WO2012157979A2

WO2012157979A2 - Portable hypermulticolor imaging optical module and kinase inhibitory drug screening system based on microbead color coding

Info

Publication number: WO2012157979A2
Application number: PCT/KR2012/003904
Authority: WO
Inventors: 송준명
Original assignee: 서울대학교 산학협력단
Priority date: 2011-05-19
Filing date: 2012-05-17
Publication date: 2012-11-22
Also published as: KR20120129182A; WO2012157979A3; KR101303815B1

Abstract

The present invention relates to developing a portable Hypermulticolor imaging (HMI) optical module and a new drug screening system based on microbead color coding. The HMI optical module is a portable new multi-wavelength imaging system that replaces a High-content screening (HCS) device, which is currently used as an important device for drug screening, and also is a portable HMI system that is attached to the side of a typical optical microscope and provides drug screening at a cell level and a molecular level. Additionally, in order to overcome limitations that Robotics based drug screening has, which is used for drug action and cellular toxicity screening of large scale library compounds through a Well type sample platform, a screening system of large scale library compounds without pipe tong by using microbeads is provided as a new drug and drug target storage. By using a portable HMI optical module attached to an optical microscope and microbeads storing a drug or drug target, more economical and rapid drug screening is possible. Through the application thereof, screening for drug toxicity, which causes a Kinase hindrance effect and drug hepatotoxicity, is provided.

Description

【Specification】

[Name of invention]

Portable kinase inhibitor drug screening system based on hypermulticolor imaging optics and microbead colorcoding

Technical Field

The present invention relates to the development and application of a portable Hypemulticolor imaging (HMI) system capable of high efficiency multicolor imaging through multi-wavelength scanning by attaching to a microscope. Specifically, the present invention is an alternative drug screening system that can replace the existing high-content screening (HCS) drug screening device to implement multi-wavelength imaging by replacing a wheel type bandpass optical filter. Rather than the existing HCS drug screening system, a portable drug screening MHI system can be obtained for each of single-wavelength images in rapid injection form. This portable drug search MHI system is a portable drug search multi-wavelength imaging system that can be used to attach to the optical microscope side that is mostly secured in existing bio research groups, including both Upright and Inverted optical microscopes. It enables multiwavelength cell imaging and multiwavelength molecular imaging at the molecular level. The drug discovery system involves the development of an HMI system consisting of an image sensor, including a c-mount lens, an acousto-optical tunnable filter (AOTF) and a CCD attached to the side of the microscope. Alternative Site (Article 26) The drug efficacy as an inhibitor was applied using microbeads suitable for rapid and large-scale drug efficacy screening without the need for pipetting. In addition, hepatotoxicity evaluation of drugs mediated by mitochondrial dysfunction was performed using microbead technology.

Background Art

Currently, drug-based cell-based assays for drug screening cannot identify on / off target effects and side effects in a single essay, but can be obtained by conducting multiple assays. This screening system is always in a state of drug failure probabilities due to drug side effects throughout the entire drug development process. There is a need for innovative drug discovery platform technology that can dramatically reduce the probability of drug fail. The core technology of the Innovative Drug Search Platform is to verify the many on / off target effects and cytotoxicity that can eventually be caused by drugs with the maximum cost-effectiveness possible in one cell-based assay. Currently, the flow cytometer is a widely used device related to drug efficacy and toxicity. Flow cytometer is a device to measure various fluorescent probe material of each cell by using laser on the part when the cell passes the detector part in the fluid state.

Alternative Site (Article 26) f low cytomete

By detecting minerals

In particular, all detection can be done at the single cell level and at the same time measure different properties of the same particle or cell. The disadvantage is that images cannot be obtained with the f low cytometer. Cell imaging is a very important technique for observing cellular responses to stimuli because it shows where, and when, and how the cellular response to external stimuli occurs. Intracellular signaling materials and numerous signaling proteins form a signaling network. F low cytometry is difficult to understand these complex signaling processes, and it is difficult to observe the movement of proteins during the signaling process. Therefore, not only methods for observing cell images but also methods for simultaneously observing organic reactions in cells have been developed. The development of computer assays in the imaging technology and the breakthrough in general cell biology have greatly increased high-throughput imaging techniques for target val id ions. Existing high-content screening (HCS) is an automated platform for performing fluorescence microscopy and quantitative imaging techniques and is currently being used as a powerful tool for multicolor cell imaging analysis. Cell imaging investigates cell dynamics and cellular kinet i cs, morphology and three alternative papers (Rule 26) Artillery movement

high-content imaging ，

Intracellular elements and reactions can be visualized simultaneously. Automated systems are essential to realize the versatility of high-content imaging. Existing representative HCS measurement instruments include BD biosciences and HCS systems released by Cellomix. The BD pathway high-content imaging system includes a number of excitation filter wheels and an emission filter wheel. Although up to eight different colors of cell imaging are possible, the luminescent filters are narrow narrow band pass filters, and the emission wavelength range is limited in the selection. The price is very expensive. In addition to such imaging technology, another major technology to be developed in the present invention is microbead technology. Microbead technology can potentially run a variety of bioassays in microbeads, and this bioassay is intended to be used as a major platform for drug screening in the present invention. That is, molecular and cell-level assays at 6, 12, 24, and 96 wells are performed in microbeads rather than wells. In the well type, each assay is distinguished by assays in spatially separated wells, but screening in microbeads, rather than wells, simultaneously monitors a large number of beads in one well or space. (Rule Article 26) The microbeads to be divided can address the reactions caused by the test substance or drug present in each microbead. Addressing of microbeads can usually be done by color coding. Therefore, for the bead addressing of microbeads, an optical system that can distinguish multicolored colors must be in place. This microbead-based drug screening can, among other things, process a single drug at the same time for each microbead containing a drug target, enabling the construction of a platform that does not require pipetting, resulting in drug screening assays involving numerous library compounds. The result is a very rapid and economical platform for sorting.

[Detailed Description of the Invention]

[Technical problem]

The present invention was devised to solve the above problems, and developed a portable optical imaging model system for color coding of microbeads using multi-wavelength high resolution color selection based on the high spectral resolution of 1 nm of A0TF. I would like to. Currently, most individual life science bio laboratories have optical microscopes, so portable HMI optical modules that can be attached to the side of existing optical microscopes are developed and replaced (Article 26). Expensive HCS

Perform drug screening

A0TF minimizes the overlap of each wavelength region in creating a sega profile of light transmitted through the microbeads in the 3-7 wavelength range which should be selected to code the color of the microbeads with lnm optical resolution. Finally, there is the advantage of being free to choose a specific wavelength region for the color of the microphone lobby and designing the wavelength profile. It is possible to obtain the transmission image of lnm through AOTF, so 3-7 selected wavelength regions can be obtained without overlapping with existing bandpass filters, and the selective wavelength region can be easily adjusted to greatly increase the color resolution for color coding. By increasing the number and quality of color coding of microbeads can be greatly improved. The present invention devised a color coding method of multicolored microbeads manufactured using various microbeads including multi-wavelength imaging optical systems based on portable HMI modules and alginate gel beads. By encapsulating the drug targets, a number of drug or drug targets are distinguished by color coding and applied to screen pharmacological efficacy and cytotoxicity at the molecular or cellular level of each drug. Therefore, the aim is to develop a new platform of drug screening based on portable HMI optical models and microbeads. Replacement Paper (Rule Article 26) Technical Solution

Techniques for addressing and coding individual microbeads in which each drug or drug target is present, especially when applied to drug screening using microbeads, especially when applied to library compounds with a number of drugs ranging from 10 to 1 million This is a very important technique. In general, when addressing with color codes, different colors can be produced by varying the ratio of pigments such as red, green, and blue. When white light is transmitted to each of these different pigment combinations and then the intensity of light detected in the different wavelengths is transmitted, an image of the color seen by the human eye can be obtained. In order for an image of such a color to be closer to the primary color, the more wavelengths that combine and combine the light intensities in different wavelength regions, the closer to the primary color. For example, the number of wavelength regions to be combined is three to five, As you increase the number from 5 to 7, you can obtain an image close to the primary color. The wavelength range also corresponds to, for example, red (630-740 nm), orange (585-620 nm), yellow (570-580 nm), green (520-570 nm), and blue (440-490 nm). In order to collect the light in each wavelength range, the intensity of transmitted light in the wavelength range corresponding to 630-740 ran should be obtained in one combination. Replacement Paper (Rule Article 26) The present invention

The company is developing a portable multiwavelength imaging model based on A0TF. A0TF is very advantageous for this microbead based color coding. Best of all, AOTF-based microbead imaging systems can be used to obtain transmission images at each wavelength of microbeads with 1 nm resolution. When the intensity of light passing through the pigment-containing microbeads is a function of wavelength, A0TF can measure the intensity of transmitted light at 500-600 single wavelengths, ranging from visible to near-infrared light with guaranteed light transmission. You can also add each of these wavelengths and combine them into a range of wavelengths to obtain an image of the light intensity and microbeads in that range. To obtain an image of the transmission intensity of light in the wavelength range of 630–740 nm, we can first obtain the transmission intensity of the microbeads at each single wavelength ranging from 630-740 nm, red, orange, yellow, green. In addition, the merit of A0TF can be clearly highlighted when measuring the transmission intensity of a microphone lobby in a specific wavelength range corresponding to blue color, and selectively specifying a specific wavelength region through A0TF. This is because, when using a bandpass filter like a conventional HCS device, there is a limit in selectively specifying a specific wavelength. Like this, A0TF can obtain the transmittance in the single wavelength in the visible and near-infrared region by scanning, which makes it easy to select the wavelength region to be combined. Article 26) Avoid hitting wavelength range

As in primary colors;

De-addressing can be enabled. In addition, since A0TF only transmits light at a specific wavelength, white light can be used as a light source for irradiating microbeads. That is, it is easy to measure the measurement time because white light can be irradiated to the whole microbead containing dye without scanning the light of a specific wavelength region as a light source and the transmitted light can be scanned at each wavelength through A0TF. Can shorten. Another advantage of the handheld HMI model is that it attaches to the side of the microscope, so in bright fly mode, white light is irradiated to the microbeads placed on the microscope stage, and the transmitted light is scanned by AOTF to achieve the desired intensity of light in the desired wavelength range. After the measurement, change the microscope's measurement mode to the fluorescence mode, and then measure the fluorescence generated through the microbeads through the AOTF. System is in place. Therefore, an object of the present invention is to provide a drug screening system of a new paradigm of drug screening at the microbead-based molecular level and cell level because portable HMI modules can be attached to an existing microscope. Drug screening of this new paradigm will provide a system that can be done at the level of a single biopharmaceutical laboratory with existing optical microscopes due to the price competitiveness of portable optical model HMIs. Replacement Paper (Rule Article 26) Advantageous Effects

Kinase inhibitor screening system based on color coding of microbeads using MI 1 and 2 systems and HMI optical fibers according to the present invention has the following effects. Firstly, the present invention provides a microbead drug screening system based on a portable HMI optical model, which can be used in a conventional optical microscope holding laboratory through the use of an HMI optical model that can be attached to the side of an existing optical microscope, an upright and an inverted microscope. Providing general purpose multi-wavelength cell and molecular imaging equipment can greatly contribute to the low changes in relevant bioimaging studies. In particular, since most bio laboratories have optical microscopes, bioimaging research can be spread by installing a modeled optical system. Second, the HMI optical mother system can be used as an HCS device, which has a cost savings of 1 / 7.4 compared to the existing HCS device, which is a great cost saving effect that can be held in individual laboratory units economically. HCS devices can be used to enable large-scale drug screening at the individual laboratory level. Thirdly, the present invention uses 14.4 minutes of image measurement per 96well plate as the HCS device, and the existing HCS device takes 40 minutes, whereas the drug target protein reservoir such as Kinase is made of microbeads and the mixtures thereof are prepared. Replacement Paper (Rule Article 26) Microscope Sample Plates ί

¾ for all microbeads

Can be very short. This greatly increases the number of drugs that can be measured and processed per day. It also saves the reagents consumed by treating all microbeads at the same time, rather than adding each reactant to each weld, like 96 welds. Fifth, since the present invention uses A0TF instead of Narrow bandpass filter, the number of applicable wavelengths is greatly increased compared to HCS devices that can use eight Narrow bandpass filters. The width becomes wider. Sixth, the present invention is a new era of drug screening platform without pipetting because it is possible to indicat ion of each drug by making microbead by inserting different dye or drug target for each drug by color coding method and encapsulat ion Can open Seventh, the present invention can be actively used in the pharmaceutical industry as a new product because it can be used as a system in charge of the essential drug screening process of the new drug development process by the microbead color coding method.

[Brief Description of Drawings]

1 is a schematic diagram of a color coding process of a microbead using a portable HMI.

^" ΓΓ Paper (Article 26 of the Rules) 2 is portable

De absorbance image. Figure 3 is (a) microbead fluorescence image by Kinase GSK3b activity, (b) GS 3b inhibited microbead fluorescence image by Methanesulfonate. 4 is a schematic diagram of an HMI 1 system composed of a c-mount lens, A0TF, and a CCD. 5 is a schematic diagram of an HMI 2 system including a beam collimator or a beam expander, A0TF, and a CCD.

[Best form for implementation of the invention]

The present invention is a portable HMI optical models that can be attached to all optical microscopes in general, and the microbead color coding method using the same, the Kinase inhibitor assay and the drug-induced hepatotoxic cell-based HCS measurement method are provided. The present invention is based on ultra-multi-wavelength imaging using HMI optics attached to a general optical microscope, and is required for utilizing microbeads as a new platform for evaluating the efficacy and toxicity of numerous library compounds. Color coding dressings provides a new paradigm of drug screening technology.

Compared to BD bioscience, an HCS device, the portable HMI optical models have a replacement for plain brown rice (rule 26). Attached to the mirror

Cheap specials from

Compared to the filter system, a much faster scanning speed is possible and the versatility of using more than one brob material in various emission wavelength ranges for drug screening is increased. In addition, the HMI system can provide not only high-efficiency imaging but also spectrum by plotting the intensity at a specific photodetector of the images obtained at each wavelength scanned with A0TF, obtained with a CCD detector, as a function of wavelength. First of all, the portable HMI is a cost-effective system compared to the existing HCS drug screening system, which can enjoy the financial burden on consumers. HMI hardware is fabricated in different modes (HMI 1 and HMI 2) according to the customer's needs. In the present invention, the HMI may be characterized by performing hepatotoxicity measurement by kinase inhibition and mitochondrial paralysis by actual drugs using microbeads. Existing drug screening to detect kinase inhibitors based on kinase inhibition assays shows that kinase and substrate fluorescence or lucifer in and lucif erase based chemiluminescence are inhibited by kinase inhibitors in wells such as 96 wells. Monitoring is in progress. In this case, large corporations, such as MNCs, have solved the screening of tens of thousands to hundreds of thousands of library compounds through an automated platform using automated robotics. have . Robotics is a par '

Compounds and reaction products The present invention encapsulates one microbead for one drug or kinase through color coding using microbeads and knows which drug is stored in which microbeads through the color coding ol of each microbead. Can be. This allows numerous drug-containing microbeads to form phosphors through the reaction between Kinase and its substrates, respectively, and monitor the fluorescence response to observe which fluorescence has an inhibitory effect on Kinase. Efficacy can be confirmed. Unlike conventional robotics, this microbead Kinase Inhibition naturally removes the need for pipetting each reactant associated with each Kinase Inhibit ion by treating all reactants at once in microbeads, and through microbead fluorescence imaging New concept innovative screening platforms can be built to quickly identify inhibitory effects. This microbead Kinase inhibition screening can highlight the efficiency and speed of screening, especially as the number of compounds to be screened increases from tens of thousands to hundreds of thousands. In particular, the HMI system is free to substitute A0TF one wavelength per second and transmit wavelength interval (Article 26). (1 nm-several tens nm- ¹

Number increased dramatically ^

Color coding and kinase reactions can be detected in microbeads via a microscopic white light Briefield and a detector HMI attached to the side of the microscope that operates identically without moving in fluorescence mode. 4 is a schematic diagram of an HMI 1 system composed of a c-mount lens, an AOTF, and a CCD, and FIG. 5 is a schematic diagram of an HMI 2 system composed of a beam collimator or a beam expander, an AOTF, and a CCD. Referring to the drawings, the present invention (portable hypermulti-imaging optical modules, i.e. HMI system), the case 10, the first lens member 20, the variable filter 40, mechanical plate, tube pass filter or band pass A filter 50 and an image sensor 60. The case 10 has a model structure in which one side thereof is fastened to the microscope. In addition, the first lens member 20 is installed at an opening of one side of the case 10 and collects a fluorescent beam from a microscope. It has a condensing lens 22. Of course, in the opening of one side of the case 10, a chaff U lens member 20 having a beam collimator or a beam expander 24 for reducing the size of the fluorescent beam emitted from the microscope is installed. May be

Alternative Site (Article 26) Arler, above

Fluorescent lamp passed 20

All. In addition, the mechanical plate (not shown) may be disposed in the case 10, and serves to mechanically remove the beam of the non-diffraction wavelength from the fluorescent beam passing through the variable filter 40. In addition, the longpass filter or the bandpass filter 50 is disposed in the case 10 to remove light from the excitation light source from the fluorescent beam passing through the variable filter 40. In addition, the image sensor 60 is installed in the case 10, and detects the fluorescent beam passing through the mechanical plate and the through-pass filter or the band-pass filter 50. At this time, the second lens unit (not shown) is configured to be disposed in front of the image sensor to obtain a high-quality image. The second lens book member may not only be mounted on the front of the image sensor by being c-mounted, but may also be installed on the front side of the image sensor separately in a case. That is, the installation structure of the second lens member can finally obtain a high-quality emission image

Alternative Site (Article 26) Image sensor

Do not. The present invention configured as described above is capable of hypermulticolor imaging (61 ^"# 1 «10 imaging, HMI), and is configured to be detachably attached to a microscope in a portable manner. And a support bar 80 for preventing and fixing the variable filter 40, the mechanical plate, the through-pass filter or the band pass filter 50, and the image sensor 60, respectively. 80) chapter is grip on the rail member 90 provided on the bottom in the housing 10. That is _yae first lens member 20 from the variable filter 40, the mechanical plate, tube pass filter or band-pass filter (50 ), And the image sensor 60 is movable in a straight direction, the lower end of the support bar 80 is movable to the rail member 90 in the longitudinal direction so that the image is focused (focusing) On the other hand, the above The case 10 replaces and repairs the first lens member 20, the variable filter 40, the mechanical plate, the through-pass filter or the band pass filter 50, and the image sensor 60 disposed therein. The opening / closing door 12 formed in one surface is provided so as to be possible.

Alternative Site (Article 26) In addition, in order to attach and detach the collection-aulle condenser lens 22 to the opening of the case 10, a thread is formed at the edge of the condenser lens 22 and the edge of the opening of the case 10, and the opening of the case 10 is opened. At least one condenser lens 22 is preferably configured to be C-mounted. Here, the present invention may further include a lens support member 30 configured to be supported by the first lens member 20 at an opening of one side portion of the case 10. In addition, the variable filter 40 is preferably an acoustic optical wavelength variable filter 40 (acousto-optic tunable filter 40 (acousto-optic tunable filter, A0TF)). In addition, the image sensor 60 is preferably a two-dimensional image sensor 60 including a charge coupled device camera and a photodiode array. On the other hand, the case 10, the water cooling ( _wa ) by the air-cooling (air-cooling) member or cooling water configured to ventilate for cooling the variable filter 40 or the image sensor 60 ( _wa ter-cooling) member may be further provided. In addition, the case 10 is opaque to block external light from entering

Alternative Site (Article 26) Made of material [form for implementation of the invention]

Example 1 HMI 1 Module The present invention provides (a) an HMI 1 system using a c-mount lens, A0TF, and a CCD, (b) a beam expander AOTF, and an HMI 2 system using a CCD, and uses the system. This study was applied to color coding and kinase inhibitor screening in microbeads and to observation of drug-induced mitochondrial dysfunction via hepatocellular cytotoxic cell imaging. In particular, these HMI modules are attached to existing microscopes and used to provide new drug screening platforms based on portable HMI and microbead technology through HCS imaging at the molecular and cellular levels of drug efficacy mentioned in existing microscope-held laboratories. It aims to be able to realize form all. In the present invention, HMI 1 modules are attached to the side of the fluorescence microscope to perform the HCS, the configuration is largely on the side of the microscope such as c-mount lens at the entrance of the HMI module attached to the side of the optical fluorescence microscope It consists of a first lens member for condensing the outgoing beam, an A0TF through which the beam from the first lens member passes, and a CCD positioned at the focal point of the outgoing beam through the A0TF to obtain the image.

In front of the CCD, better light from the light source of a fluorescence microscope typically passes through an excitation filter and is then collected through an objective lens through a dichroic mirror to reach a sample on the sample stage of the microscope. Replacement Paper (Rule Article 26) Reached Light is ¾

The material emits light, and the emitted light is focused again on the lens. Condensed light exits through the dichroic mirror to the side of the microscope, where light from the side of the microscope has different diameters depending on commercial microscopes. The diameter of this particular category is generally larger than that of A0TF, which has a window of lxl cm ² . Therefore, the HMI module has a first lens member, such as a c-mount lens, which can be attached from a microscope, to condense the light from the microscope side. The A0TF in the module is located behind the modal inlet c-mount first lens member so that the light collected through the lens passes through the window of the A0TF. Of the light passing through A0TF, light of a certain wavelength is diffracted in the A0TF crystal so that it passes through a certain angle (within the range of about 3-15 ⁰ ) from the main axis of A0TF and the transmitted incident beam and the light of the remaining wavelengths cannot pass.

Light of a certain wavelength passing through A0TF is detected by the CCD. In addition to the light of a specific wavelength diffracted and passed through A0TF, there is also a background light that is directly transmitted without diffraction. This background light usually passes through a crystal in A0TF in the same direction as the axial direction of the incident beam, taking advantage of the angular difference (within the range of about 3-15 ⁰ ) between the background light and the particular wavelength light that is diffracted and transmitted. Substitute sheet by installing mechanical plate in front of CCD behind AOTF (Rule Article 26) Selective removal of light only On the other hand, the specific wavelength light diffracted from A0TF at a certain angle comes out of the excitation light source in the fluorescence microscope, so a longpass filter or bandpass filter is installed in front of the CCD as a means to remove the light from this excitation light source To get a clearer image. In addition, a c-mount lens is attached in front of the CCD for clear, high-quality images, so that AOTF-transmitted specific wavelength light can be smoothly collected. This HMI optical module structure allows scanning of all fluorescent light excited by the microscope, such as intracellular or probe material, microbeads, and the like at 1 nm spectral resolution on the microscopic sample stage to obtain images at each wavelength. In addition, by linking the intensities of the CCD at a specific location of the pixel at each wavelength, the spectrum of a certain wavelength range can be obtained simultaneously. At this time, the scanning speed is 1 frame / sec. The image thus obtained is analyzed by software such as Metamorph. These optical modules are two-dimensional image detectors, including the inlet c-mount first lens element, AOTF, Longpass filter (or bandpass filter), and CCD, which detect the condensation by the inlet system one lens element with high sensitivity. Alternatives, in which each component can be moved through rails or similar moving devices for fixing in order to be secured through specific supports present in HMI modules (rule 26) It is possible. The optical modules are all in one package and are designed to be attached to the side of the optical microscope, including all commercial fluorescence microscopes. QHMI models are equipped with an image detector including a CCD and an air-cooling or water-cooling system to prevent overheating due to prolonged use of the A0TF. Example 2 HMI 2 System Another optical mode system designed for mods with different optical characteristics from the HMI 1 system. HMI 1 has a structure in which the beam from the side of the microscope is focused by a c-mount 1 lens member located at the entrance of the optical modules. As a result, the HMI 1 optical models are relatively small in image size, while the images are high in intensity and high in resolution. The HMI 2 system has different characteristics by different optical components and arrangement than HMI 1. In this case, the image size is increased and more detailed morphology of the material, including the cells, can be observed.

The optical module inlet on HMI 2 attaches a beam collimator or expander to make the large radius beam from the side of the microscope smaller than the parallel beam (the radius of which remains almost constant relative to the straight direction).

Replacement Paper (Rule Article 26) Collimator ：

Parallel pans

By reorienting and installing at the inlet of the HMI 2 module, when the beam coming from the side of the microscope enters the inverted expander, the output beam decreases rather than increases its diameter.

The inlet with the collimator or expander was attached to the c-mount, which also coupled the two HMIs to the side of the microscope. The collimator or expander is fabricated so that the radius of the parallel beam with reduced radius can enter the 1cm X lcm window of A0TF so that a parallel beam with a radius of less than 1 cm is obtained through the collimator or expander. The parallel beam of this radius passes through the A0TF only when the acoustic field is applied, and only the transmitted light of the specific wavelength is selectively removed from the transmitted light without diffraction through the mechanical plate. Then, the mgpass filter or the bandpass filter is used. The excitation light source is reduced and the image is acquired by a two-dimensional image detector with a CCD. In addition, a C-mount lens (second lens member described above) is attached directly in front of the CCD for a clear high-quality image, so that the A0TF transmission specific wavelength light can be smoothly collected.

The HMI 2 optics are all in one package and are designed to be attached to the side of an optical microscope, including all commercial fluorescent microscopes. Alternative Site (Article 26) HMI 2 mods

Air-cooling or water-cooling system is installed to prevent heat. In addition, all components can be moved around each component, including the rails, to achieve an optimized optical arrangement and can be fixed at a specific location using a support. The image thus obtained is analyzed by software such as metamorph. Example 3 Addressing Microbeads by Color Coding of Microbeads Using HMI Systems Microbeads are prepared using sodium alginate and calcium chloride. Sodium alginate in powder form is dissolved in autoc leaved water to form a 2% sodium alginate solution.

Sodium alginate solution is prepared by adding a variety of colors such as polystyrene microsphere dye or organic dye, sodium alginate solution of different colors. Spherical microbeads are prepared by adding a sodium alginate solution containing dye to a syringe and placing it in a syringe pump, then dropping the sodium alginate solution into 2% calcium chloride solution. The distance between the syringe needle and calcium chloride solution was 5 cm, the voltage applied to the syringe needle was 10.0 kV, and the dissolution rate of the syringe gel was mL / min. Ca ²⁺ acts as a bridge to the polymer sodium alginate, so that the solution is a gel substitute (Article 26) It changes to gel state. Sodium alginate solutions containing different dyes were prepared using gel electrolytic microbeads using electrospray methods, and then using an optical microscope with HMI optical modules. Identification can be performed based on. Each of the other colors can be made from a mix of green, red and blue dyes.

Among the HMI system components, a method for color coding through a microbead image formed on an image sensor including a CCD may be represented by a schematic diagram as shown in FIG. 1. First, a microbead sample of a specific color is placed on the microscope sample platform. Set the microscope to brightfield mode and irradiate the microbeads with white light. A0TF sets the scanning speed of each wavelength to 1 second and performs scanning to obtain microbead image for each wavelength. The wavelength range scanned is red (630-740 nm), orange (585-620 nm), yellow (570-580 nm), green (520-570 nm) and blue (440-490 nm). Each wavelength from 440 nm to 740 nm is scanned 1 nm per second to cover the entire region. When the A0TF is scanned, the CCD image sensor also performs a coincident operation, and when the AOTF is scanned by 1 nm, the CCD has an exposure time of 1 second and the CCD captures an image of light through the AOTF at that wavelength. This coincident operation already replaces the microbeads at each wavelength (art. 26). After a long time

Same image of Seo ^ᅵ

Obtain the microbead absorbed image at each wavelength by subtracting the background signal image from the unknown. Thereafter, five wavelengths are summed together with the intensity of the microbead absorbed images at respective wavelengths corresponding to the predetermined wavelength region to produce the microbead absorbed images of the five red, orange, yellow, green, and blue wavelength ranges. Obtain microscopic absorbance images of the region. Subsequently, the absorption images of these five wavelengths of microbeads are input into the software including Metamorph, and the image of the microbeads with the color corresponding to the color of the microbeads seen through the eyepiece of the microscope is displayed on a computer. You can get it. Figure 2 shows an image of Alginate microbeads containing red Polystyrene microspheres obtained through HMI optical modules through this process. Example 4 Screening Kinase Inhibitors Using Nase-encapsu 1 at ed Microbead The present invention encapsulated kinase in microbeads, and then screened the efficacy of the drug as Kinase inhibi tor using the microbeads. . Microbeads were prepared using sodium alginate solution.

Replacement Paper (Rule Article 26) 2% sodium alg

polystyrene dye ¾

One color polystyrene dye with kinase was added to indicate the kinase. After mixing the mixture well, the mixture was placed in a 30 Gauge needle syringe and dropped into an aqueous calcium chloride solution by electrospray method (10.0 kV, 1.5 mL / min, 5 cm) to prepare 150 i microbeads. The microbeads were collected and washed three times with distilled water. Thereafter, the obtained microbeads were reacted overnight after drug treatment by concentration. Since the microbeads are in a gel state, the reactions were carried out overnight to allow enough time for the drug to enter the microbeads and react with the kinase. After drug reaction, the microbeads were washed three times with distilled water, and then reacted at 37 ° C. for 8 hours in an ATP, D1T, kinase buffer, and kinase substrate. After the reaction was washed three times with distilled water and the microbeads were obtained through a microscope -HMI system. The kinase substrate is Sox die 1 at ion-enhanced f luorophore (CHEF) 8-hydr oxy-5- (, -di me t hy 1 su 1 f onam i do) -2-methlyquinol ine °]. In the absence of the drug, kinase was added to a mixture of ATP, DTT, kinase buffer, and kinase substrate, and the reaction was carried out. Kinase substrate is phosphorylated by kinase, and Mg ²⁺ forms Sox and phosphote groups. It emits light (excitation wavelength = 360 nm, emission wavelength = 485 nra). Kinases replaced by drugs (Article 26 of the Rule) If it becomes inhibition ^

do. The following figure is the result of measuring the inhibition of Kinase GSK3b by Methanesulfonate Inhibitor using HMI-fluorescence microscopy system.

To monitor the inhibition of Kinase GS 3b by the methanesulfonate inhibitor, microbeads were exposed to a 360 nm UV beam, and then the 485 nm wavelength was set to the transmission wavelength of HMI A0TF to obtain fluorescence images of the microbeads. (a) shows microbeads showing brighter Kinase-induced fluorescence obtained without treatment with Inhibitor, and (b) shows greatly reduced microbead fluorescence images of approximately the same size as background signal after 1 mM treatment with Inhibitor Methanesulfonate. Indicates. As shown in Figure 3, the kinase inhibition by each drug can be determined through the fluorescence image of the microbeads through the microscope -HMI system. In addition, in order to identify a specific Kinase to which the drug reacted, after changing the mode of the microscope to the Brightfield mode and executing Example 3, the corresponding Kinase can be identified based on the color of the microbead. The method of screening drug efficacy of Kinase inhibitor of tens of thousands to hundreds of thousands of library compounds using HMI-optical microscope is as follows. When a specific Kinase and a specific dye are added to an aqueous solution of Alginate, and the microbeads are made using the Electrospray method, the microbeads are identified or replaced. The Kinase reservoir microbeads are mixed with different Kinase storage microbeads to a specific Kinase that is possible, and then a specific drug is applied to the microbead mixture, and the fluorescence image of each microbead is then captured in the HMI optical module—microscope. When measured in the fluorescent mode using the microbeads to know the on / off according to the brightness of the microbeads, and then immediately after the absorption image corresponding to the dye containing the microbeads in the brightfield mode, as shown in Example 3 The microbead absorption image of can be obtained so that one drug can quickly confirm the efficacy as an inhibitor against various kinds of kinases by one image measurement. Consecutive measurements of microbead complex fluorescence images in different regions of the microbead solution will readily show that a new generation of platforms for imaging-based drug efficacy screening without pipetting is realized. have. In the case of drug screening based on the existing 96-well plate, the drug is added and reacted with the reaction material including the kinase and its substrate in each well. In this case, the pipette is used to deliver to the well by automated pipetting. . Provides a very simple and rapid screening platform when screening a large number of library compounds as the Kinase inhibitor of the present invention. Example 5: Screening Kinase Inhibitors with Drug-encapsulated Microbeads

Alternative Site (Article 26) The present invention is kin

Microbeads

The lobby is made using 2% sodium alginate solution.

One drug and one colored polystyrene microsphere dye were added to a 2% aqueous sodium alginate solution. After mixing the mixture well, place the mixture in a 30 Gauge needle syr inge, and electrospray method with 1-10.0 kV, 1-5 mL / min gel flow rate, and distance between 1-20 cm syr inge needle and alginate solution. 10.0 kV, 1.5 mL / min, 5 cm preferred) was added to the aqueous solution of cal cium chloride to prepare 150 / m microbeads. The microbeads were collected and washed three times with distilled water. Thereafter, the obtained microbeads were reacted overnight with Kinase GSK3b. In addition to GSK3b, all Kinases can be applied to this essay. After reacting with the drug and kinase in the microbeads, the microbeads were washed three times with distilled water, and then mixed with ATP, DTT, kinase buffer, and kinase substrate in a mixed solution for 8 hours at 37 ⁰ C. After the reaction was washed three times with distilled water and the microbeads were obtained through a microscope -HMI system. Example 3 is then performed to determine the drug present in the microbeads. To screen for thousands and tens of thousands of kinase inhibitors of various drugs, the microbeads's mixed solution as a drug reservoir containing each different drug is placed on the microscope sample stage and repeated.

Replacement Paper (Rule Article 26) Example 6: Luciferin-luciferase assay using microbeads can be used as an effective drug screening method as an experiment for determining the action of lucifer as a Kinase inhibitor. In luminescent reactions, luciferin reacts with oxygen to oxidize to oxyluciferin, producing light (luciferin + ¾ → oxyluciferin + light). In most luminescence reactions, carbon dioxide is released as a product. The reaction rate between luciferin and oxygen is very slow and is catalyzed by luciferase and sometimes by cofactors such as calcium silver or ATP. The reaction catalyzed by firefly luciferase occurs in two stages. First, luciferin reacts with ATP to produce luciferyl adenylate and PPi (luciferin + ATP → luciferyl adenylate + PPi), and luciferyl adenylate reacts with oxygen to produce oxyluciferin, AMP and light (luciferyl adenylate + 0 ₂ → oxyluciferin + AMP + light). This reaction is a very effective reaction, in which almost all the energy put into the reaction is transformed into light (560 nm). Kinase phosphorylates the substrate using ATP in cells.

Alternative Site (Article 26) Thus Kinase

Because luciferin-lu 入

However, if the drug acts as a Kinase inhibitor, ATP remains stable because the drug inhibits the kinase phosphorylation of the substrate even after reacting the drug with Kinase first and then reacting the substrate with ATP. Thus, luciferin-luci Perase reactions occur and light is generated. The present invention introduces a drug screening method using 1 luciferin-luciferase assay using microbeads in which different Kinases are encapsulated with this logic. A 2% sodium alginate gel containing one kinase and one colored polystyrene bead in microbeads is prepared by electrospray method. The prepared microbeads are placed in a solution in which the drug is dissolved and incubated for a certain time so that the drug enters the microbeads and reacts with the drug. After washing the microbeads, the substrate and ATP are added, reacted, and washed again with water. Finally, luciferin and luciferase are added and reacted for a while. After completion of the final reaction, the fluorescence can be detected using the HMI system and microbead color coding can be used to determine the efficacy of various drugs on various kinases. Example 7: Drug-induced mitochondrial dysfunction hepatocyte replacement paper using microbeads (Rule 26) Toxicity Measurement The present invention provides a drug toxicity screening method using microbeads in which cells are encapsulated. This method involves capturing cells in microbeads, coating them to improve microbead stability, and detecting toxicity after drug treatment. The process of encapsulating cells in microbeads was performed by adding 8xl0 ⁶ cell / mL liver cell (HepG2, etc.) to sodium alginate solution in 1-10.0 kV, 1-5 mL / min gel flow, 1-20 cm syringe needle and alginate. The solution was encapsulated in 1-8% (2% preferred) sodium alginate gel by curing in BaCl ₂ using electrospray (10.0 kV, 1.5 mL / min, 5 cm preferred) with distance between solutions. Sodium alginate gels are coated to stabilize cells in culture medium. The collected microbeads were placed in 0.W Poly-L-lysine and treated for 7 minutes, followed by 5 minutes in 0.125% sodium alginate solution. Coated microbeads are incubated in 37 ⁰ C, 5% C0 ₂ in cell culture media. Drug toxicity screening is performed using microbeads that have completed cell encapsulation. Substitutes increased by intracellular calcium concentrations due to drug-induced cytotoxicity (art. 26) Swords Mitochondria

spec i es (R0S) increases

Permeabi lithium transit ion (MPT) occurs, which releases Cytochrome C from the mitochondria, and subsequently caspase 9 and caspase 3, resulting in cell death. Each of these phenomena can be observed using fluorescent probe material. An increase in calcium is detected through calcium markers, which combine with the scab to generate fluorescent material. Mitochondrial MPT can be measured by Cal cein-AM. Calcein- 細 accumulates in the mitochondrial membrane. When MPT changes, that is, the mitochondrial membrane is damaged, Calcein-AM accumulated in the mitochondrial membrane diffuses into the cytoplasm. The intensity of fluorescence by calcein-AM is reduced in vivo. Caspase-3 can be detected using Caspase-3 substrate, and Caspase-3 substrate generates fluorescent substance when it meets Caspase-3, and it can be understood that Caspase-3 is generated through fluorescence detection. In order to observe the hepatotoxicity in the microbeads due to the mitochondrial dysfunction mechanism of the drug, hepatocyte-containing microbeads were first reacted in a solution containing three fluorescent markers, calcium markers and Cal cein-AM and Caspase-3 substrates. Replacement paper to inject fluorescent markers into the hepatocytes present in the microbead (Article 26) After the drug (e.g. yes

If the drug of each marker has hepatotoxicity, the increase of calcium marker fluorescence, the decrease of Calcein-AM fluorescence according to mitochondrial MPT, and the increase in Caspase-3 substrate caused by the increase of Caspase-3 An increase in fluorescence can be seen. As a result of monitoring three fluorescence changes, it can be confirmed that cytotoxicity caused by drug may be due to mitochondrial dysfunction. As described above, although the present invention has been described by way of limited embodiments and drawings, the present invention is not limited thereto and is intended by those skilled in the art to which the present invention pertains. Of course, various modifications and variations are possible within the scope of the claims to be described below.

[Description of the code]

10 ： Case 12 ： Opening and closing door

14 ： Air cooling member 20 ： First lens member

22 ： Condenser lens 24 ： Pan collimator or beam expander

30 ： Lens support member 40 ： Variable filter

50 ：： Pass-pass filter or van: de-pass filter 60 ： Image sensor

80: support bar 90: rail member

【Industrial Availability】 Alternative Sites (Rule Article 26) According to the present invention ^

based on color coding

Has an effect. Firstly, the present invention provides a microbead drug screening system based on a portable HMI optical model, which can be used in a conventional optical microscope holding laboratory by using HMI optical models that can be attached to the side of an existing optical microscope, an upright and an inverted microscope. Providing general-purpose multiwavelength cell and molecular imaging equipment can greatly contribute to the lowering of related bioimaging studies. In particular, since most bio laboratories have optical microscopes, bioimaging research can be spread by installing a modeled optical system. Second, the HMI optical mother system can be used as an HCS device, which has a cost savings of 1 / 7.4 compared to the existing HCS device, which has a significant cost saving effect, which can be economically retained by individual laboratory units. It can be a device that enables large-scale drug screening at the individual laboratory level. Third, the present invention is an HCS device, the time required for image measurement per 96well plate is 14.4 minutes, while the existing HCS device takes 40 minutes, but the drug target protein reservoir such as Kinase is made of microbeads and the combinations thereof. Can be placed on the microscope sample platform and the reagents required for reaction can be added to the sample platform to produce the same reaction for all microbeads, so that the drug efficacy screening time is replaced by the replacement paper (Rule 26). Can be very shortened

It is effective. 2

All microbeads are processed at the same time, which saves a lot of reagents. Fifth, since the present invention uses A0TF instead of Narrow bandpass filter, the number of applicable wavelengths is greatly increased compared to HCS devices that can use eight Narrow bandpass filters. The width becomes wider. Sixth, the present invention is a new era of drug screening platform without pipetting because it can indicat ion each drug by encapsulating different dyes or drug targets by color coding method and encapsulat ion Can be opened. Seventh, the present invention can be actively used in the pharmaceutical industry as a new product because it can be used as a system for the indispensable drug screening process of the drug development process by the microbead's method of coding. .

Replacement Paper (Rule Article 26)

Claims

[Range of request]

[Claim 1]

A model case having one side coupled to the microscope;

A first lens member disposed in an opening of one side of the case and having a condenser lens for condensing a type light beam emitted from the microscope;

A variable filter disposed in the case and scanning wavelengths of the fluorescent beams passing through the first lens member for each wavelength;

A mechanical plate disposed in the case and mechanically removing a fluorescence beam including a wavelength undiffracted in the fluorescence band passing through the variable filter;

A pass-pass filter or a band-pass filter disposed in the case to remove light of the excitation light source from the fluorescent beam passing through the variable filter;

An image sensor installed in the case and detecting a fluorescent beam passing through the mechanical plate and the through-pass filter or the band pass filter; And

Hypermulticolor imaging (HMI) is possible, including a second lens member disposed at the front of the image sensor and configured to obtain a high quality image.

Portable hypermulticolor imaging optical fibers characterized in that they are configured to be detachable to the microscope in a portable manner.

[Claim 2]

A model case having one side coupled to the microscope;

Installed in the opening of one side of the case, type replacement paper coming out of the microscope (Article 26 of the rule) Enjoy the size of the light beam ^

1st with expander)

A mechanical full plate disposed in the case and mechanically removing a fluorescent beam including a wavelength undiffracted from the fluorescent beam passing through the variable filter;

An image sensor installed in the case and detecting a fluorescent beam passing through the mechanical plate and the through-pass filter or the bandpass filter; And

And a second lens member disposed at the front side of the image sensor and configured to obtain a high quality image. Hypermulticolor imaging (HMI) is possible,

A portable hypermulticolor imaging optical module, characterized in that it is configured to be detachable to the microscope in a portable manner.

[Claim 3]

The method according to claim 1 or 2,

The variable filter, the mechanical plate, the through-pass filter or the band-pass filter, and a support bar for fixing the image sensor to prevent the image is provided, respectively,

It is installed on the lower surface of the case, the variable filter, the mechanical plate, the through-pass filter or the band-pass filter, and the image sensor from the first lens member is a straight replacement sheet (Article 26) Moveable in the direction

Rail member locked

Opticians with gongs.

[Claim 4]

The method according to claim 1 or 2,

The case includes an opening and closing door formed on one surface of the first lens member, the variable filter, the mechanical plate, the through-pass filter or the band-pass filter, and the image sensor disposed therein so that the image sensor can be replaced and repaired. Optical models with.

[Claim 5]

Article: The method of claim 1,

The condensing lens is composed of at least one,

In order to attach and detach the condensing lens to the case opening, a screw thread is formed at an edge of the condensing lens and an edge of the case opening so that at least one condensing lens is C-mounted in the case opening. Optical fibers, characterized in that the configuration.

[Claim 6]

The method according to claim 1 or 2,

And a lens support member configured to be supported by the first lens member in an opening of one side portion of the case.

Replacement Paper (Rule Article 26)

[Claim 7]

Claim 1 or Article:

The variable filter is an optical optical wavelength tunable filter (A0TF) characterized in that the optical model.

[Claim 8]

The method according to claim 1 or 2,

The image sensor,

Optical modules characterized in that it is a two-dimensional image sensor including a charge coupled device camera, a photodiode array.

[Claim 9]

The method according to claim 1 or 2,

The case is

Optical cooling further comprises an air-cooling member or a water-cooling member by cooling water configured to ventilate for cooling the variable filter or the image sensor. Modle.

[Claim 10]

The method according to claim 1 or 2,

The case is

Alternative paper, characterized in that it is made of an opaque material to block external light (Article 26) Optical models with.

[Claim 11]

Pigmented microbeads were placed on a microscopic sample stage, and a white light beam was irradiated onto the microbeads. The optical optically tunable filter (acousto-opt ic tunable filter) with lnm resolution in the wavelength range from 400 nm to 800 nm was applied. Scanning AOTF) to obtain a transmission image of white light corresponding to the pigment of the microbead at each wavelength (step 1);

Irradiating a beam of white light on the microbead-free aqueous solution in the aqueous microbead solution of step 1, and then performing step 1 to obtain a transmission image corresponding to a background signal (step 2);

Subtracting the signal strengths of the transmission images by wavelength obtained in step 1 through the meta amor ph software to the signal strengths of the transmission images by wavelength obtained in step 2 (step 3);

Wavelengths corresponding to red (630-740nm), orange (585-620nm), yellow (57 ()-580nm), green (520—570nm) and blue (440-490nm) in the wavelength-specific transmission image obtained in step 3 Using the Metamorph software (step 4) to merge images of respective wavelengths corresponding to color wavelength ranges to obtain a transmission image of a region (step 4); And

Inputting the color image obtained by the step 4 through the DOver lay function of the software to add the respective wavelengths to obtain a microbead image of the color corresponding to the pigment in the microbead to complete the microbead color coding ol ( Step 5) My Alternative Paper Based on Microbead Color Coding, comprising: Rule 26 Method of discriminating crobyd.

[Claim 12]

The method of claim 11,

In step 1 above,

The microbead, alginate, poly lactide (PLA), photocurable photopolymer polymer (polyethylene glycol diacrylate, PEG-DA), polypropylene fumarate (Poly propylene fumarate, PPF), Poly propylene fumarate (PPF) I diethyl fumarate (DEF), pentaerythritol triacrylate (Pentaerythritol tr iacrylate), or trimethylolpropane tree It is made of acrylate (Trimethylolpropane triacrylate) polymer

The pigment includes a polystyrene microsphere bead polymer pigment, an organic pigment, an inorganic pigment quantum dot, a pigment-doped silica nanoparticle, and gold,

The software is applicable to software related to Metamorph, Metaview, or ImagePro image analysis, and is identical to the step-by-step execution function executed in the function increase function of the software. Microbead discrimination method based on microbead color coding, characterized in that by executing a function.

[Claim 13]

12. Alternative paper according to claim 11 (Article 26) One specific

Number of lobbyists

Incubating for one hour so that one specific drug enters the microbeads and reacts with each kinase (step 1); after the step 1, the microbeads are washed three times with distilled water. particular between adenosine tri phosphate (adenosine triphosphate, ATP), dithiothreitol roll (dithiothreitol, DTT), kinase buffer (kinase buffer), and kinase substrate (substrate kinase) is a freshmen for the liquid heunhap 10-45 ^o C Reacting with time in a step (step 2); After the step 2, the microbeads were washed with distilled water, placed on a microscope sample stage, and irradiated with a beam of white light to the microbeads, with an lnm resolution in the wavelength range from 400 nm to 800 nm (acoust optic). scanning a tunable filter (AOTF) to obtain a transmission image of white light according to the pigment of the microbead at each wavelength (step 3);

After the step 3, the microscope is switched to the fluorescent mode, and the phosphor formed in each microbead is excited through the microscopic HMI system, and then the wavelength of the acoustic-optic tunable filter (AOTF) is transmitted at the maximum emission wavelength of the phosphor. Fixing and obtaining a fluorescence image of the microbeads (step 4);

Obtaining a fluorescence image of the blank solution free of microbeads after step 4 through the same procedure as in step 4 (step 5);

After completing step 5, changing the microscope mode to the brightfield mode and irradiating white light to the background solution to obtain an absorbance image (step 6);

Alternative paper obtained in step 5 from the fluorescent images of the microbeads obtained in step 4 (Article 26) Century of image ^' 1 delight

System (step 7);

Obtaining an absorbing image of the microbeads from which the background signal of each wavelength is removed by subtracting the intensity of the image obtained in the step 6 from each of the absorbing images of the microbeads obtained in step 3 for each wavelength (step 8); And

Completing the step 3 and step 5 of claim 3 to complete the kinase inhibitory drug efficacy screening in the microbead (step 9); microbead color coding based kinase (Kinase) comprising the Inhibitory Drug Screening Room

[Claim 14]

The method of claim 13,

In step 1 above,

The microbead, alginate, poly lactide (PLA), photocurable photopolymer polymer (polyethylene glycol diacrylate, PEG-DA), polypropylene fumarate (Poly propylene fumarate, PPF), Poly propylene fumarate (PPF) I diethyl fumarate (DEF), pentaerythritol triacrylate (Pentaerythritol tr iacrylate), or trimethyl propane tree Made of acrylate (Trimethylolpropane triacrylate) polymer,

In step 2 above,

The kinase substrate, in addition to Sox, is any organic or inorganic kinase replacement paper capable of reacting with kinases in the microbeads to generate phosphors (Article 26) Substrate (Kinase substrat '

Half kinase.

[Claim 15]

An aqueous solution of a mixture of microbeads, each containing one specific pigment and one specific drug, is placed on the microscope sample stage, and then one specific kinase is added to the microbeads to react with the drug. Incubate for about one hour to provide (step 1);

After washing the microbeads with distilled water three times after step 1, adenosine triphosphate (ATP), dithiothreitol (DTT), kinase buffer (Kinase buffer), and kinase substrate (Kinase substrate) ) Was added to the mixed solution and reacted for a sufficient time at a specific silver content between 10 and 45 ^° C. (step 2); After the step 3, the microbeads were washed with distilled water, placed on a microscope sample stage, and then irradiated with a bead of white light on the microbeads, with an lnm resolution in the wavelength range of 400 nra-800 nm (acousto-optic filter). scanning a tunable filter (AOTF) to obtain a transmission image of white light according to the pigment of the microbead at each wavelength (step 3);

After the step 3, the microscope mode is changed to the fluorescence mode, and the phosphor formed in each microbead is excited through the microscope-HMI system, and then the acoustic-optic tunable filter is applied to the maximum emission wavelength of the phosphor. AOTF) fixing the transmission wavelength and then obtaining a fluorescence image of the microbeads (step 4);

After step 4, the fluorescent image of the background solution in which the microbeads are not present

Alternative Site (Article 26) Same as step 4 above

Step 5 above

Irradiating the substrate solution to obtain a light absorption image (step 6);

Obtaining a fluorescent image of the microbeads from which the background signal is removed by subtracting the intensity of the image obtained in the step 5 from the fluorescent image of the microbeads obtained in step 4 (step 7);

Obtaining the absorbed image of the microbeads from which the background signal of each wavelength is removed by subtracting the intensity of the image obtained in the step 6 from each of the absorbed images of the microbeads obtained in step 3 for each wavelength (step 8); And

Completing the step 3 and step 5 of claim 3 to complete the screening kinase inhibitory drug efficacy in the microbead (step 9); Kinases based on drug-containing microbead color coding comprising (Kinase) inhibitor drug screening method.

[Claim 16]

The method of claim 15,

In step 1 above,

The microbead, Al iginate, Poly lactide (PLA), Photocurable photopolymer polymer, Polyethylene glycol diacrylate (PEG-DA), Polypropylene Poly propylene fumarate (PPF), Poly propylene fumarate (PPF) / diethyl fumarate (DEF), Pentaerythri tol tr iacrylate, or Trimethylolpropane triacrylic substitute paper (Rule Article 26) Trimethylolpr

Water-containing microbi ί

Way.

[Claim 17]

An aqueous solution of a specific pigment or a mixture of specific pigments and a mixture of microbeads each containing one specific kinase is placed on a microscope sample stage, followed by incubation of the specific drug in the solution for a sufficient time. Allowing the drug to enter the microbeads and react with the drug (step 1);

After the step 1, the microbeads are washed, and the kinase substrate to be phosphorylated by phosphorylation of Adenosine triphosphate (ATP) and the drug target kinase (Kinase) is added to the microbead solution, and the reaction is repeated with water. Step K) 2) to wash;

After step 2, luciferin and luciferase are added to the microbead solution, and the reaction is carried out for one hour, and the chemiluminescence generated from the microbeads is measured using the optical fibers of claim 1, step 3);

After the step 3, a beam of white light is irradiated to the microbeads, and an acoustic-optic tunable filter (AOTF) is scanned at Iran resolution in a wavelength range ranging from 400 nm to 800 nm to dye the microbead at each wavelength. Obtaining a transmission image of white light according to (step 4);

Obtaining a fluorescence image of the blank solution free of microbeads after step 4 through the same procedure as in step 3 (step 5);

After completing Step 5, change the microscope mode to Brightfield mode and replace the white light paper (Rule 26). When investigating the background solution

In step 3 above

Obtaining a chemiluminescent image of microbeads from which the background signal is removed by subtracting the intensity of the image (step 7);

Obtaining the absorption images of the microbeads from which the background signals of the respective wavelengths have been removed by subtracting the intensity of the image obtained in the step 6 from the absorption images of the microbeads obtained in the step 4 for each wavelength (step 8); And

Comprising the steps 4 and 5 of claim 3 to complete the kinase inhibitory drug efficacy screening in the microbead (step 9); Luciferin based on drug-containing microbead color coding, characterized in that it comprises a Method of screening kinase inhibitor drug using luciferase assay.

[Claim 18]:

The method of claim 17,

The microbeads of step 1, wherein the microbeads are polyethylene glycol diacrylate, which are alginate, poly lactide, PLA, and photocurable photopolymer polymer. PEG-DA), Poly propylene fumarate (PPF), Poly propylene fumarate (PPF) I Diethyl fumarate (DEF), Pentaerythritol triacrylate (Pentaerythritol kinase inhibitor drug replacement paper using luciferin-luciferase assay based on drug-containing microbead color coding, characterized in that it is made of trimethyl) or trimethylolpropane triacrylate polymer Article 26) Cleaning method.

Replacement Paper (Rule Article 26)