JP2006250953A - Microcircuit for analyzing protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microcircuit for analyzing a protein capable of enhancing sensitivity, precision and reproducibility, by controlling adsorption of the protein onto a circuit wall face and by enhancing smoothness of a circuit surface. <P>SOLUTION: In this microcircuit for analyzing the protein, at least a surface of the circuit contacting with a protein solution is coated with an ultra-hydrophilic polymer, in the microcircuit having 1 mm<SP>2</SP>or less of cross section contacting with the protein solution. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a microcircuit that performs the flow, reaction, or analysis of a protein solution among apparatus / equipment used for protein structure / function analysis and reaction using a protein.

  At present, microcircuits for performing various chemical reactions are a technology that is attracting attention from the viewpoint of reaction efficiency, speed, and reagent saving, and is already formed on a glass chip of several centimeters square called “lab-on-a-chip”. In general, the concept of new analytical methods for chemical reaction and analysis in a circuit is well established.

  In the future, with the progress of biotechnology, the use of microcircuits is an indispensable technology in the field of biochemistry, and in particular, the application of microcircuits to protein structure / function analysis and reactions using proteins is expected (for example, Patent Document 1).

  A major problem when flowing a protein solution through a microcircuit is protein adsorption on the circuit surface, and trace amounts of protein are not only greatly affected by the decrease and structural change caused by adsorption, but also when the circuit is used repeatedly. However, there is a problem that the adsorbed protein remains as a history, and that the circuit itself is very small, so that the circuit may be blocked by the adsorbed protein.

  In addition, when the reaction is performed with two kinds of solutions flowing in a microcircuit, it is important that the two kinds of solutions form a phase flow in one circuit. The fact that irregularities are formed on the circuit wall surface, resulting in the occurrence of turbulent flow and reduced reaction efficiency is a very big problem for microcircuits.

  Most of the substrates currently used for microcircuits are glass (quartz glass) or plastics, but proteins exhibit high adsorptivity to these substrates.

Further, as a problem related to the processability of the microcircuit, there are cases where fine irregularities are generated on the surface when the microcircuit is formed on the substrate, and such irregularities are not preferable for the above-mentioned reasons.
International Publication No. 01/47637 Pamphlet

  An object of the present invention is to provide a microcircuit for protein analysis that improves sensitivity, accuracy, and reproducibility. Adsorption of protein onto a circuit wall surface and improvement in smoothness of the circuit surface are problems for achieving the above object. I thought.

That is, the present invention is a microcircuit for protein analysis in which a microcircuit having a cross-sectional area of 1 mm ² or less in contact with a protein solution is coated with a superhydrophilic polymer at least on the surface of the circuit in contact with the protein solution.

  As described above, according to the present invention, the following excellent effects can be obtained.

  First, since there is no protein adsorption on the circuit surface and no reduction or denaturation of the protein to be analyzed occurs, analysis with a very small amount and high accuracy becomes possible.

  Second, there is no protein adsorption on the circuit surface, and the used protein does not remain as a history in the circuit, and analysis with excellent repeatability can be performed.

  Thirdly, there is no protein adsorption on the circuit surface, the occurrence of turbulence in the circuit is suppressed, and there is no problem of blockage.

  Fourth, the smoothness of the circuit surface is expected to be improved by coating and swelling with the superhydrophilic polymer.

  Conventional protein adsorption to glass or plastic occurs in a short time of contact, and its adsorption rate (ratio of protein adsorbed among proteins in the contacted protein solution) in a low concentration region (about 1 ng to 100 ug / ml). Reaches a maximum of about 50%, and once adsorbed protein undergoes irreversible structural changes (denaturation), the denatured protein induces secondary protein adsorption, resulting in the formation of a multilayer adsorption layer of proteins. The

  Therefore, in the present invention, the surface that is in contact with the protein solution is coated with a superhydrophilic polymer to reduce the hydrophobic interaction, which is the largest factor causing protein adsorption, and to prevent initial protein adsorption. Yes.

  A super hydrophilic polymer is a polymer that has a very good affinity with water, and is a polymer material that maintains a uniform free water layer on its surface when immersed in water, with a water contact angle of 0 to 1 degree. Point to.

  Examples of superhydrophilic polymers include polyhydroxyalkyl methacrylate, polyoxy C2-C4 alkylene group-containing methacrylate polymer or a copolymer containing the same, or polyvinylpyrrolidone, a phospholipid / polymer complex (Japanese Patent Laid-Open No. 5-161491). And 2-methacryloyloxyethyl phosphorylcholine copolymer (hereinafter abbreviated as MPC) or a copolymer containing the same (Japanese Patent Laid-Open No. 9-3132).

  The superhydrophilic polymer coating has effects other than prevention of protein adsorption. Superhydrophilic polymers swell with free water when in contact with a liquid, and the surface becomes smooth.

  That is, unevenness during processing that causes turbulence on the circuit surface is reduced by coating and swelling of the superhydrophilic polymer. A point to be noted when coating the superhydrophilic polymer is the thickness of the coating layer.

  If the coating layer of the super-hydrophilic polymer is too thick, it may clog the circuit when it swells in contact with the protein solution, and the protein adsorption performance also decreases, so the coating layer thickness is 5 μm or less. It is preferable that the layer be as thin as possible if the surface is completely covered.

  The method of coating the superhydrophilic polymer is not particularly limited, but the circuit is filled with a solution obtained by dissolving the superhydrophilic polymer in a solvent, and a suction nozzle connected to a suction pump is applied to the open end of the circuit. A method of sucking the filled hydrophilic polymer solution and continuing the suction until the hydrophilic polymer remaining on the circuit surface is dried is preferable.

  By the coating method, a superhydrophilic coating layer is formed without clogging the circuit, and the thickness thereof can be easily adjusted by the viscosity of the hydrophilic polymer solution. In addition, a smooth coating surface can be obtained by aeration drying.

  Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1
A linear circuit having a diameter of 0.5 mm and a length of 30 mm was formed using a drill in the long axis direction of a polystyrene plate having a thickness of 2 mm and a 20 × 30 mm square. A 2.5 Wt / vol% methanol solution of polyhydroxyethyl methacrylate (SIGMA P-3932) was injected from one open end of the circuit. At the time of injection, a 2.5 ml syringe with a 0.5 mmφ blunt needle attached to the tip was used.

  After the injection, the opening end was covered with a tape and allowed to stand for 30 minutes, and then a nozzle connected to a suction pump-trap was pressed against the opening end to perform suction for 1 minute. It was further dried overnight.

When the thickness of the coating layer after drying was cut out with a circuit portion and measured with an electron microscope, the thickness was about 1.3 μm.
(Example 2)
A 0.5 wt / vol% ethanol solution of MPC polymer was injected from one open end of the same circuit as that used in Example 1.

  MPC polymer is a co-polymer of MPC and BMA (butyl methacrylate) ratio = 3/7 in accordance with the content of “Drug Release from Hydrogel Membranes Having a Phospholipid-like Structure, 46,591-595 (1989)”. A coalescence was synthesized and used.

  After the injection, the opening end was covered with a tape and allowed to stand for 30 minutes, and then a nozzle connected to a suction pump-trap was pressed against the opening end to perform suction for 1 minute.

  Furthermore, it dried overnight and was set as Example 2.

When the thickness of the coating layer after drying was cut out by a circuit portion and measured with an electron microscope, the thickness was about 0.5 μm.
(Comparative Example 1)
Comparative Example 1 was obtained by forming a linear circuit having a diameter of 0.5 mm and a length of 30 mm using a drill in the long axis direction of a polystyrene plate having a thickness of 2 mm and a 20 × 30 mm square.
(Comparative Example 2)
Comparative Example 2 was obtained by forming a linear circuit having a diameter of 0.5 mm and a length of 30 mm using a drill in the long axis direction of a glass plate having a thickness of 2 mm and a square of 20 × 30 mm.
(Comparison of protein adsorption)
After circulating 200 ul of a 50 ng / ml bovine albumin (BSA) solution 30 times repeatedly in the circuit portions of Example 1, Example 2, Comparative Example 1, and Comparative Example 2, the concentration of the BSA solution was measured, and the rate of change in the concentration Asked.

  The concentration of BSA solution was measured according to the following procedure.

  The recovered BSA solution is dispensed onto an ELISA plate (Sumiton Bakelite Sumilon ELISA plate H) and incubated at 37 ° C. for 1 hour. Thereafter, washing was repeated 3 times using a plate washer. In addition, 0.05% Tween20-containing phosphate buffer (Nissui Pharmaceutical Dulbecco PBS-pH 7.4) was used as the washing solution.

  Thereafter, 3% skim milk (manufactured by Cosmo Bio) phosphate buffer solution was dispensed at 250 μL / well and incubated at 37 ° C. for 1 hour. Thereafter, washing was repeated 3 times using a plate washer.

  Next, a 1.5 ug / ml phosphate buffer solution of peroxidase-labeled bovine albumin antibody (manufactured by Cosmo Bio) was dispensed at 100 ul / well, allowed to stand at room temperature for 30 minutes, and then washed three times using a plate washer. Was repeated.

  Next, color was developed using a TMBZ substrate buffer (Sumitomo Bakelite's Sumilon Peroxidase Coloring Kit T), then the absorbance was measured with a plate reader, the concentration was determined from the calibration curve, and the rate of change from the initial concentration was determined.

  The results are shown in Table 1, and it was confirmed that the changes in the concentration of the protein in the protein solution were greatly suppressed as compared with Comparative Examples 1 and 2 in both Examples 1 and 2.

(Comparison of circuit occlusion)
In order to compare the influence of the protein adsorbed and retained on the circuit, the following examination was conducted using Example 1 and Comparative Example 1.

  Example 1 and Comparative Example 1 By adhering and connecting a silicon tube to the open end of each circuit, filling it with 500 mg / ml BSA phosphate buffer solution, and connecting the tube to a peristaltic pump. A device capable of continuously circulating the BSA solution through the circuit was produced.

  Example 1 and Comparative Example 1 The peristaltic pumps connected to each were operated for 1 hour, then washed with pure water and dried, and then connected to the pump and circulated the BSA solution for 1 hour repeatedly for 5 days (30 times). The state of the luminal surface of the circuit after observation was observed.

As a result, in Example 1, the BSA adsorption layer was not confirmed, and the circuit lumen surface maintained the state at the start of the study, but in Comparative Example 1, BSA was uniformly adhered to the lumen surface. It was clear that a lump-like lump was confirmed in some places, and the flow of the BSA solution in the circuit was obstructed.

Claims (1)

A microcircuit for protein analysis in which at least a surface of a circuit that is in contact with a protein solution is coated with a superhydrophilic polymer in a microcircuit having a cross-sectional area of 1 mm ² or less in contact with the protein solution.