KR20030008340A - Lab on a chip for the analysis of amine compound - Google Patents

Abstract

PURPOSE: A lap on a chip system for analysis of amine compounds is provided, thereby successively carrying out reaction and separation, and producing the lap on a chip system simply. CONSTITUTION: The lap on a chip system for analysis of amine compounds comprises a reactor for fluorescence induction of amine compounds and a separation channel for separation of each amine compound, wherein the reactor and the separation channel are sequentially connected on a board; the inner volume of the reactor ranges from 0.01 picoliter(pL) to 10 milliliter(mL); the separation channel has the width ranging from 10 nm to 10 cm, the height of 10 nm to 1 cm, and the length of 10 nm to 100 cm; the amine compound is at least one compound selected from the group consisting of alkylamine, aromatic amine, amino acid, biogenic amine and ammonia; and the lap on a chip is made of glass, crystal, melted silica or plastic. The lap on a chip system comprises a sample vessel(1), a reagent vessel(2), a reactor(3), a buffer solution vessel(4), a reacted solution discharging vessel(5), a buffer solution discharging vessel(6) and a separation channel(7).

Description

LAB ON A CHIP FOR THE ANALYSIS OF AMINE COMPOUND

The present invention relates to a lab-on-a-chip capable of analyzing the amine compound at high speed and high sensitivity. The chip of the present invention integrates a reactor and a separation channel for fluorescence derivatization of the amine compound so that the reaction and separation can be continuously performed within minutes. It can be done with

Amine compounds such as alkylamines, aromatic amines, ammonia, amino acids, biogenic amines and the like are physiologically and industrially important substances. For example, alkylamines and aromatic amines are widely used as raw materials for various medicines and pesticides, and amino acids are proteins that are important components of physiological activity, and biogenic amines are amino acids. It is a slightly toxic substance produced by fermentation or degradation, and it is contained in many foods, fermented foods and alcoholic beverages. These amine compounds are substances to be analyzed frequently for quality control, etc. of the produced products, in particular, the analysis of amino acids is an essential process to determine the composition of the protein or to investigate the nutritional components of the food.

In analytical methods currently used to analyze various amine compounds in liquid or gas phase, the compounds are separated using high pressure liquid chromatography (HPLC) or capillary electrophoresis (CE) devices, followed by absorption detectors or fluorescence. Many methods of detecting using a detector are used.

These amine compound analyzers have the advantage of excellent separation and reproducibility. In these devices, detection methods using a light absorption detector may be used for a variety of samples detected no restriction on the nature of the sample, but the detection limit is 10 ^-5 to 10 ^-7 molar degree fluorescence detection method (detection limit: 10 ^-9 - 10 ^{- 15} molar concentration) is not good sensitivity. On the other hand, the fluorescence detection method has a high sensitivity, but only a substance which shows fluorescence by irradiated light can be detected. A derivatization reaction in which a fluorescent label is reacted with a fluorescence substance is required.

There are three methods of attaching fluorescent labels to analytes. The first method is to react in advance in a test tube, and the second is precolumn reaction, which reacts in advance before separating a sample in the analyzer. And finally, a postcolumn reaction in which the sample is separated and then reacted in the analyzer. Among these methods, the method of reacting in a test tube has disadvantages in that the consumption of samples and reagents required for the reaction is large and the reproducibility of the reaction is poor. On the other hand, the pre-column and post-column reactions have the advantages of easy automation and good reproducibility, but additional devices (pumps, valves, and line connecting devices) to transfer the fluorescent solution to be reacted are required. There is a fear that a dead volume is generated at the connection part of the transfer line and the degree of separation is lowered. In addition, the conventional analytical device has a disadvantage in that it consumes a lot of samples, reagents, and the like, and takes a long time for analysis.

Therefore, in the present invention, by connecting the reactor for the phosphorylation reaction of the amine compounds and the separation channel for separation between them to perform the fluorescence derivatization reaction and separation of the amine compound at once and to perform the detection of the phosphorized material continuously We developed a lab-on-a-chip analysis system.

1 is a plan view of an example of a lab-on-a-chip according to the present invention having a structure in which a pre-column reactor and a separation channel are connected;

2 is a view showing a glass-on-a-chip manufacturing process,

3 is a view showing a lab-on-a-chip manufacturing process of plastic material,

4 is a block diagram of an amine compound analysis system using a lab-on-a-chip;

5 is a graph showing the results of analyzing the amine compound using a lab-on-a-chip made of glass and plastic.

The present invention provides a system for separating and analyzing each amine compound in a sample containing at least one amine compound, wherein a reactor for fluorescent derivatization of the amine compound and a separation channel for separation of each compound are integrally connected to the substrate. The present invention provides a lab-on-a-chip for amine compound analysis, which is located in a phase.

In the lab-on-a-chip according to the present invention, the fluorescent derivatization reactor is connected to the separation channel and has a structure of a precolumn reactor that performs the reaction before sample separation. In the lab-on-a-chip according to the present invention, the internal volume of the reactor can be variously applied depending on the application in the range of 0.01 picoliter (pL) to 10 milliliters (mL).

In the lab-on-a-chip form analysis system of the present invention, the derivatization reaction and separation of the amine compound are continuously performed, and the amine compound reacts with the fluorescent reaction reagent in a pre-column reactor in the chip to generate a fluorescent derivative. As the fluorescent reaction reagent, any reagent capable of reacting with an amine compound to form a fluorescent derivative may be used.

The analysis system of the present invention is based on the separation and analysis method by capillary electrophoresis using capillary electroosmotic phenomenon to apply a voltage across a channel filled with a solution to create a flow of the solution. Therefore, the analysis system of the present invention can perform the movement of the solution and the separation of the sample in the microchannel inside the lab-on-a-chip with only a high voltage without using a mechanical pump or valve. It has the advantage of being small and relatively inexpensive, and there is no dead volume at the connection because no other connection device is required to connect the reactor and the separation channel. In addition, the analytical system of the present invention is very small in the amount of samples, reagents and solvents required for the reaction and separation, from several picoliters to several hundred microliters, and the reaction and separation time is very short from several seconds to several minutes. Research is possible.

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

1 is a view showing the structure and operation of a lab-on-a-chip according to the present invention, in which a pre-column reactor and a separation channel are connected, and shows a plan view of the lab-on-a-chip connected to the precolumn reactor ③ and the separation channel ⑦. The figure shows the structure of the reactor. Inside the reactor, the reaction channel is curved, so that the mixing and reaction of the sample solution and the reaction solution can be performed quickly and with high efficiency. The structure of the reactor can be modified depending on the application.

The channel width and depth of the lab-on-a-chip according to the present invention are in the range of 10 nm to 10 cm and 10 nm to 1 cm, and the length of the separation channel varies between channel width and length depending on the application within the range of 1 μm to 500 cm. You can.

In the chip of the type shown in FIG. 1, for example, an amine compound is placed in a sample container ①, a fluorescent reaction reagent is placed in a reagent container ②, and a voltage is applied to the samples ① and ②. Fluorescent derivatization reaction takes place in the reactor. At this time, the amount and reaction time of the sample and reagent entering the reactor can be adjusted by changing the voltage of each vessel. In other words, if the same voltage is applied to the two vessels, the amount of solution flowing into the reactor from the two vessels is the same, and a 1: 1 reaction is performed. The lower the voltage applied to the two vessels, the slower the solution flows through the reactor. The longer the voltage and the higher the voltage, the faster the flow rate, the shorter the reaction time.

In Fig. 1, the buffer solution ④, the reaction solution discharge container ⑤, and the buffer solution discharge container ⑥ are each filled with a buffer solution. Before sample injection, the buffer solution ④ is 0.5 to 2.0 times the voltage applied to the sample container ①. , ⑤ to 0.1 ~ 0.5 times the voltage to ⑥, -1.0 to 0.5 times the voltage is applied to ⑥, so that the reacted sample is released only to the reaction liquid discharge vessel ⑤ and does not flow into the separation channel ⑦. When the sample is injected, the voltage applied to the buffer solution container ④ is shut off for a predetermined time, for example, about 0.01 seconds to 600 seconds using a high voltage relay, and then connected again so that the reaction solution reacted in the precolumn reactor is introduced into the separation channel. do. The sample injected into the separation channel is separated by being transferred by the buffer solution of the buffer container ④.

The sample separated through the separation channel is then fluoresced by the light irradiated from the detector at the end of the separation channel, and the fluorescence is detected by the photomultiplier detector to complete the sample analysis.

The lab-on-a-chip according to the present invention can be produced by engraving a channel according to a conventional method on a plate made of a material such as glass, quartz, fused silica, or plastic. In order to make the channel, photolithography, chemical etching, molding, stamping, machining and laser processing can be used to engrave the desired channel on the glass plate. Lap-on-a-chip made of plastic is made of various plastic materials such as PDMS (polydimethylsiloxane), PMMA (polymethylmethacrylate), PC (polycarbonate), PE (polyethylene), PP (polypropylene), PS (polystyrene), etc. Can be produced with The method of making a channel in a plastic plate is a molding technique in which a mold is poured into a mold and then solidified, or a hot embossing method in which a flat substrate is pressed into a heated mold or processed by mechanical means or light or heat. Methods can be used.

By using the lab-on-a-chip of the present invention, it is possible to detect various amine compounds in liquid or gaseous form, ie, biogenic amines, alkylamines, aromatic amines, amino acids, ammonia and primary amine compounds such as drugs and pesticides at high speed and high sensitivity. Can be.

Examples of the present invention are presented below, but the scope of the present invention is not limited by the examples.

Example 1

In this embodiment, an example of fabricating a lab-on-a-chip according to the present invention on a glass plate by a photolithography technique and a chemical etching technique is illustrated.

As shown in FIG. 2, after the photoresist ② was coated on the clean slide glass ①, the photomask ③ was engraved with the shape as shown in FIG. 1 and exposed to ultraviolet rays. After exposure, the slide glass was immersed and developed in a developing solution to remove the photoresist of the channel portion, and then the glass was immersed in a hydrofluoric acid solution and only the channel portion was etched to a depth of 10 nm to 1 cm. Punch a hole for inserting the container into the glass plate ④ where the etching is completed, cover the other slide glass ⑤, and heat it at 550 to 600 ℃ to bond the two glass plates. Finally, cut the 200 μl pipette tip ⑥ into an appropriate size and insert it into the hole. The glass-on-a-chip was completed by fixing with.

Example 2

In this embodiment, a method of manufacturing a lab-on-a-chip according to the present invention is illustrated by molding using PDMS.

As shown in FIG. 3, SU-8 ① as a negative photoresist was coated on the silicon wafer ② at a height of 10 nm to 1 cm, and the photomask ③ was covered and exposed to ultraviolet rays. The wafer is developed to obtain an embossed frame ④ having the desired pattern. Pour the PDMS ⑤ into the mold, crosslink and remove it to obtain a PDMS plate ⑥ having the desired intaglio pattern. After surface treatment with an arc discharge formed using a Tesla coil, another PDMS plate ⑦ of the same surface treatment was bonded. At this time, the bonding through the surface treatment of the PDMS plate using the Tesla coil may be made in the glass plate, silicon wafer, etc., as well as other PDMS plate. Finally, a 200 μl pipette tip ⑧ was cut into a suitable size and inserted into a hole and fixed with an adhesive to complete a lab-on-a-chip made of PDMS.

Example 3

4 shows a configuration of an analysis system using a lab-on-a-chip according to the present invention and an operation example thereof manufactured as in Example 1 or 2. As shown in Figure 4, the movement of the solution in the chip, sample reaction, injection and separation was performed using an electroosmotic flow, for this purpose, a high voltage supply ①, a voltage divider ② and a high voltage relay ③. Sample injection was performed using an open-close injection method, and the voltage applied to each container was controlled using a voltage divider. Before sample injection, a voltage in the range of 0.1 kV to 50 kV in the sample container, 0.1 to 5 times the voltage applied to the sample container in the buffer container, 0 to 1 times the voltage in the sample discharge container, and 0 V in the buffer discharge container. When the voltage was applied to the sample and the sample was injected, the voltage applied to the buffer container was shut off for a predetermined time, for example, 0.01 seconds to 60 minutes using a high voltage relay ③, and then connected again so that the sample was injected into the separation channel.

The detection of the separated reaction sample concentrates the light source from the laser ⑤ at the end of the separation channel, collects the fluorescence generated from the sample with the fluorescent label through the objective lens ⑥, passes the filter ⑦, and then increases the intensity of the fluorescence with the photomultiplier detector ⑧. It was performed by measuring. The computer 9 was used to regulate the voltage of the high voltage supply and the high voltage relay and to record and store the signal from the photomultiplier detector.

Example 4

In this embodiment, using an analysis system according to the present invention prepared as in Example 3, after reacting a biogenic amine sample such as histamine, tyramine, putrescine and tryptamine in a reactor in a chip with a fluorescent labeling reagent An example of a high resolution, high sensitivity separation analysis is shown. Ortho-phthaldialdehyde (OPA) was used as a fluorescent labeling reagent for reacting with biogenic amines.

Fluorescent labeling reaction between the amine compound (R ¹ -NH ₂ ) and OPA is carried out as follows.

At this time, R ² -SH acts as a reducing agent, mainly mercaptoethanol (CH ₃ CH ₂ -SH) is used.

The reaction completes very quickly in minutes and the resulting OPA-amines are excited at wavelengths near 330 nanometers and fluoresce around 450 nanometers.

In the reactors of the LabonA chip of the glass material and PDMS material prepared in Examples 1 and 2, respectively, the biogenic amine mixture was reacted with OPA to form a fluorescent derivative, and then the synthesized OPA-biogenic amines were separated from the separation channel. Detection was by laser induced fluorescence, and the results are shown in FIGS. 5B and 5A, respectively. As shown in FIG. 5, in the analytical apparatus of the present invention of glass material and PDMS material, fluorescent labeling reactions of four biogenic amines (1: histamine, 2: tyramine, 3: putrescine, and 4: tryptamine) Separation assays can be performed at very high speeds within 1 minute.

The amine compound analysis lab-on-a-chip according to the present invention integrates a reactor and a separation channel for fluorescence derivatization of the amine compound, so that the reaction and separation of the amine compound and the fluorescent material can be continuously performed within several minutes.

In addition, the lab-on-a-chip device according to the present invention is simple in structure and easy to manufacture, and the small size of the device can drastically reduce the consumption of samples, reagents and solvents required for reaction and analysis, and enables multiple simultaneous analysis and automation. It has the advantage of being easy, and can detect various amine compounds in liquid or gas phase, that is, primary amine compounds such as alkylamines, aromatic amines, amino acids, biogenic amines, ammonia and pharmaceuticals and pesticides at high speed and high sensitivity.

Therefore, the lab-on-a-chip device of the present invention can be widely used as equipment for analysis, quality control, real-time monitoring, etc. in medicine, food, pesticide, life, environment and forensic medicine research fields, and the time required for research in related research fields, Significant savings in labor costs and labor can dramatically increase research efficiency.

Claims (8)

In a system for separating and fluorescence of each amine compound from a sample containing at least one amine compound, a reactor for fluorescence derivatization reaction of the amine compound and a separation channel for separation of each compound are sequentially connected on a substrate. Lap-on-a-chip for the analysis of amine compounds, characterized in that located in. The method of claim 1, Lap-on-a-chip, characterized in that the internal volume of the reactor ranges from 0.01 picoliters (pL) to 10 milliliters (mL). The method of claim 2, A separation channel has a size in the range of 10 nm to 10 cm in width, 10 nm to 1 cm in height, and 10 nm to 100 cm in length. The method of claim 1, The amine compound is characterized in that at least one selected from the group consisting of alkylamines, aromatic amines, amino acids, biogenic amines and ammonia, lab-on-a-chip. The method of claim 1, The lab-on-a-chip, characterized in that consisting of a substrate formed with a channel on one side and another substrate that can cover the channel. The method according to claim 1 or 5, Lab-on-a-chip, characterized in that made of glass, quartz, fused silica or plastic material. The method of claim 6, Glass, quartz, fused silica material, characterized in that it is manufactured by photolithography, chemical etching, molding, stamping, machining or laser processing. The method of claim 6, A plastic on-chip, characterized in that it is manufactured by molding, stamping, machining or laser processing.