JP2005077396A - Optical waveguide for analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and an optical waveguide for analyzing simply and inexpensively a biotechnology-related material such as DNA or an environment-related material, qualitatively or quantitatively by an optical waveguide method. <P>SOLUTION: Many extremely-fine materials (nano-dots, nano-particles, nano-lines or the like of various materials including gold) are arranged at intervals in the light-passable degree on the optical waveguide surface, and thereby evanescent light oozes out from the clearance to the surface and the evanescent light acts on a component bonded to the surface of the fine material. Hereby the evanescent light oozing out from the waveguide surface is absorbed by DNA, and shows an absorbance in proportion to the DNA concentration. Namely, qualitative/quantitative analysis is enabled from the absorbance. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention provides a method for qualitatively and quantitatively measuring a sample to be measured (DNA or the like) immobilized on the surface of a substance (such as gold) that does not transmit light with a small amount of sample by an optical waveguide method at low cost. .

  (1) In a system in which a thin film layer such as gold is provided on the entire surface of the optical waveguide substrate, and the specimen to be measured is fixed and coupled thereto, the evanescent light is absorbed by the thin film and cannot reach the surface of the thin film. For this reason, there is a drawback in that the method of measuring by combining a sample to be measured with a substance that does not transmit light cannot be applied to the optical waveguide method.

(2) A specific example is shown below.
A DNA probe in which a thiol group (-SH) is modified at one end is immobilized on a gold thin film by bonding of -S and Au (111) surface. A commonly used DNA chip has a base layer made of Cr, Ti, etc. on a substrate made of glass, plastic, silicon single crystal, etc., and a thin gold layer is laminated on it, and the Au (111) surface is the outermost surface. It has the structure formed in. When a thin layer having this structure is formed on the entire surface of the optical waveguide, the evanescent light is absorbed and does not reach the gold surface because the film thickness is large. Therefore, it is not possible to excite a fluorescent substance modified with DNA immobilized on the gold surface. Similarly, absorption of evanescent waves by DNA immobilized on the gold surface cannot be measured.

  (3) The target DNA in the unknown sample is hybridized to the DNA probe fixed on the Au (111) surface on the gold thin layer. There is a method in which a specific intercalating agent is intercalated into this double-stranded DNA and the current flowing between the electrode substrate and the electrode substrate is measured. This method has a drawback that the cost of the chip becomes very high because the structure of the electrode is complicated and requires advanced manufacturing techniques.

  (4) A DNA probe is immobilized on the Au (111) surface on the gold thin layer via a thiol group, and the target DNA is hybridized thereto. There is a method for measuring the double-stranded DNA thus formed by surface plasmon resonance (SPR). This method has the disadvantage of using a large amount of sample.

  (5) A DNA probe is immobilized on gold nanoparticles via a thiol group, and a target DNA is hybridized thereto. There is a method of aggregating the plurality of gold nanoparticles and detecting DNA from the density thereof. This method cannot identify multiple types of DNA. In addition, a large amount of sample is required.

  It is an object to provide a method for qualitatively and quantitatively measuring a sample to be measured (DNA, etc.) immobilized on the surface of a substance (such as gold) that does not transmit light with a small amount of sample by an optical waveguide method at low cost. To do.

The basic means for solving the above problems and specific application examples are shown below.
The substance (gold or the like) that binds the specimen is made into nano-order fine particles, thin-film dots or thin lines, and is arranged on the surface of the optical waveguide so as to have a distance that allows light to pass through each other. A sample to be measured (DNA or the like) is bound to the surface of a substance to which the sample is bound. In the case of fine particles, it is also possible to place the analyte on the surface and then place it on the surface of the optical waveguide.

A specific method will be described below by taking a DNA measurement method as an example.
In order to allow evanescent light to reach the DNA immobilized on the gold surface, a plurality of gold nanodots or nano-order lines are formed on the surface of the optical waveguide at a certain interval, or nanoparticles are arranged, and thiol is placed on the gold. The DNA probe is immobilized via the group. A sample is dropped here, the target DNA is hybridized to the probe, and then the sample is washed away so that only the hybridized double-stranded target DNA is fixed to gold. Thereafter, a fluorescent substance is adsorbed on the double-stranded DNA, and excitation light is incident on the optical waveguide. Excitation light reaches the DNA fixed on the gold surface from the waveguide surface through dots or particles, and excites the fluorescent substance attached to the DNA. The fluorescence intensity is measured by a detector provided to oppose the waveguide surface. Qualitatively or quantitatively determine DNA by fluorescence emission intensity.

In addition, after fixing only the target double-stranded DNA to the gold surface, ultraviolet light is incident on the optical waveguide, and this light is made to reach the DNA fixed on the gold surface from the waveguide surface through the dots, fine wires, or particles. Then, the absorbance is detected by a detector provided on the exit side of the optical waveguide, and DNA is qualitatively or quantitatively determined by the magnitude of the absorbance.

{Circle around (1)} It is possible to measure not only an object bonded to a light-transmitting substance but also an object bonded to the surface using a non-light-transmitting substance by the optical waveguide method.
Specific examples are as follows:
In general, a method of immobilizing DNA on gold by a bond between gold and a -SH group, that is, a thiolate bond is generally used. In a general method of forming a gold thin film on the surface of an optical waveguide and immobilizing DNA on this, the thickness of the gold thin film is several hundred to several thousand nm, and evanescent waves cannot penetrate this film, so DNA immobilized on the gold surface Cannot be measured. That is, optical waveguide spectroscopy cannot be applied to DNA measurement. However, the present invention enables optical waveguide spectroscopy to be used for DNA measurement.

(2) Optical waveguide spectroscopy is very inexpensive and can provide an inexpensive measurement method.
(3) Measurement is possible with a small amount of sample.
(4) Measurement is easy.
(5) A plurality of samples can be measured with one waveguide chip.

(6) The cost of the chip is low.
For example, the current detection method that has already been developed and the method of detecting DNA by the electrochemiluminescence method have a very expensive measuring chip, but this method may reduce the cost to half or less.
(7) The method according to the present invention can be used in a wide range of fields such as biotechnology and environment-related substances, and can greatly expand the analysis range by an optical waveguide.

BEST MODE FOR CARRYING OUT THE INVENTION

Examples according to the present invention will be described below.
Examples relating to the optical waveguide DNA detection chip are shown below.

(1) Optical waveguide nanodot DNA evaluation chip for fluorescence detection method:
Figure 1 shows a schematic diagram.
A large number of gold nanodots are fixed on an optical waveguide (6) made of glass, quartz glass, plastic or the like at intervals. A DNA probe is fixed to the gold dot, a target DNA in an unknown sample is hybridized, and a fluorescent dye is further adsorbed. In this state, the excitation light (1) is incident from the side surface of the waveguide (6). An evanescent wave leaches out of the waveguide surface through the gap between the gold nanodots to excite the fluorescent dye (5) adsorbed on the DNA (3). The fluorescence (4) thus excited is detected by a detector (FIG. 9- (14)) installed so as to face the waveguide surface. Since the fluorescence intensity is proportional to the number of ds-DNAs immobilized on the gold nanodots formed on the waveguide surface, qualitative and quantitative analysis is possible.

(2) Waveguide for multiple simultaneous measurement:
The above waveguide can be branched as shown in FIG. 2 to provide a plurality of detectors. As shown in the figure, the waveguide is branched into a plurality of parts, a plurality of DNA probe fixing regions are provided, and the target DNA is hybridized there. Further, a fluorescent dye is adsorbed, and fluorescence is generated by excitation light incident from the left side. Although an example of four branches is shown here, the number of branches is not limited.

(3) Absorbance measurement method DNA chip:
FIG. 3 is for measuring the light absorption of DNA without adsorbing the fluorescent dye (5) in the fluorescence measuring chip described in FIG. When the incident light (1) -1 passes through the optical waveguide (6), its evanescent wave (eg, wavelength λ≈260 nm) is absorbed by DNA. Therefore, if the absorption spectrum of the outgoing light (7) is measured, the DNA concentration on the surface of the waveguide can be measured.

(4) Absorbance measurement method for multiple simultaneous measurement DNA chip:
As shown in Fig. 4, it is possible to fix a plurality of DNAs on a single chip by using a waveguide DNA chip having the structure shown in Fig. 3 as the DNA fixing region, and to measure by a light absorption measurement method. is there.

(5) Optical waveguide DNA nanoparticle chip for fluorescence measurement;
As shown in FIG. 5, a DNA probe immobilized on the surface of a gold nanoparticle {circle around (2)}-1 via a thiol group (—SH), etc., by thiolate bonding, and hybridized with the target DNA is light. It is placed on the surface of the optical waveguide with an interval that allows transmission of light, or is adhered or adsorbed. The incident evanescent light of the excitation light (1) propagates through the gaps between the nanoparticles, excites the fluorescent dye (5) adsorbed on the DNA, and emits fluorescence. If this fluorescence intensity is measured by a fluorescence detector (FIG. 9- <14>), its concentration can be measured.

(6) Gold nanoparticle DNA chip for measuring absorbance:
As shown in FIG. 6, after the evanescent light of the incident light (1) -1 propagates through the gold nanoparticles (2) -1, and receives the absorption of the DNA (3) immobilized on the nanoparticles. Then, the light exits from the waveguide exit (7) -1. Nanoparticles can be measured even in liquid. That is, a liquid containing DNA-immobilized nanoparticles may be placed on the surface of the optical waveguide.

(7) Gold nanoline chip for fluorescence measurement;
Gold nanodots {2} and nanoparticles {circle around (2)}-1 shown in FIGS. 1, 3, 5 and 6 are converted into gold nanolines having a line width, height (thickness), and interval of several nanometers to several hundred nanometers. The optical waveguide chip replaced with 2 ▼ -2 can be applied for the same purpose. An example is schematically shown in FIG. A plurality of gold thin film wires {circle around (2)}-2 are provided on the surface of the optical waveguide {circle around (6)} so as to satisfy the above conditions at right angles to the light traveling direction or at an inclination angle. On this gold wire thin film, DNA {circle around (3)} is fixed by the method already described, and further fluorescent dye {circle around (4)} is adsorbed. Excitation light (1) is made incident on the optical waveguide chip thus formed, and the evanescent wave that has exuded to the surface through the gold nanoline (gold thin film line) excites the fluorescent dye (4), and the DNA concentration Fluorescence (5) corresponding to is generated. The intensity of fluorescence (4) is detected by a detector (FIG. 9- (14)) installed so as to face each other on the surface of the waveguide.

(8) Gold nanoline chip for absorbance measurement;
As shown in FIG. 8, the evanescent wave of the incident light (1) incident on the DNA (3) without adsorbing the fluorescent dye (4) in FIG. It oozes out through (2) -2, is absorbed by DNA (3) immobilized on the gold nanoline surface, and is emitted from the end face of the optical waveguide as outgoing light (7). DNA can be measured by measuring the absorbance of this light with a spectroscope (Fig. 9-12).

Chips having the structure shown in FIGS. 5, 6, 7 and 8 can be provided in the DNA fixing regions of FIGS. 2 and 4, respectively, to form a chip for measuring a plurality of DNAs.

An example of a measurement system using the above chip is shown below.
FIG. 9 shows an outline of a measurement system for both the fluorescence measurement method and the absorbance measurement method using the chip according to the present invention.

First, a fluorescence measurement method will be described. In the case of fluorescence measurement, the excitation wavelength of the fluorescent dye is generally in the visible light region. Therefore, a visible light region (VIS) light source (8); a tungsten or tungsten halogen lamp, or an LED is often used. The light generated by the light source is collected by the condenser lens (9) and enters the end face of the optical waveguide chip (10). As shown in the figure, this light travels in the waveguide while repeating total reflection in the waveguide, and is emitted from the opposite end face. On the way, the evanescent light that has oozed out on the surface of the waveguide excites the fluorescent dye (5) adsorbed on the DNA (3) fixed to the gold fine particles or the nanodot (2) or the like to generate fluorescence. This fluorescence intensity is counted by the detector (14) and converted into a concentration using a calibration curve or the like prepared in advance by the computer (13).

Next, an absorbance measurement method will be described. The gold nanoparticles fixed on the surface of the optical waveguide, or the DNA {circle around (3)} fixed to the nanodot {circle around (2)} by a thiolate bond has an ultraviolet (wavelength λ≈260 nm) absorption spectrum. Therefore, qualitative quantitative measurement can be performed by measuring the absorbance at this wavelength. Light emitted from the light source (8) (UV light source; halogen lamp, etc.) shown in FIG. 9 is incident on the end face of the optical waveguide chip (11) as in the fluorescence measurement method. The light travels through the waveguide while being repeatedly reflected, and is emitted as transmitted light from the right end. On the way, the evanescent light oozes out on the surface and is absorbed by the gold nanodot (2) fixed on the optical waveguide surface (lower surface) or the DNA (3) fixed on the nanoparticle (2) -1. The transmitted light emitted from the optical waveguide chip (10) is collected by the lens (11) and the absorption spectrum is measured by the spectroscope (12). The data is processed by a computer (13) to display a spectrum and convert it to a concentration.

The above shows the measurement of DNA using Au-S bond (thiolate bond). However, this method can be measured not only by thiolate binding but also by binding bio-related substances in general and environment-related substances (environmental hormones, residual agricultural chemicals, etc.) by other methods. The example is shown below.

Highly sensitive detection is performed by binding chemical substances in the environment to various nanoparticles using an antigen-antibody reaction peculiar to living organisms and arranging them on the surface of the optical waveguide. That is, an antibody or an antigen is bonded to nanoparticles, and a harmful chemical substance is fixed to the nanoparticle by an antigen-antibody reaction and disposed on the surface of the optical waveguide. Measure qualitatively and quantitatively by measuring the light intensity using light absorption spectroscopy in that state, or by modifying the fluorescent substance and measuring the fluorescence intensity.

Dioxins and the like can be detected with high sensitivity by synthesizing peptides with nanoparticles, modifying a dye complex in which a fluorescent dye is bound to dioxins, arranging the complex on the surface of an optical waveguide, and measuring the fluorescence.

As described above, the present invention has the potential to be applied to the measurement of various substances.

"Effect of the embodiment"
According to this embodiment, it is possible to measure the absorbance of DNA immobilized on the surface of a substance such as gold that does not transmit light, or to excite the fluorescent dye adsorbed on the DNA immobilized on the surface of gold or the like to emit light. And DNA can be measured.

FIG. 1 is a schematic diagram of a waveguide DNA nanodot chip for fluorescence measurement. FIG. 2 is a schematic diagram of a waveguide DNA chip for simultaneous measurement. FIG. 3 is a schematic diagram of a light absorption measurement type DNA nanodot chip. FIG. 4 is a schematic diagram of an absorbance measurement method DNA nanodot chip for multiple simultaneous measurement. FIG. 5 is a schematic diagram of an optical waveguide DNA nanoparticle chip for fluorescence measurement. FIG. 6 is a schematic diagram of an optical waveguide DNA nanoparticle chip for absorbance measurement. FIG. 7 is a schematic diagram of an optical waveguide DNA nanoline chip for fluorescence measurement. FIG. 8 is a schematic diagram of an optical waveguide DNA nanoline chip for absorbance measurement. FIG. 9 shows a schematic configuration diagram of a DNA measurement system using an optical waveguide chip.

Explanation of symbols

(1) Excitation light, (1) -1 incident light, (2) Nanodot, (2) -1 Nanoparticle, (2) -2 Gold nanoline, (3) DNA, (4) Fluorescence, (5) Fluorescent dye , (6) Optical waveguide, (7) Transmitted light, (7) -1 Outgoing light, (8) Light source, (9) Condensing lens, (10) Optical waveguide chip, (11) Condensing lens, (12) Spectrometer, (13) Calculator, (14) Fluorescence detector

Claims (8)

  It is characterized in that a substrate with a size of several to several hundreds of nanometers on which the sample to be measured (DNA, bio-related substances, environmental hormones, etc.) is fixed is placed on the surface of the optical waveguide at an interval that allows light to pass. Detection element   2. The detection element according to claim 1, wherein the substrate on which the sample to be measured is fixed is a gold nanodot.   2. The detection element according to claim 1, wherein the substrate on which the sample to be measured is fixed is gold nanoparticles.   2. The detection element according to claim 1, wherein the substrate to which the analyte is measured is a gold thin film nanoline.   The detection element according to claim 1, wherein the substrate for fixing the sample to be measured is made of a material other than gold.   2. The detection element according to claim 1, wherein in the optical waveguide branched into a plurality, the fluorescent substances contained on the surface of the waveguide each adsorbing an arbitrary analyte (DNA or the like) are simultaneously excited.   7. The detection system according to claim 6, wherein an area sensor such as a semiconductor is used as a light receiving unit, and the light emission intensities of a plurality of waveguides are simultaneously measured.   A detection system for measuring absorbance absorbed by a waveguide in which an arbitrary analyte (DNA or the like) is adsorbed in an optical waveguide branched into a single or plural optical waveguides