Stably preserved microspheres

Inactive Publication Date: 2006-12-28
HITACHI SOFTWARE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019] It is an objective of the present invention to provide a technique whereby activated carboxylic acid ester groups are stabilized on the surfaces of microspheres without fluorescent dye seeping so as to simplify protocols for the conjugation between biomolecules and microspheres, extend the type of applicable peptide.
[0027] In the reaction of the activated carboxyl group a reaction buffer conducting a coupling reaction are substituted for lower alcohol. Then, the microparticles are coupled with biomolecules on the surfaces of the microspheres. Thus, biomolecules such as antibodies, antigens, nucleic acids, sugar chains, and peptides can be coupled under the respective optimum conditions. In addition, side reactions caused by nucleic acids can be significantly suppressed.

Problems solved by technology

Thus, for example, peptides that are soluble only in a basic solvent cannot be coupled, resulting in a limitation in terms of the selection of biomolecules that can be coupled.
However, the conjugation reaction between those esters and biomolecule which contain primary amino group is very slow.

Method used

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Examples

Experimental program
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example 1

[0045] A coupling reaction was performed on biotinylated oligonucleotide 20-mers, in which primary amino group had not been introduced, using microspheres activated in accordance with the present invention, as shown in FIG. 6. The results were compared with those obtained by a conventional method. Then, the side reaction was found to have been significantly suppressed compared with that in the conventional method (FIG. 8).

example 2

[0046] Nucleic acids were immobilized using microspheres produced in accordance with the present invention, as shown in FIG. 6. Conjugation of primary amino group introduced in nucleic acids with activated microspheres were performed in the various condition such as before, immediately after, and 3 days, 1 week, and 1 month after substitution using an isopropanol solvent. The experiment was carried out to confirm the presence or absence of fading of fluorescent dyes in isopropanol and the presence or absence of activity retention of active carboxylic acid ester groups (FIG. 9). The identification of bead #01 and #97 were performed ordinarily as in those without isopropanol treatment. Therefore, fluorescent dyes do not seep out during the preservation in isopropanol. Even after one month, about half of the activated ester had been maintained.

example 3

[0047] Nucleic acids were immobilized as shown in FIG. 6, using microspheres produced in accordance with the present invention. Conjugation of primary amino group introduced in nucleic acids with activated microspheres was performed in the various condition such as before, immediately after, and 3 days after substitution using an ethanol solvent. The experiment was carried out to confirm the presence or absence of fading of fluorescent dyes in ethanol and the presence or absence of activity retention of active carboxylic acid ester groups (FIG. 10). The identification of bead #6 and #85 were performed ordinarily as in those without ethanol treatment. However, about half of the activated ester had been degraded even in 3 days.

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Abstract

In accordance with the present invention, active carboxylic acid ester groups are coupled on the surfaces of microspheres so as to reduce protocols for microsphere processing, control side reactions, and stably preserve beads containing active carboxylic acid ester groups. Further, microspheres labeled with at least one fluorescent dye cage in the microspheres, and the microspheres are preserved in lower alcohol.

Description

BACKGROUND OF THE INVENTION [0001] 1. Field of the Invention [0002] In general, the present invention is applied to flow cytometry technique. The present invention relates to microspheres, which usually has a diameter of 100 μm or less and is filled with multicolor fluorecent. More specifically, the present invention relates to microspheres that activated esters are held and stabilized on the surface without fluorescence seeping and polymer molecules filled with at least one type of fluorescent dye. [0003] 2. Background Art [0004] Polymer particles, filled with fluorescent dye, are often used as a marker or indicator in various biomedical assays. “Microspheres” indicates minute particles basically having total diameters within micrometer-size. Microspheres can be analyzed with manual techniques or other methods known in the art. Preferably, automation technologies such as flow cytometry disclosed in U.S. Pat. No. 4,665,024 described below, the patent for which was granted to Mansour...

Claims

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Application Information

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IPC IPC(8): G01N33/53C04B14/00
CPCG01N33/54393
Inventor UKAWA, HISASHIYAMANE, AKIO
Owner HITACHI SOFTWARE ENG
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