JP2009174954A - Encoded particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an encoded particle for enabling precise analysis using beads, and to provide an analyzer. <P>SOLUTION: The encoded particle 100 is constituted of two transparent flat plate layers 102 and 104, having an outer shape comprising a circle with a diameter or a polygon of which one side has a length of 1-100 μm, and a thickness of 1-30 μm the light-reflecting film or light-shielding film 110, provided in between two transparent flat plate layers and patterned corresponding to a particle kind discriminating code. Biological surface modification is applied to the encoded particle 100, and then the encoded particle 100 is used in the analyzer. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to coded particles used as beads in an analysis technique using beads. The present invention also relates to a method for producing coded particles, an analysis apparatus using the coded particles, and an analysis method.

  Analytical techniques such as immunoassay using beads, DNA (Deoxyribo Nucleic Acid) analysis, SNPs (Single Nucleotide Polymorphisms) analysis, drug discovery and the like are known. In this analysis technique, for example, beads subjected to biological surface modification are suspended in a sample, and the beads and a target labeled (labeled) with a fluorescent substance are subjected to a binding reaction.

  Here, the recognition of the kind of bead is generally performed by changing the mixing ratio of a plurality of fluorescent substances to be mixed in the bead and measuring the fluorescence intensity ratio for each observed fluorescence wavelength. However, since this recognition process is an analog and statistical process, there is a problem that the noise is large and the resolution is low.

  There is also an example in which a bead with a code, that is, a coded particle is used as a bead. In the example shown in FIG. 6, the type of particle 602 is identified by marking the solid support particle 602 with a through hole 604, a recess 606, a groove 608, or a notch 610 (see Patent Document 1 below). Reference numeral 612 denotes an orientation mark.

  FIG. 7 is a diagram for explaining conventional particles coded by bleaching a fluorescent agent in a cord form. FIG. 7 (a) is a diagram of codes of various colors bleached. 7 (b), (c) and (d) schematically illustrate various intensities in the code using graphs (see Patent Document 2 below). ).

  FIG. 8 is a diagram showing an example of a conventional code reading device (see Patent Document 1 below). In the figure, reference numeral 802 denotes encoded particles, reference numeral 804 denotes a capillary flow path, reference numeral 806 denotes a reading station, reference numeral 808 denotes a charge coupled device (CCD) camera, and reference numeral 810 denotes a calculation system. In this example, the coded particles are flowed and washed, and the code is read by photographing the coded particles in the flow.

  Even in the prior art shown in FIG. 6, FIG. 7 and FIG. 8, a good recognition of signal-to-noise ratio (signal-to-noise ratio) is an issue, and depending on the bead orientation, the code is read. There is a problem that it cannot be cleaned and cannot be cleaned cleanly.

JP 2000-516195 gazette JP 2002-542484 A

  The present invention has been made in view of the above-described problems, and an object of the present invention is to provide a coded particle, a method for producing the same, and an analysis apparatus using the same, which can perform analysis using beads with high accuracy. And providing an analysis method.

  In order to achieve the above object, according to the first aspect of the present invention, there are two transparent flat layers having a circular or polygonal outer shape, and the particle type identification between the two transparent flat layers. There is provided a coded particle comprising a light reflecting film or a light shielding film patterned according to a code for use.

  Moreover, according to the 2nd surface of this invention, what carried out bio-surface modification to the coded particle | grains by said 1st surface is provided.

  According to a third aspect of the present invention, there is provided a method for producing a coded particle according to the first surface, wherein the release layer and the first transparent flat layer are formed on the semiconductor substrate, A step of applying one resist layer, a step of exposing and developing the first resist layer in a pattern according to a particle type identification code, and then forming a vapor deposition film; and After peeling, by forming a second transparent flat layer, applying a second resist layer, exposing and developing the second resist layer in the form of particles, and etching or ion milling There is provided a method for producing coded particles, comprising the step of dividing into particle pieces and the step of dissolving the release layer and the second resist layer to obtain a plurality of coded particles.

  According to the fourth aspect of the present invention, the capturing means for capturing the encoded particles in a plane aligned state by adsorption from the suspension of the encoded particles according to the second surface, and the capturing means Based on the irradiation means for irradiating light to the coded particles and the reflected light or transmitted light from the coded particles irradiated with light by the irradiation means, the particle type identification code of the coded particles is read. And a reading means.

  Furthermore, according to the present invention, there are provided an analysis method having the same technical features as the above-described analytical device and a program for causing a computer to function as the control unit of the above-described analytical device.

  According to the disclosed encoded particle and analyzer, the code is read and recognized by the reflected light or transmitted light generated by the light reflecting film or the light shielding film, so the SN ratio is good. Further, since the encoded particles are captured in an aligned state by adsorption, they do not flow out by washing, and the code can be read easily.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. FIG. 1 is a diagram showing an encoded particle 100 according to an embodiment, where (a) is a plan view, (b) is a side view, and (c) is a bottom view. The coded particle 100 was patterned between two transparent flat layers 102 and 104 having a circular outer shape with a diameter of 26 ± 1 μm and a thickness of 4 μm according to the particle type identification code. The light reflecting film 110 is provided. The transparent flat layers 102 and 104 are made of glass, ceramics, or the like such as Si, SiO ₂ , SiN, Si ₃ N ₄ or the like. The light reflecting film 110 is made of Al, Ti, or the like.

  The light reflecting film 110 includes one reference mark 112 and a plurality (N) of bit marks 114. The reference mark 112 indicates the starting point and the arrangement direction, and this makes it possible to correctly identify the code when viewed from either the front surface or the back surface. That is, it can be recognized as the same particle type in any of the states of FIGS.

Further, the bit mark 114 represents information of “1” or “0” depending on whether or not it exists. When the number N of bit marks 114 is 14 (N = 14), a particle set capable of displaying 2 ^N = 16384 types of codes can be prepared. The recognition of the code is realized by reading strong light generated by reflecting the incident illumination light by the light reflection film 110.

  Each of the transparent flat layers 102 and 104 shown in FIG. 1 has a circular structure with a diameter of 26 ± 1 μm and a thickness of 4 μm, but the diameter is in the range of 1 to 100 μm and the thickness is in the range of 1 to 30 μm. It can be changed as appropriate. Furthermore, it is not necessary to be circular, and it is good also as two transparent flat plate layers which have the external shape which consists of a polygon whose 1 side length is 1-100 micrometers, and are 1-30 micrometers in thickness. With these dimensions, the coded particles are arranged on a plane according to the aspect ratio.

  In the example shown in FIG. 1, the light reflecting film 110 is disposed between the two transparent flat layers 102 and 104 to represent the particle type identification code. However, instead of the light reflecting film, the code is used. A light shielding film patterned in accordance with the above may be provided. In this case, the code can be recognized by illuminating the coded particle from one side of the coded particle, observing the transmitted light on the other side of the coded particle, and reading the shadow. Regardless of which one of the light reflecting film and the light shielding film is employed, the code can be read with a contrast having a high light intensity.

FIG. 2 is a diagram showing an example of a method for producing the coded particle shown in FIG. First, as shown in FIG. 2A, a release layer 204 made of polysilicon (Poly-Si) and a first transparent (ie, lower layer side) transparent made of SiO ₂ on a semiconductor (Si) substrate 202. The flat layer 206 is formed by PECVD (Plasma Enhanced Chemical Vapor Deposition), and then the first resist layer 208 is applied.

  Next, as shown in FIG. 2B, after the first resist layer 208 is exposed and developed in a pattern corresponding to the particle type identification code, a vapor deposition film 210 made of Al or Ti is formed.

Next, as shown in FIG. 2C, after the first resist layer 208 is peeled off, a second (that is, the upper layer) transparent flat plate layer 212 made of SiO ₂ is formed by PECVD.

  Next, as shown in FIG. 2D, after the second resist layer 214 is applied, the second resist layer 214 is exposed and developed in the form of particles.

  Next, as shown in FIG. 2E, the particles are divided into particles by etching or ion milling.

  Finally, as shown in FIG. 2 (f), the plurality of coded particles 100 are peeled from the semiconductor (Si) substrate 202 by dissolving the peeling layer 204 and the second resist layer 214. In this way, 6M coded particles can be produced per 4 inch substrate.

  FIG. 3 is a diagram for explaining the biological surface modification applied to the encoded particle shown in FIG. 1, wherein (a) is a coated encoded particle, and (b) is a biological sample after the coating. Coded particles with surface modification, (c) schematically show the binding state between the bio-surface-modified coded particles and a target labeled with a fluorescent substance (labeled).

  As shown in FIG. 3A, various functional groups are introduced by coating the surface of the coded particle 100 with a silane coupling agent or polymer 302. After the functional group is introduced, as schematically shown in FIG. 3B, biosurface modification 304 (for example, immunoassay) related to immunoassay, DNA analysis, SNPs analysis, drug discovery, etc. Antibody-modified), and the encoded particle 306 having a biological surface modification for analysis is completed.

At this time, a code may be assigned for each type of biological surface modification. For example, as shown in FIG. 1, if the code is arranged in 14 bit patterns, 2 ¹⁴ = 16384 types of biological surface modification. It will be possible to cope with. For example, as shown in FIG. 3C, the bio-surface-modified encoded particle 306 can cause a binding reaction with a target 310 that is labeled with a fluorescent material 308 (labeled). In the analysis experiment, it is possible to detect the presence of the target in the sample by discriminating the code of the coded particle that emits fluorescence.

  FIG. 4 is a diagram illustrating a schematic configuration of an embodiment of the analyzer. FIG. 5 is a diagram for explaining the usage form of the coded particles in the analysis. FIG. 5A is a diagram for explaining how the coded particles are captured by the petri dish and code recognition under visible light. (B) is a figure for demonstrating fluorescence observation.

  4, reference numeral 402 is a 96-well plate, reference numeral 404 is an electric pipette, reference numeral 406 is a capture petri dish, reference numeral 408 is a bead capture / release pressure regulator, reference numeral 410 is a buffer tank, reference numeral 412 is a syringe pump, reference numeral 414 is a purge pressure regulator, 416 is a washing water tank, 418 is a waste liquid tank, 420 is a microscope, 422 is a fluorescent excitation lamp, 424 is a fluorescent filter turret, 426 is a cooled CCD (Charge Coupled Device). Element) camera, reference numeral 430 indicates a personal computer (PC), and reference numeral 440 indicates a display.

  The PC 430 is a control unit in the analysis apparatus and includes a processor, a memory, and the like, and functions as a mechanism control unit 432, an image processing unit 434, an analysis processing unit 436, and the like by causing the processor to execute a program loaded in the memory. To do.

  First, in each hole of the 96-well plate 402, various beads (bio-surface-modified encoded particles) 306 are suspended in the sample (analyte). Furthermore, if necessary, a fluorescent label reagent for labeling the detection target substance is also suspended. The beads 306 react with and bind to the target (protein, antigen, DNA, etc.) in the sample corresponding to the biological surface modification. The beads 306 bound to the target acquire fluorescence.

  This suspension is transferred to the capture petri dish 406 by the electric pipette 404. As shown in FIG. 5A, the capture petri dish 406 is provided with about 1000 counterbore holes (concave portions) 502 in a matrix. Each counterbore 502 has substantially the same size as the bead 306 and is provided with a suction hole 504 at the bottom thereof. The PC 430 controls the bead capture / release pressure regulator 408 and sucks the bead 306 to the counterbore hole 502 by suction from the back surface of the capture petri dish 406 via the buffer tank 410, the suction hole 504, and the like. To do.

  Next, the PC 430 controls the syringe pump 412 and the purge pressure regulator 414 in such an adsorption state so that the washing water flows from the washing water tank 416 to the capture petri dish 406 and binds to beads and beads not captured by the petri dish. The components that are not present are poured into the waste liquid tank 418.

  Thus, the beads (bio-surface-modified encoded particles) 306 can be observed in a state where the beads 306 are captured in an aligned state on the surface by adsorption and an excess component is removed by washing.

  In the observation, first, the code of the bead 306 captured in each hole of the capture petri dish 406 is read. As shown in FIG. 5A, the PC 430 reads the code of the bead 306 through the microscope 420 under incident visible light illumination, and sets the code, that is, the bead type, for each of the holes 502 provided in a matrix. Remember.

  Next, the PC 430 performs the fluorescence observation shown in FIG. In FIG.5 (b), the mode that the fluorescence has emitted is represented by the oblique line for convenience. That is, the PC 430 uses a fluorescence excitation lamp 422, a fluorescence filter turret 424, etc., performs luminance measurement for each fluorescence filter under epi-illumination, and within the matrix of beads exhibiting the luminance due to the binding reaction with the target. Determine the position. If the position in the matrix is known, the type of bead at that position, that is, the target component, has been investigated in advance, so that the PC 430 can identify the component contained in the sample.

  Regarding the above embodiment, the following supplementary notes are disclosed.

(Supplementary Note 1) Two transparent flat plate layers having a circular or polygonal outer shape,
A light reflecting film or a light shielding film between the two transparent flat layer layers and patterned according to the particle type identification code;
A coded particle comprising:

  (Supplementary note 2) The encoded particle according to supplementary note 1, which has been subjected to biological surface modification.

(Additional remark 3) It is a manufacturing method of the coded particle of Additional remark 1, Comprising:
Applying a first resist layer after forming a release layer and a first transparent flat layer on a semiconductor substrate;
A step of forming a deposited film after exposing and developing the first resist layer in a pattern according to a particle type identification code;
After peeling off the first resist layer, forming a second transparent flat layer;
After applying the second resist layer, exposing and developing the second resist layer in the form of particles; and
Dividing into particle pieces by etching or ion milling;
Dissolving the release layer and the second resist layer to obtain a plurality of coded particles;
A method for producing coded particles, comprising:

(Supplementary Note 4) Capture means for capturing the coded particles from the suspension of coded particles according to Supplementary Note 2 in an aligned state on a plane by adsorption;
Irradiating means for irradiating the encoded particles captured by the capturing means with light;
A reading unit that reads a particle type identification code included in the encoded particle based on reflected light or transmitted light from the encoded particle irradiated with light by the irradiation unit;
An analysis apparatus comprising:

(Appendix 5) Capturing the encoded particles from the suspension of encoded particles according to Appendix 2 in an aligned state on a plane by adsorption;
Irradiating the captured encoded particles with light;
Reading a particle type identification code of the encoded particle based on reflected light or transmitted light from the encoded particle irradiated with the light; and
An analysis method comprising:

(Supplementary note 6) The computer provided in the analyzer is
Means for controlling the capturing unit to capture the encoded particles from the suspension of encoded particles according to appendix 2 in an aligned state by adsorption;
Means for controlling the irradiating unit to irradiate light onto the encoded particles captured by the capturing unit;
Means for reading a particle type identification code of the encoded particle based on reflected light or transmitted light from the encoded particle irradiated with light by the irradiation unit;
Program to function as.

It is a figure which shows the encoding particle | grains concerning one Embodiment, Comprising: (a) is a top view, (b) is a side view, (c) is a bottom view. It is a figure which shows the example of the manufacturing method of the coding particle | grains shown by FIG. FIG. 2 is a diagram for explaining a biological surface modification applied to the encoded particle shown in FIG. 1, wherein (a) is a coated encoded particle, and (b) is a biological surface modification after the coating. (C) is a diagram schematically showing the binding state between the bio-surface-modified encoded particles and the target labeled (labeled) with a fluorescent substance. It is a figure which shows schematic structure of one Embodiment of an analyzer. It is a figure for demonstrating the usage form of the coding particle | grain in an analysis, Comprising: (a) is a figure for demonstrating a mode that the coding particle | grain is trapped by petri dish, and the code recognition under visible light, (b). These are the figures for demonstrating fluorescence observation. FIG. 4 shows conventional particles encoded by through holes, depressions, grooves or notches. It is a figure for demonstrating the conventional particle | grains coded by bleaching a fluorescent agent to code form. It is a figure which shows the example of the conventional code reader.

Explanation of symbols

100 Coded Particles 102, 104 Transparent Flat Layer 110 Light Reflective Film 112 Reference Mark 114 Bit Mark 202 Semiconductor (Si) Substrate 204 Peeling Layer 206 First (Lower Layer) Transparent Flat Layer 208 First Resist Layer 210 Deposition Film 212 Second (upper layer) transparent flat layer 214 Second resist layer 302 Silane coupling agent or polymer 304 Biological surface modification 306 Biological surface modified coded particle 308 Fluorescent substance 310 Target 402 96-well plate 404 Electric pipette 406 Capture Petri dish 408 Bead capture / release pressure regulator 410 Buffer tank 412 Syringe pump 414 Purge pressure regulator 416 Wash water tank 418 Waste liquid tank 420 Microscope 422 Fluorescence excitation lamp 424 Fluorescence filter turret 426 cooled CCD camera 430 a personal computer (PC)
432 Mechanism control unit 434 Image processing unit 436 Analysis processing unit 440 Display 502 Counterbore hole (concave portion)
504 Suction hole 602 Solid support particle 604 Through hole 606 Depression 608 Groove 610 Notch 612 Orientation mark 802 Coded particle 804 Capillary flow path 806 Reading station 808 Charge coupled device (CCD) camera 810 Calculation system

Claims (6)

Two transparent flat layers having a circular or polygonal outer shape;
A light reflecting film or a light shielding film between the two transparent flat layer layers and patterned according to the particle type identification code;
A coded particle comprising:   The encoded particle of claim 1, which has been subjected to biosurface modification. A method for producing a coded particle according to claim 1,
Applying a first resist layer after forming a release layer and a first transparent flat layer on a semiconductor substrate;
A step of forming a deposited film after exposing and developing the first resist layer in a pattern according to a particle type identification code;
After peeling off the first resist layer, forming a second transparent flat layer;
After applying the second resist layer, exposing and developing the second resist layer in the form of particles; and
Dividing into particle pieces by etching or ion milling;
Dissolving the release layer and the second resist layer to obtain a plurality of encoded particles;
A method for producing coded particles, comprising: Capture means for capturing the encoded particles from the suspension of encoded particles according to claim 2 in an aligned state by adsorption;
Irradiating means for irradiating the encoded particles captured by the capturing means with light;
A reading unit that reads a particle type identification code of the encoded particle based on reflected light or transmitted light from the encoded particle irradiated with light by the irradiation unit;
An analysis apparatus comprising: Capturing the encoded particles from a suspension of encoded particles according to claim 2 in an aligned manner by adsorption;
Irradiating the captured encoded particles with light;
Reading a particle type identification code of the encoded particle based on reflected light or transmitted light from the encoded particle irradiated with the light; and
An analysis method comprising: A computer provided in the analyzer
Means for controlling the capture unit to capture the encoded particles in an aligned state by adsorption from the suspension of encoded particles according to claim 2;
Means for controlling the irradiating unit to irradiate light onto the encoded particles captured by the capturing unit;
Means for reading a particle type identification code of the encoded particle based on reflected light or transmitted light from the encoded particle irradiated with light by the irradiation unit;
Program to function as.

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