Biosensor and method for immobilizing a physiologically active substance
a biological sensor and substance technology, applied in chemical methods analysis, chemical indicators, instruments, etc., can solve the problems of physiologically active substances, inability to immobilize, and inability to obtain preconcentration effects,
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example 1
[0082]This example relates to preparation of a sensor chip for immobilizing proteins.
(1) Preparation of Sample 1 (Comparative Example)
[0083]A Biacore sensor chip CM-5 (research grade) was used as a surface to which carboxymethyl dextran had been bound.
(2) Preparation of Sample 2 (the Present Invention)
[0084]100 μl of a 1:1 mixed solution of a 2.8 mM HODhbt (3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine) aqueous solution and a 0.4M EDC aqueous solution was caused to come into contact with the surface of a Biacore sensor chip CM-5 (research grade), followed by 10 minutes of reaction at room temperature. The surface was washed with water and then dried at room temperature for 10 minutes using a vacuum dryer. 100 μl of 1,2-bis(2-aminoethoxy)ethane was caused to come into contact with the surface, followed by 10 minutes of reaction at room temperature and then washing with water. The surface of interest was thus obtained.
example 2
[0085]This Example relates to preconcentration of proteins having pHs that are equivalent to or higher than the isoelectric points on a surface (sample 2) modified with 1,2-bis(2-amino ethoxy)ethane. Proteins used herein were pepsin (produced by Wako Pure Chemical Industries, Ltd.), BSA (Bovine Serum Albumin: produced by SIGMA), and CA (Carbonic Anhydrase: produced by SIGMA).
[0086]Sample 1 (comparative example) and the sample 2 (the present invention) prepared in Example 1 were set in a Biacore 3000 (a surface plasmon resonance device produced by Biacore). Preconcentration was examined by applying each protein solution (pH7.4, 1.0 mg / ml) for 5 minutes. FIGS. 1 to 3 show the thus obtained sensorgrams.
[0087]In the case of sample 1 to which carboxymethyl dextran had been bound, no proteins were observed to have been preconcentrated thereon. In contrast, in the case of sample 2 of the present invention, all proteins were observed to have been preconcentrated thereon. The degrees of the ...
example 3
[0088]This Example relates to immobilization of proteins preconcentrated on the surface having thereon a primary amino group. Sample 3 was prepared by washing without allowing an EDC (0.4 M) / NHS (0.1 M) aqueous solution (a carboxylic acid activator) to come into contact with sample 2, upon which pepsin (1 mg / ml, pH 7.4) had been preconcentrated for 5 minutes. Sample 4 was prepared by washing after causing an EDC (0.4 M) / NHS (0.1 M) aqueous solution to come into contact with sample 2 and remain in contact therewith for 5 minutes. The amounts of pepsin immobilized on samples 3 and 4 were examined using Biacore 3000. Washing was performed through injection of a 1% EDTA aqueous solution (1 minute×2) and a glycine buffer (pH 1.5, produced by Biacore) (1 minute each×2). FIG. 4 shows the thus obtained results.
[0089]It was confirmed that after washing, preconcentrated pepsin had been dissociated from the surface (sample 4) that had been prepared by washing without contact with the carboxyli...
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