. At this time, 5% (v / v) of trehalose was used as a preservative.

And bioluminescence intensity and coefficient of variation (*) of the biochip stored for 90 days at -20 ℃ condition was measured and shown in FIG.

Referring to FIG. 8, all of the amino acid-required Escherichia coli prepared according to the present invention had excellent bioluminescence intensity and coefficient of variation even after 90 days.

The biochip for quantitative analysis of amino acids according to the present invention can quantify concentrations of amino acids such as arginine, isoleucine, lysine, leucine and methionine in a sample, and express them as luminescence intensity.

Quantitative analysis of amino acids using these microorganisms, stability of storage is a very important factor. For example, degradation of diagnostic straightness, depending on storage period, storage temperature, and / or storage environment, results in unreliable biochip diagnostic properties at the actual diagnostic site and eventually unusable.

In particular, the actual diagnosis site does not have the facilities to maintain the ultra low temperature (for example, -80 ℃) to prevent the deterioration of the activity of the biochip, and only has a storage facility of -20 ℃ level. In addition, the preservation period should be able to be preserved for as short as 30 days to as long as 100 days.

The present inventors have applied them to biochips under various conditions from various preservative candidates, so that they can be used at the actual diagnosis site, that is, even if stored for more than 90 days at -20 ° C, the diagnostic straightness is not lost, and excellent diagnostic emission is achieved. A biochip capable of indicating strength and diagnostic slopes was completed.

Through this, it is possible to provide a biochip, a diagnostic kit, and a sensor device using the same, which can be mass-produced and quantify amino acids in a sample in a relatively simple manner. Accordingly, it is possible to reduce the blood test costs of patients including newborns, and to contribute to the health of the public by quickly and economically diagnosing the nutritional status of amino acids, blood sugar, and vitamins in the blood. The concentration of amino acids is also related to the degree of decay of the bacteria and / or food in the food. Therefore, the present invention may be used as a label for detecting the state of food.

Although described above with reference to the embodiments of the present invention, which is merely an example and not limiting the present invention, those skilled in the art to which the present invention pertains without departing from the essential characteristics of the embodiments of the present invention. It will be appreciated that various modifications and applications are not possible. For example, each component specifically shown in the embodiment of the present invention may be modified. And differences relating to such modifications and applications will have to be construed as being included in the scope of the invention defined in the appended claims.

Claims (11)

An amino acid quantitative analysis biochip for solving the problem of lowering diagnostic linearity when stored at -20 ° C, compared to when stored at -80 ° C,
Arginine requirement strains were selected by inserting a KAN-2 transfer factor (Tn5) into E. coli ATCC11105 strains, and pTAC-luc plasmids expressing luciferase or fluorescent protein were inserted into the selected strains. As the required Escherichia coli, Arginine requirement Escherichia coli expressing luciferase or fluorescent protein;
Trehalose 4.9% (v / v) to 5.1% (v / v); And
Including agarose,
Amino acid quantitative biochip with a diagnostic linearity (R ² ) of at least 0.980 when stored at −20 ° C. for more than 90 days. delete delete delete delete The method of claim 1,
The quantitative analysis biochip is an amino acid quantitative analysis biochip containing, as an active ingredient, dimethyl sulfoxide, glycerol, ethylene glycol, polyethylene glycol and sucrose. delete Board; And
Amino acid quantitative analysis kit comprising an amino acid quantitative analysis biochip according to claim 1 disposed on the substrate. Amino acid quantitative analysis biochip to solve the problem of lowering diagnostic linearity when stored at -20 ° C, compared to when stored at -80 ° C, and induced by inserting KAN-2 transfer factor (Tn5) into E. coli ATCC11105 strain Arginine-required Escherichia coli prepared by screening the purified arginine-required strains and inserting pTAC-luc plasmid expressing luciferase or fluorescent protein into the selected strains, and expressing luciferase or fluorescent protein. E. coli with requirement; Trehalose 4.9% (v / v) to 5.1% (v / v); And preparing an amino acid quantitative analysis biochip comprising agarose;
Contacting the amino acid quantitative analysis biochip with a sample; And
Comprising the step of quantifying the amino acid content of the sample by measuring the luminescence of the biochip,
Amino acid quantitative analysis method having a diagnostic linearity (R ² ) of 0.980 or more when stored at −20 ° C. for 90 days or more. The method of claim 9,
Preparing the amino acid quantitative analysis biochip,
Administering and culturing nutrient-forming Escherichia coli and antibiotics to LB medium,
After incubation, centrifugation, removing the supernatant and washing with M9 minimal medium, and
After washing, mixing with agarose and trehalose to solidify,
Amino acid quantitation method. The method of claim 10,
Between preparing the amino acid quantitative analysis biochip and contacting the biochip with a sample,
Amino acid quantitative analysis method comprising the step of storing the amino acid quantitative analysis biochip at -20 ℃.

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