JP2009265067A - Silica nanoparticle labeled with molecular recognition fluorescence - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for preparing silica nanoparticles labeled with molecular recognition fluorescence for selectively recognizing specific molecules and provide a method for evaluating molecular recognition abilities. <P>SOLUTION: By coating the surfaces of silica nanoparticles including a fluorescent substance with polymers (molecular recognition polymers, Molecularly Imprinted Polymers, MIPs) which recognize specific molecules, silica nanoparticles labeled with molecular recognition fluorescence having molecular recognition abilities are prepared. Their specific molecule recognition abilities are evaluated by the pseudo-immunoreaction between the silica nanoparticles labeled with molecular recognition fluorescence and specific molecules immobilized to the surface of a solid. The effectiveness of this method as a method for evaluating the molecular recognition abilities of IMPs is shown. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

Detailed description

  The present invention relates to a method for preparing and evaluating molecule-recognized fluorescently labeled silica nanoparticles for detecting and analyzing specific molecules such as trace antibacterial substances, environmental pollutants and biological substances with high selectivity and high sensitivity.

Biological antigen-antibody reaction is widely used to detect and analyze specific trace substances with high selectivity and high sensitivity. However, the method using the above antibody often requires skill because of the instability of the antibody or complicated reaction operation using the antibody. In addition, there are disadvantages such as expensive antibodies and too high cost per measurement. In order to compensate for these drawbacks, it is known to use molecularly recognized polymers (MIPs) using a specific molecule as a template (see, for example, Patent Document 1).
JP 2005-533146 A

Furthermore, in an antigen-antibody reaction, an antibody is labeled with a fluorescent molecule or an enzyme in order to increase measurement sensitivity. Alexa Fluor® protein labeling kit (manufactured by Molecular Probes), HiLyFluor natural antibody labeling series (manufactured by Cosmo Bio), or QuantaBlu ^™ Fluorogenic Peroxidase Substrate (manufactured by Takara Bio) Kits are commercially available. However, all of the above labeled molecules have disadvantages such as being unstable and expensive.

  The present invention has been proposed to cope with such various situations, and is a stable and inexpensive molecule that replaces the unstable and expensive fluorescently labeled antibody used in the conventional antigen-antibody reaction. It was an object to prepare a recognition fluorescently labeled silica nanoparticle and to establish a method for evaluating its molecular recognition ability.

  In order to solve the above-mentioned problems, in the invention of claim 1, fluorescently labeled silica nanoparticles that recognize specific molecules were prepared by coating the surfaces of the fluorescently labeled silica nanoparticles with MIPs that recognize specific molecules.

  The molecular recognition ability evaluation according to claim 2 was performed by causing a pseudoimmune reaction between the molecular recognition fluorescent-labeled silica nanoparticles and a specific molecule immobilized on another solid surface (for example, silica gel, φ = about 100 μm).

  As described above, according to the invention of claim 1, an adsorption site for recognizing a stable and inexpensive specific molecule is formed on the surface of the fluorescently labeled silica nanoparticle, and therefore the specific molecule in the sample is captured by a pseudo-immune reaction. Possible artificial antibody preparations are possible.

  According to the evaluation method of claim 2, it is possible to easily evaluate the molecular recognition ability of MIPs targeting a wide variety of specific molecules.

In addition, after extracting and concentrating a small amount of specific molecules contained in a dilute solution to a solid, quantitative measurement of the specific molecules is enabled by the method of Patent Document 2.
Japanese Patent Application No. 2007-41414

  In the following, embodiments of the present invention relating to the preparation of molecularly recognized fluorescently labeled silica nanoparticles that recognize penicillin G (PG) and evaluation of their recognition ability will be described with reference to the drawings.

FIG. 1 is an estimated schematic diagram of molecularly recognized fluorescently labeled silica nanoparticles according to the present invention. It consists of silica nano particles 1 (RuBpy-SiO ₂ ) having a particle system of about 100 nm encapsulating a coating film (PG-MIPs) 2 of MIPs that recognizes PG and a fluorescent ruthenium complex. The preparation process of the PG recognition fluorescently labeled ruthenium silica nanoparticles 3 (PG-MIPs / RuBpy- SiO 2) is described below.

Fluorescent RuBpy-SiO ₂ (φ = about 100 nm) in which a fluorescent ruthenium complex (Tris (2,2′-bipyridine) dichlororuthenium (II) hexahydrate, ruby dye) is encapsulated in silica nanoparticles is prepared by a sol-gel method.

The fluorescent RuBpy-SiO ₂ is added to a saturated solution of PG-MIPs in acetonitrile, reacted at room temperature for 1 hour, separated from the acetonitrile solvent by suction filtration, and dried in air.

  Hereinafter, embodiments of the present invention will be described in more detail with reference to examples.

Preparation of fluorescently labeled silica nanoparticles RuBpy-SiO ₂

The fluorescent RuBpy-SiO ₂ was prepared according to the method of Tan et al. (Ref. 1 W. Tan et al., J. Biomed. Opt., 6 (2001), 160).

That is, cyclohexane (7.5 ml), n-hexanol (1.8 ml), Triton X-100 (1.77 ml), 1.2 mM ruby dye (Tris (2,2′-bipyridyl) dichlororuthenium (II) -6H ₂ O) / water (0.48 ml) and TEOS (Tetraethoxysilane) (0.1 ml) were mixed and shaken at room temperature for 20 minutes.

Thereafter, NH ₄ OH (60 μl) was added and shaking was continued for 24 hours at room temperature.

After the reaction for 24 hours, 11 ml of (CH ₃ ) ₂ CO is added and vortexed.

This was centrifuged (3000 rpm, 10 min), and the precipitated product was washed three times with 95% ethanol and then dried in air at room temperature to obtain fluorescent RuBpy-SiO ₂ .

The fluorescent RuBpy-SiO ₂ prepared in this way is a sphere having a diameter of about 100 nm, and is considered to have a structure in which rubpy dye, which is a fluorescent substance, is encapsulated in layered silica (FIG. 1). 3).

  Here, Ruby dye was used as a fluorescent substance for fluorescent labeling, but the present invention is not limited to this.

Preparation of PG molecule recognition fluorescent labeled silica nanoparticles PG-MIPs / RuBpy-SiO ₂

_{On the} surface of RuBpy-SiO ₂ prepared as in Example 1, PG was prepared as a template, PG was coated with MIPs having molecular recognition ability (PG-MIPs), and PG was labeled with fluorescence having molecular recognition ability Prepare MIPs.

  Here, although the example using MIPs produced using penicillin G which is an antibacterial substance as a template is shown, the present invention is not limited to this.

  MIPs using PG as a template (PG-MIPs) were prepared according to the method of Urraca et al. (Reference 2 JL. Urraca et al., Anal. Chem., 79 (2007), 695). Add 10 mg of PG-MIPs into 10 ml of acetonitrile and prepare the saturated solution while warming at 60 ° C.

In 1 ml of the obtained saturated solution, 10 mg of RuBpy-SiO ₂ prepared in Example 1 is added, and the mixture is allowed to stand at room temperature for 1 hour.

Thereafter, the acetonitrile solvent was removed by suction filtration, and a sample dried in air was designated as PG-MIPs / RuBpy-SiO ₂ 6 (FIG. 1).

Evaluation of molecular recognition ability of PG-MIPs / RuBpy-SiO ₂

The molecular recognition ability of PG-MIPs / RuBpy-SiO ₂ prepared in Example 2 was evaluated by performing a pseudoimmune reaction with PG7 (FIG. 1) immobilized on a silica gel surface having a particle size of about 100 μm.

That is, silica gel (FIG. 213) with PG immobilized on its surface was added to the buffer solution to which PG-MIPs / RuBpy-SiO ₂ (FIG. 211) prepared in Example 2 was added, and 10 minutes at room temperature. React.

PG7 on the PG-immobilized silica gel surface binds (pseudoimmune reaction) to the PG adsorption site 5 (FIG. 1) on the PG-MIPs / RuBpy-SiO ₂ surface.

After the reaction, unreacted PG-MIPs / RuBpy-SiO ₂ is washed to leave only those bound to PG-immobilized silica gel (FIG. 216).

  After washing, the silica gel is taken out on a glass plate and a photograph observed with a fluorescence microscope is shown in FIG.

For comparison, FIG. 332 shows the results observed after the same operation (FIGS. 218 and 19) without adding PG-MIPs / RuBpy-SiO ₂ .

  Further, FIG. 333 shows the results observed after the same operation using silica gel not fixed with PG (FIGS. 21 and 22).

As a result of binding PG-MIPs / RuBpy-SiO ₂ of the present invention to PG-immobilized silica gel (FIG. 331), strong fluorescence due to RuBpy-SiO ₂ was observed on the silica gel. On the other hand, such strong fluorescence is not observed on the PG-immobilized silica gel to which PG-MIPs / RuBpy-SiO ₂ is not added (FIG. 332), and the fluorescence observed in FIG. It was confirmed that it was due to RuBpy-SiO ₂ .

Moreover, only weak fluorescence was observed in the result of reacting PG-MIPs / RuBpy-SiO ₂ with PG unfixed silica gel (FIG. 333). Therefore, the strong fluorescence observed in FIG. 331 should confirm that the PG immobilized on the silica gel surface was bound to the PG adsorption site on PG-MIPs / RuBpy-SiO _{2 by a} pseudoimmune reaction. I can do it.

Such PG molecular recognition ability of PG adsorption sites with the PG-MIPs / RuBpy-SiO ₂ were evaluated by the method.

How to Use

  MIPs coated on the surface of silica nanoparticles encapsulating a fluorescent substance have an adsorption site that recognizes the specific template molecule used in preparing the MIPs, and thus highly selectively capture the specific molecule by a pseudo-immune reaction. It can be used as an artificial antibody capable of

  In addition, it is possible to quantitatively measure the specific molecule by extracting and concentrating the trace specific molecule contained in the dilute solution into a solid, and then performing a pseudo-immune reaction using the molecular recognition fluorescently labeled silica nanoparticles as an artificial antibody. .

It is the figure which showed the coating by the molecular recognition artificial antibody (MIPs) of the silica nanoparticle which included the fluorescent substance, and the fluorescent silica nanoparticle for preparation of the fluorescent labeling molecular recognition artificial antibody in this invention. In addition, a method for measuring a specific molecule using a fluorescently labeled molecule recognition artificial antibody is also shown. All figures are shown as cross-sectional views. It is the figure which showed the evaluation method of the fluorescence labeled molecule recognition artificial antibody prepared in this invention. It is a fluorescence micrograph which shows the evaluation result of the fluorescence labeled molecular recognition artificial antibody prepared in this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fluorescent substance 2 Silica gel layer 3 Fluorescent silica gel 4 Molecular recognition artificial antibody layer 5 Compound molecule recognition site 6 Fluorescent labeling MIPs
7 Compound Molecule 8 Penicillin G-immobilized silica gel 31 Photomicrograph of a conjugate of fluorescently labeled MIPs and penicillin G-immobilized silica gel 32 Microphotograph of penicillin G-immobilized silica gel 33 Binding of fluorescence-labeled MIPs to silica gel not immobilized with penicillin G Photomicrograph

Claims (3)

  Silica nanoparticles (fluorescence-labeled silica nanoparticles, φ = approx. 100 nm) prepared by encapsulating a fluorescent substance are coated with molecular recognition polymers (MIPs) that recognize specific molecules. Preparation method.   A method for evaluating molecular recognition ability, wherein the molecule-recognized fluorescent-labeled silica nanoparticles are subjected to a pseudoimmune reaction with a specific molecule immobilized on a solid surface.   The method for evaluating the molecular recognition ability of MIPs according to claims 1 and 2.