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Nuclein acid fixing method and method for producing biosensor by said method

A technology of biosensors and immobilization methods, applied in the field of nucleic acid immobilization, can solve the problems of long film formation time, difficulty in nucleic acid density, and density changes.

Inactive Publication Date: 2004-09-29
SEIKO EPSON CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reaction time is an extremely unstable parameter, and it is extremely difficult to effectively control the nucleic acid density.
In addition, it is documented that in the embedding process of spacer molecules, the density of the nucleic acid probe adsorbed first changes [A.B.Steel et al., Biophysical Journal, Vol.79, pp.975-981(2000)], Not suitable method for density adjustment
Moreover, the displacement method has the problem that the film formation time is too long and is not practical.

Method used

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  • Nuclein acid fixing method and method for producing biosensor by said method
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  • Nuclein acid fixing method and method for producing biosensor by said method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086]

[0087] (1) Nucleic acid probes: nucleic acid probes for adsorption on solid substrates can be purchased from the formula (dA)20-(CH 2 -O-CH 2 ) n PO 4 -(CH 2 ) 6 Nucleic acid probe represented by -SH. Here, dA refers to deoxyadenosine 5'-phosphate.

[0088] (2) Spacer molecule: The spacer molecule can be purchased as 6-mercapto-1-hexanol (MCH).

[0089] (3) Preparation of buffer: prepare PBS buffer (50mM KPO 4 , 5mM EDTA, 1M NaCl, pH7.0). Nucleic acid probes and spacer molecules were dissolved in this PBS buffer, and two solutions of 1 μM and 10 μM were used, respectively.

[0090] (4) Preparation of solid substrate: According to this manufacturing method, a glass substrate with a gold thin film on the surface is prepared as a solid substrate.

Embodiment 2

[0092]

[0093] (1) Co-adsorption: Prepare a mixed solution by mixing the nucleic acid probe solution prepared in Example 1 and the spacer molecule solution (respectively 1 μM and 10 μM), and the ratio of nucleic acid / MCH=25 / 75, Three ratios of 50 / 50 and 75 / 25, 1 μM nucleic acid probe solution and 1 μM spacer molecule solution were mixed, and 10 μM probe solution was mixed with 10 μM spacer molecule solution to prepare a mixed solution. Dip the solid substrate with a clean surface into the mixed solution and incubate for 1-3 hours. The reflected light intensity of the substrate was measured by the SPR method with the viewing angle fixed.

[0094] (2) Results:

[0095] figure 2 Shown is the result of evaluating the adsorption process of the nucleic acid probe by the amount of change in reflected light measured by SPR. Changes in the intensity of reflected light are associated with changes in the refractive index and, furthermore, with increases and decreases in film thick...

Embodiment 3

[0098]

[0099] (1) The preparation of nucleic acid probes, spacer molecules, buffers, and solid substrates is as follows

[0100] The record of embodiment 1.

[0101] (2) Co-adsorption: the nucleic acid probe solution prepared in Example 1 and the spacer molecule solution (respectively 10 μM) were mixed in molar % ratio with nucleic acid / MCH=25 / 75, 50 / 50, 80 / 20, 90 / 10 The four ratios were mixed to prepare a mixed solution. Dip the solid substrate with a clean surface into the mixed solution and incubate for 1-3 hours.

[0102] (3) Hybridization reaction: The complementary DNA (target DNA molecule: dT20) was dissolved in PBS buffer at a DNA concentration of 1 μM, and the DNA solution was brought into contact with the co-adsorbed membrane substrate prepared in (2) above to cause a hybridization reaction.

[0103] (4) Results:

[0104] image 3 Shown are the results of evaluating the degree of adsorption of nucleic acid probes and the level of hybridization using the amoun...

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Abstract

A superior adsorption method for immobilizing nucleic acid probe on the surface of a solid phase substrate is provided. A nucleic acid immobilization method for immobilizing nucleic acid on a solid phase substrate comprising: bringing the above-mentioned solid phase substrate into contact with a composition comprising a total concentration of 0.1 to 2 mu M of a nucleic acid as a probe and a compound or a salt thereof, the compound being represented by the following formula: HS-L<1>-L<2>-R wherein L<1> is a single bond or a C1-15 alkylene group; L<2> is a single bond, nucleic acid, a polyethylene glycol group, -CO-NH-, or -NH-CO-; R is a hydroxyl group, an amino group, a ferrocenyl group, or a carboxyl group; provided that neither L<1> nor L<2> is a single bond, and incubating the composition in contact with a surface of the solid phase substrate.

Description

technical field [0001] The invention relates to a method for immobilizing nucleic acid, a method for manufacturing a biosensor and a method for detecting nucleic acid using the method. Background technique [0002] As methods for detecting target nucleic acid molecules, analyzing sequences, and gene mapping, biosensors such as microarray technology using nucleic acid probes have been widely used in recent years. A typical hybridization method uses a nucleic acid probe that utilizes the interaction (hybridization) of complementary nucleic acid strands to capture a target nucleic acid molecule, and directly or indirectly (for example: detection of the luminescence intensity of a fluorescent molecule attached to a sample) discriminates the target nucleic acid molecule. All or part of the nucleic acid sequence is analyzed for the presence of the target nucleic acid molecule. Generally, a single-stranded nucleic acid molecule having all or part of the target nucleic acid molecul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53B05D3/00C07H21/00C12M1/34C12N15/09C12Q1/68G01N33/543G01N33/566
CPCB82Y15/00C07H21/00B82Y30/00
Inventor 泷口宏志福岛均
Owner SEIKO EPSON CORP
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