KR20120129182A - Potable hypermulticolor imaging optical module and Microbead colorcoding kinase inhibition drug screening system - Google Patents

Abstract

PURPOSE: A portable hypermulticolor imaging optical module is provided to screen drugs of a cellular level or molecular level. CONSTITUTION: A portable hypermulticolor imaging optical module(HMI system) comprises a case; a first lens member(20); a variable filter(40), a mechanical plate, long pass filter, or band pass filter(50); and an image sensor(60). The case has a module structure which is coupled to a microscrope at one side. The first lens member is mounted at an opening part at one side of the case and collects fluorescent beam. The variable filter is placed in the case and is able to control the wavelength of the fluorescent beam by each shore wave length ever second.

Description

Portable hypermulticolor imaging optical module and Microbead colorcoding kinase inhibition drug screening system

The present invention relates to the development and application of a portable Hypermulticolor 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, and implements the existing HCS for implementing multi-wavelength imaging by replacing a wheel-type bandpass optical filter. Rather than a drug screening screening system, the inventors have invented a portable drug screening MHI system that can each obtain a single-wavelength image of a rapid injection form.

This portable drug search MHI system is a portable drug search multi-wavelength imaging system that can be attached to the side of an optical microscope that is mostly secured in existing bio research groups, including both upright and inverted optical microscopes. Imaging and multi-wavelength 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.

As a scope of application, the drug efficacy of the library compounds at the molecular level as a Kinase inhibitor was applied using a microbead suitable for rapid and large-scale drug efficacy screening that does not require pipetting. In addition, hepatotoxicity evaluation of drugs mediated by mitochondrial dysfunction was performed using microbead technology.

Currently, drug-based cell-based assays for drug screening cannot be identified with a single essay on / off target effect and side effect, 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 discovery platform is to verify as many cost-effectively as possible on-off target effects and cytotoxicity in a single cell-based assay, which can eventually be caused by drugs.

Currently, the flow cytometer is a widely used device related to drug efficacy and toxicity. The flow cytometer is a device that measures various fluorescent probe substances of each cell by shooting a laser on the part when the cell passes through the detector part in the fluid state.

The advantage of the flow cytometer is that it allows quantitative measurement of numerous cells by detecting fluorescent substances attached to many cells in a short time. 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.

However, the disadvantage is that images cannot be obtained with flow cytometers. Cell imaging is a very important technique for observing cellular responses to stimuli because it shows where, when, and how the cellular response to external stimuli occurs.

Intracellular signaling materials and numerous signaling proteins form a signaling network. Flow 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.

Advances in computer analysis and rapid advances in general cell biology in imaging techniques have greatly increased high-throughput imaging techniques for target validation. Existing high-content screening (HCS) is an automated platform capable of performing fluorescence microscopy and quantitative imaging techniques and is currently being actively used as a powerful tool for multicolor cell imaging analysis.

Cell imaging has numerous advantages in investigating cell dynamics and cell kinetics and observing cell characteristics such as morphology and cell movement. High-content imaging is a type of multicolor cell imaging technology that allows simultaneous visualization of cellular elements and responses, including cell morphology. Automated systems are essential to realize the versatility of high-content imaging.

Representative HCS measuring instruments currently exist include BD bioscience and HCS system released by Cellomix. The BD pathway high-content imaging system includes a number of excitation filter wheels and emission filter wheels. Although cell imaging of up to eight different colors is possible, the luminescent filters are narrow narrow band pass filters, and the emission wavelength range is limited in the selection. In addition, the purchase price is very expensive to purchase as a laboratory equipment in a private laboratory.

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 utilized 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 through the assays in spatially separated wells, but screening in microbeads, rather than the wells, simultaneously monitors a large number of beads in one well or space. Addressing of microbeads is essential.

Through the addressing of these microbeads, it is possible to distinguish reactions due to test substances or drugs present in each microbead.

Addressing of microbeads can usually be done by color coding. Therefore, for the addressing of microbeads, an optical system that can distinguish multicolored colors must be equipped.

This microbead-based drug screening, among other things, can simultaneously process one drug on each of the microbeads containing drug targets, allowing for the construction of a platform that does not require pipetting, thereby screening numerous library compounds through drug screening assays. A very fast and economical platform can be realized.

The present invention was devised to solve the above problems, and aims to develop a portable optical imaging module system for color coding of microbeads using multi-wavelength high resolution color selection based on 1 nm high spectral resolution of AOTF. .

Currently, most individual life science bio laboratories have optical microscopes, so when a portable HMI optical module is developed that can be attached to the side of an existing optical microscope, microbead-based molecular and cell-level drugs are not required without expensive HCS devices. Screening can be performed.

AOTF can minimize the overlap of each wavelength region and finally create an intensity profile of light transmitted through the microbeads in the 3-7 wavelength region that should be selected for coding the color of the microbeads with 1 nm optical resolution. The advantage is the freedom to choose a specific wavelength range for the microbeads by specifying the specific wavelength range needed to produce the color.

It is possible to obtain the transmission image of 1nm through AOTF so that 3-7 selected wavelength regions can be obtained without overlapping with existing bandpass filter, and the selective wavelength region can be easily adjusted to greatly increase the color resolution for color coding. The number and quality of color coding of microbeads can be greatly improved.

The present invention has devised a color coding method for multicolored microbeads manufactured using various microbeads including alginate gel beads and multi-wavelength imaging optical systems based on portable HMI modules. By encapsulation of 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 modules and microbeads.

When applied to drug screening using microbeads, particularly when applying to a library compound with a screening quantity of 10 to 100 million, the technique of addressing and coding individual microbeads in which each drug or drug target is present is very high. It is an important technology.

In general, when addressing with a color code, different colors may be produced by varying the ratio of pigments such as red, green, and blue.

When white light is transmitted to each other where these different pigments are combined, and then the intensity of light detected in each of the different wavelengths transmitted is combined to obtain an image of the color seen by the human eye.

In order for the image of this color to be close to the primary color, the number of wavelength regions combining and combining the intensity of light in different wavelength regions is closer to the primary color. For example, the number of wavelength regions to be combined is from 3 to 5 and 5 As you increase the number to seven, you can get an image close to the primary color.

In addition, the wavelength range is 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 light in the wavelength range, the intensity of transmitted light in the wavelength range corresponding to 630-740 nm should be obtained in one combination.

The present invention is to develop a portable multi-wavelength imaging module based on AOTF as an optical system capable of high resolution color coding of such primary colors. AOTF is very advantageous for such 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.

This indicates that when the intensity of light passing through the pigment-containing microbeads is a function of wavelength, AOTF can measure the intensity of transmitted light at 500-600 single wavelengths, from visible to near-infrared, where light transmission is guaranteed. It is also possible to combine each of these wavelengths and bundle them into a range of wavelengths to obtain an image of light intensity and microbeads in that wavelength range.

In order to obtain the transmission intensity image of light in the wavelength range of 630-740 nm, we can obtain the transmission intensity of microbeads in each single wavelength ranging from 630-740 nm, and then red, orange, yellow, green and blue. Even when measuring the transmission intensity of the microbead in the specific wavelength range corresponding to AOTF, the advantages of AOTF can be clearly highlighted even if the specific wavelength region is selectively designated through the AOTF.

This is because when using a bandpass filter like a conventional HCS device, there is a limit in selectively specifying a specific wavelength. As such, AOTF can obtain the transmittance in single wavelength in the visible and near-infrared region by scanning, which makes it easy to select the wavelength region to be combined, and above all, the pure color corresponding to the pigment avoiding the overlapping wavelength region by absorption of pigments. By measuring the intensity of light in the wavelength range, microbead addressing can be made possible through a greater number of color combinations, which is closer to the primary color.

In addition, since AOTF transmits only light at a specific wavelength, it may have an advantage of using white light as a light source irradiating the microbeads. That is, it is easy to measure and shorten the measurement time because white light can be irradiated to the whole microbead containing dye without scanning light of specific wavelength range as a light source and the transmitted light can be scanned at each wavelength through AOTF. You can.

Another advantage of the portable HMI module is that it is attached to the side of the microscope, so in bright field mode, white light is irradiated to the microbead placed on the microscope stage, and the transmitted light is scanned by AOTF to measure the intensity of light in the desired wavelength range. When the measurement mode of the rear microscope is changed to the fluorescence mode and the fluorescence generated through the microbeads is measured again through the AOTF, the system can quickly and conveniently measure the fluorescence caused by the color coding of the microbeads and the drug reaction in the microbeads. Will be equipped.

Accordingly, 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 the portable HMI module 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 the portable optical module HMI.

Kinase inhibitor screening system based on color coding of microbeads using HMI 1 and 2 systems and HMI optical modules according to the present invention has the following effects.

First, the present invention is a microbead drug screening system based on a portable HMI optical module is a low-cost and versatile in the existing optical microscope holding laboratory through the use of an HMI optical module that can be attached to the side of the existing optical microscope, upright and inverted microscope Providing wavelength cellular and molecular imaging equipment can greatly contribute to the low change in relevant bioimaging research. In particular, since most bio laboratories have optical microscopes, bioimaging research can be spread through modular optical systems.

Second, the HMI optical module 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. This can enable large-scale drug screening at the individual laboratory level.

Third, the present invention is an HCS device, the time required for image measurement per 96 well plate is 14.4 minutes, while the existing HCS device takes 40 minutes. By placing the reagents required for the reaction on the sample platform and putting the same reaction on all the microbeads, the drug efficacy screening time can be greatly reduced. This greatly increases the number of drugs that can be measured and processed per day. In addition, since all the microbeads are treated at the same time without putting the reactants in each well, such as 96 wells, the reagents consumed can be greatly saved.

Fifth, since the present invention uses AOTF instead of Narrow bandpass filter, the number of applicable wavelengths is greatly increased compared to HCS devices that can use eight Narrow bandpass filters. Thus, the width of the selection region of the fluorescent probe for drug efficacy screening is increased. Widens

Sixth, the present invention can open the era of a new concept of drug screening platform without pipetting because each drug can be indicated by making microbeads by inserting different dyes or drug targets for each drug and encapsulation by color coding method. have.

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 drug development process by the microbead color coding method.

1 is a schematic diagram of a color coding process of a microbead using a portable HMI.
2 is a microbead absorbed image obtained by the color coding process of a portable HMI microbead.
Figure 3 is (a) microbead fluorescence image by Kinase GSK3b activity, (b) GSK3b inhibited microbead fluorescence image by Methanesulfonate.
4 is a schematic diagram of an HMI 1 system composed of a c-mount lens, an AOTF, and a CCD.
5 is a schematic diagram of an HMI 2 system composed of a beam collimator or a beam expander, an AOTF, and a CCD.

The present invention provides a portable HMI optical module that can be attached to all optical microscopes in general, and provides a microbead color coding method using the same, a Kinase inhibitor assay, and a drug-induced hepatotoxic cell-based HCS measurement method.

The present invention is based on ultra-wavelength imaging using an HMI optical module attached to a general optical microscope, which eliminates the bead addressing required when utilizing microbeads as a new platform for evaluating the efficacy and toxicity of numerous library compounds. Color coding provides a new paradigm of drug screening technology.

Compared with BD bioscience, an HCS device, the portable HMI optical module is attached to a conventional microscope for high resolution (1 nm) multiwavelength imaging, which is convenient for use and cost-effective, and features a wheel of a bandpass filter set in terms of performance. Compared to the filter system used, 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 particular photodetector of the images obtained at each wavelength scanned with AOTF, 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 reduce the financial burden on consumers. The HMI hardware can be fabricated in different modes (HMI 1 and HMI 2) according to customer requirements.

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 assay proceeds to monitor kinase and substrate-induced fluorescence or luciferin and luciferase-based chemiluminescence inhibition by kinase inhibitors in wells such as 96 wells. have.

In this case, large corporations such as multinationals are solving the screening of tens of thousands to hundreds of thousands of library compounds through an automated platform using automated robotics. Robotics is a system that automates and pipettes the delivery and reaction of various compounds and reactants for the Kinase inhibition assay.

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 color coding of each microbead.

This allows numerous drug-containing microbeads to produce 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 eliminates the need for pipetting each of the reactions associated with each Kinase inhibition by treating all the reactants at once in microbeads, and very quickly through microbead fluorescence imaging. A new concept innovative screening platform can be built to demonstrate this.

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 to hundreds of thousands.

In particular, the HMI system can reduce the color coding load caused by the rapid increase in the number of compounds to be screened by the AOTF's rapid adjustment of the wavelength at one wavelength per second and the transmission wavelength freely (1 nm-tens of nm-hundreds of nm). have. In addition, the detection of color coding and kinase reaction fluorescence in the microbeads can be quickly detected by 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 hypermulticolor imaging optical module, that is, HMI system) is a case 10, the first lens member 20, variable filter 40, mechanical plate, long pass filter or band pass filter 50, and an image sensor 60.

The case 10 has a modular structure in which one side can be fastened to the microscope.

In addition, the first lens member 20 is provided in an opening of one side portion of the case 10 and has a condenser lens 22 for condensing a fluorescent beam emitted from the microscope.

Of course, the first lens member 20 having a beam collimator or beam expander 24 for reducing the size of the fluorescent beam emitted from the microscope is installed in the opening of one side of the case 10. May be

In addition, the variable filter 40 is disposed in the case 10 and has a configuration in which the wavelength of the fluorescent beam that has passed through the first lens member 20 can be adjusted for scanning for each short wavelength.

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 of 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 a fluorescent beam passing through the mechanical plate and the long pass filter or the band pass filter 50.

In this case, a second lens member (not shown) is disposed in front of the image sensor to obtain a high quality image.

The second lens member may not only be mounted on the front of the image sensor in a C-mount, but may also be separately installed in the case in front of the image sensor.

That is, the installation structure of the second lens member is not limited by the present invention as long as the position of the second lens member is firmly fixed in front of the image sensor so as to obtain a high quality emission image.

The present invention configured as described above is capable of hypermulticolor imaging (HMI), and is characterized in that it is configured to be detachably attached to the microscope as a portable.

Furthermore, the present invention includes a support bar 80 for fixing the variable filter 40, the mechanical plate, the long pass filter or the band pass filter 50, and the image sensor 60 to prevent the phases.

At this time, the support bar 80 is mounted on the rail member 90 installed on the lower surface of the case 10.

That is, in order to move the variable filter 40, the mechanical plate, the long pass filter or the band pass filter 50, and the image sensor 60 from the first lens member 20 in a straight direction, the rail member 90 The lower end of the support bar 80 in the longitudinal direction is fastened to be movable, and thus the image can be focused.

Meanwhile, the case 10 replaces the first lens member 20, the variable filter 40, the mechanical plate, the long pass filter or the band pass filter 50, and the image sensor 60 disposed therein. And an opening / closing door 12 formed on one surface thereof so as to be repairable.

In addition, the condenser lens 22 may include at least one.

In addition, in order to attach and detach the 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, so that at least one condensing light is formed at the opening of the case 10. Preferably, lens 22 is configured to be C-mount.

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-optic tunable filter 40 (acousto-optic tunable filter 40).

In addition, the image sensor 60 may be a two-dimensional image sensor 60 including a charge coupled device camera and a photodiode array.

On the other hand, the case 10, in order to cool the variable filter 40 or the image sensor 60, the water cooling (air-cooling) by an air-cooling member or cooling water configured to ventilate Cooling) may be further provided.

In addition, the case 10 is preferably made of an opaque material to block the light from the outside.

Example 1 HMI 1 Module

The present invention provides (a) an HMI 1 system using a c-mount lens, AOTF, and a CCD, (b) a beam expander AOTF, and an HMI 2 system using a CCD, and using this system, color coding in microbeads. And Kinase inhibitor screening and drug-induced mitochondrial dysfunction via hepatocellular cytotoxic cell imaging observation. In particular, this HMI module is used to attach to existing microscopes, and through this module, a new drug screening platform based on portable HMI and microbead technology through HCS imaging at the molecular and cellular levels mentioned in the existing microscope-held laboratory. It aims to be able to realize it.

In the present invention, the HMI 1 module is a system that can be attached to the side of the fluorescence microscope to perform the HCS, the configuration is largely beams coming from the side of the microscope, such as c-mount lens at the inlet of the HMI module attached to the side of the optical fluorescence microscope A first lens member for condensing light, an AOTF through which a beam from the first lens member passes, and a CCD positioned at a focal point of the beam passing through the AOTF to obtain an image thereof.

In front of the CCD, 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.

The reached light reaches the sample containing the fluorescent probe material to emit the fluorescent probe material, and the emitted light is focused again on the lens.

The focused light exits through the dichroic mirror to the side of the microscope, where the light from the side of the microscope is a beam with different diameters, depending on the commercial microscope. The diameter of this particular beam is generally larger than that of the AOTF, which has a window of 1 x 1 cm ² .

Therefore, in order to pass the beam from the side of the microscope to the AOTF, the inlet of the HMI module is attached with a first lens member such as a c-mount lens that can be attached from the microscope and uses it to condense the light from the side of the microscope.

The AOTF in the module is located behind the module inlet c-mount first lens member so that the light collected through the lens passes through the window of the AOTF. Of the light passing through the AOTF, light of a certain wavelength is diffracted in the AOTF crystal so that it passes through a certain angle (within the range of about 3-15 ^o ) from the principal axis of the AOTF and the transmitted incident beam, and the light of the remaining wavelengths cannot pass.

The light of a specific wavelength passing through the AOTF is detected by the CCD. In addition to the light of a specific wavelength diffracted and passed through the AOTF, there is also a background light that is directly transmitted without diffraction. This background light usually passes through the crystal in the AOTF in the same direction as the axial direction of the incident beam, and there is an angular difference (within about 3-15 ^o range) between the background light and the particular wavelength light that is diffracted and transmitted. The plate is placed in front of the CCD behind the AOTF to selectively remove only the background light and detect only specific transmission wavelength light through the CCD.

On the other hand, since the specific wavelength light diffracted from the AOTF at a certain angle comes out with the light of the excitation light source in the fluorescence microscope, a longpass filter or a bandpass filter is installed in front of the CCD as a means to remove the light of the excitation light source to obtain a clearer image. It was made.

In addition, a c-mount lens is attached in front of the CCD for clear, high-definition images so that AOTF-transmitted specific wavelengths of light can be smoothly collected. This HMI optical module structure allows scanning of all fluorescent light excited in the microscope with intracellular fluorescent material or probe material, microbeads, etc. at 1 nm spectral resolution to obtain an image at each wavelength.

In addition, by connecting the intensities of the CCD at a specific location of the pixel (pixel) by wavelength, it is possible to simultaneously obtain a spectrum of wavelengths. At this time, the scanning speed is 1 frame / sec. The image thus obtained is analyzed by software of Metamorph et al.

The optical module is a two-dimensional image detector including an inlet c-mount first lens member, an AOTF, a longpass filter (or a bandpass filter) and a CCD. It is possible for each component to be movable through a rail or similar moving device for the optical arrangement for this purpose and to be fixed via a specific support present in the HMI module.

The optical module is a complete product in one package and is designed to be attached to the side of an optical microscope, including all commercial fluorescence microscopes. The QHMI module is 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 AOTF.

Example 2 HMI 2 System

In the present invention, the HMI 2 system is another optical module system manufactured for a module having an optical characteristic different from that of the HMI 1 system. HMI 1 has a structure in which the beam coming from the side of the microscope is focused by the c-mount first lens member located at the entrance of the optical module.

As a result, the HMI 1 optical module has a relatively small image size, while the image is high in intensity and high resolution. The HMI 2 system has different characteristics with different optical components and arrangements than HMI 1. This increases the size of the image and allows us to observe more detailed morphology of the material, including cells.

The optical module inlet on HMI 2 attaches a beam collimator or expander to make a large radius beam from the side of the microscope a parallel beam (where the radius remains almost constant with respect to the straight direction) or its radius.

Collimators are combination lenses that produce parallel beams with reduced diameters as they enter the parallel microscope side. At this time, the beam expander is installed upside down, that is, the input and output directions are installed at the inlet of the HMI 2 module so that when the beam from the side of the microscope enters the expander arranged upside down, the output beam is not increased but decreased in diameter.

The entrance with the collimator or expander was attached to the c-mount, which also coupled the HMI 2 module 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 1cm window of the AOTF. 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 AOTF only through the Acoustic field. Only the transmitted light of the specific wavelength is selectively removed from the transmitted light without diffraction through the mechanical plate. After reducing the light source, the image is acquired through a two-dimensional image detector with a CCD. In addition, a C-mount lens (second lens member described above) is attached in front of the CCD for a clear high-quality image, so that the AOTF transmission specific wavelength light can be smoothly collected.

The HMI 2 optical module is a complete product in one package and is designed to be attached to the side of an optical microscope, including all commercial fluorescence microscopes.

The HMI 2 module is equipped with an air-cooling or water-cooling system to prevent overheating due to prolonged use of image detectors and CCDs, including CCDs. 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 by Color Coding of Microbead Using HMI System

Microbeads are prepared using sodium alginate and calcium chloride. Sodium alginate in powder form is dissolved in autocleaved 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 dye sodium alginate solution. Spherical microbeads are prepared by adding sodium alginate solution containing dye to the syringe and placing it in the 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 1.5 mL / min. Ca ^{2 +} solution to the bridge role of sodium alginate polymer is changed to a gel (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 blending ratio of green, red and blue dyes.

The method for color coding through the microbead image formed on the image sensor including the CCD among the HMI system components 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. The AOTF sets the scanning speed of each wavelength to 1 second and performs scanning to obtain microbead images 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 by 1 nm per second to include all of them. When the AOTF 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 obtains the microbead image at each wavelength, and obtains the image at each wavelength corresponding to the background signal in the absence of microbeads through the same coincident operation, and then extracts the background signal image from the microbead image. Subtract the microbead absorption images at each wavelength. Then, in order to create the microbead absorbed images of the five red, orange, yellow, green, and blue wavelength ranges, the microbeads of the five wavelength ranges are summed together by intensities of the microbead absorbed images at the respective wavelengths corresponding to the predetermined wavelength ranges. Bead absorbance images are obtained.

Subsequently, the absorbance images of the microbeads of these five wavelengths are inputted 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 can be obtained through a computer. .

Figure 2 shows an image of Alginate microbeads containing red Polystyrene microspheres obtained through the HMI optical module through this process.

Example 4 Screening Kinase Inhibitors Using Kinase-encapsulated Microbeads

In the present invention, after encapsulation of kinase in microbeads, the efficacy of the drug as a kinase inhibitor was screened using the microbeads. Microbeads were prepared using sodium alginate solution.

After preparing a 2% sodium alginate solution, one kinase and one polystyrene dye were added to the solution. In the concept of one kinase-one color, one kinase contains a polystyrene dye with one kinase that can indicate that kinase. After mixing the mixed solution 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 micrometer microbeads.

The microbeads were collected and washed three times with distilled water. Thereafter, the obtained microbeads were reacted overnight after drug treatment for each concentration. Since the microbeads are gelled, overnight reactions were performed to allow sufficient 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 a mixture of ATP, DTT, kinase buffer, and kinase substrate.

After the reaction was washed three times with distilled water and the image of the microbead was obtained through a microscope-HMI system. The kinase substrate is a sox chelation-enhanced fluorophore (CHEF) 8-hydroxy-5- (N, N-dimethylsulfonamido) -2-methlyquinoline. When the kinase in a drug-free state ATP, DTT, kinase buffer, and placed in a solution with a kinase substrate mixture sikyeoteul the reaction, the kinase substrate and phosphorylation by the kinase, by Mg ^{2 +} is connected to the Sox as phosphote group fluorescence Light emission (excitation wavelength = 360 nm, emission wavelength = 485 nm). When kinase is inhibited by drugs, the fluorescence intensity emitted by kinase inactivation is reduced.

The following figure shows the result of measuring Kinase GSK3b inhibition by Methanesulfonate inhibitor using HMI-fluorescence microscope system.

To monitor kinase GSK3b inhibition by methanesulfonate inhibitor, microbeads were exposed to 360 nm UV beam, and then 485 nm was set as the transmission wavelength of HMI AOTF. (a) shows microbeads showing brighter Kinase-induced fluorescence obtained without treatment with Inhibitor, and (b) shows a greatly reduced microbead fluorescence image of approximately the same size as the background signal after 1 mM treatment of Inhibitor Methanesulfonate. .

As shown in FIG. 3, kinase inhibition by each drug can be determined through fluorescence images of microbeads through a microscope-HMI system. In addition, in order to identify a specific Kinase to which the drug has 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 as 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 then microbeads are made using the Electrospray method, the microbeads serve as a reservoir for a specific Kinase that can be identified or addressed.

The Kinase reservoir microbeads are mixed with different Kinase storage microbeads to form a mixture, and a specific drug is treated in the microbead mixture. Fluorescence images of the microbeads are then fluoresced using an HMI optical module-microscope as described above. When measuring, the on / off of each of the microbeads was determined according to the brightness of the microbeads. Then, as shown in Example 3, the absorbance image corresponding to the dye containing the microbeads was immediately measured in the brightfield mode. One drug can quickly identify the efficacy of an inhibitor as an inhibitor of various kinds of kinases.

Continuous measurement of microbead mixture fluorescence images in different regions of the microbead solution provides a new generation of platform for imaging-based drug efficacy screening without the need for pipetting. In the case of drug screening based on the existing 96-well plate, the drug is added and reacted with the reactants including the kinase and its substrate in each well. In this case, the pipette is used to deliver to the well by automated pipetting. . It 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

The present invention relates to a drug screening method using a kinase inhibition assay using microbeads in which different drugs are encapsulated. Microbeads are prepared 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 syringe and electrospray method with a distance of 1-10.0 kV, 1-5 mL / min gel flow rate, 1-20 cm syringe needle and alginate solution (10.0 kV, 1.5 mL / min, 5 cm preferred) to 150 micrometers microbeads were prepared by dropping in an aqueous solution of calcium chloride. 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 may be applied to this assay. After the reaction with the drug and the kinase in the microbeads, the microbeads were washed three times with distilled water, and then reacted at 37 ° C. for 8 hours in an ATP, DTT, kinase buffer, and kinase substrate. After the reaction was washed three times with distilled water and the image of the microbead was obtained through a microscope-HMI system.

Example 3 is then performed to determine the drug present in the microbeads. To screen for kinase inhibition of thousands and tens of thousands of different drugs, place an aqueous solution of a mixture of microbeads as drug reservoirs containing different drugs on the microscope sample stage and repeat the above procedure.

Example 6 Drug Screening Using Luciferin-Luciferase Essay

As an experiment to determine the action of the drug as a Kinase inhibitor, luciferin-luciferase assay using microbeads can be used as an effective drug screening method.

In luminescent reactions, when luciferin reacts with oxygen, it is oxidized to oxyluciferin, producing light (luciferin + O ₂ → oxyluciferin + light). In most luminescence reactions, carbon dioxide is released as a product. The reaction between luciferin and oxygen is very slow and catalyzed by luciferase and sometimes by cofactors such as calcium ions or ATP.

The reaction catalyzed by firefly luciferase occurs in two steps. 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 + O ₂ → oxyluciferin + AMP + light).

This reaction is very effective and almost all the energy put into it is transformed into light (560 nm). Kinase phosphorylates the substrate using ATP in cells.

Therefore, when kinase and substrate are first reacted with ATP, ATP is consumed, so no light is formed because luciferin-luciferase reaction does not occur. However, when the drug acts as a Kinase inhibitor, even if the drug reacts with Kinase first and then reacts with the substrate and ATP, the ATP is stably present because the drug inhibits the kinase phosphorylation of the substrate and thus luciferin-luciferase reaction. This happens and light is generated.

The present invention introduces a drug screening method using lluciferin-luciferase assay using microbeads in which different Kinases are encapsulated.

A 2% sodium alginate gel containing one kinase and one colored polystyrene bead in microbeads was 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 detection and microbead color coding method using the HMI system enables specific drugs to determine the efficacy of various kinases.

Example 7: Preparation of drug-induced mitochondrial dysfunction, liver toxicity measured using microbeads

The present invention provides a drug toxicity screening method using microbeads in which cells are encapsulated.

This method involves encapsulating the cells in microbeads, coating them to improve the stability of the microbeads, and detecting the toxicity after drug treatment. Encapsulating the cells in microbeads was performed by placing 8x10 ⁶ cell / mL hepatocytes (HepG2, etc.) in sodium alginate solution, 1-10.0 kV, 1-5 mL / min gel flow rate, 1-20 cm syringe needle and alginate solution. Cured in BaCl ₂ using an electrospray technique with a distance between them (10.0 kV, 1.5 mL / min, 5 cm preferred) and encapsulated in 1-8% (2% preferred) sodium alginate gel.

Sodium alginate gels are coated to stabilize cells in culture medium. The collected microbeads were placed in 0.1% Poly-L-lysine and treated for 7 minutes, followed by 5 minutes in 0.125% sodium alginate solution.

The coated microbeads are placed in a cell culture medium and incubated at 37 ° C. and 5% CO ₂ . Drug toxicity screening is performed using microbeads with complete cell encapsulation.

When intracellular calcium concentrations are caused by drug-induced cytotoxicity, the increased calcium enters the mitochondria, which increases the reactive oxygen species (ROS) in the mitochondria, which changes the permeability of the mitochondria. Cytochrome C is released from the mitochondria and caspase 9 and caspase 3 are generated in succession, 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 calcium to generate fluorescent material.

Mitochondrial MPT can be measured by Calcein-AM. When Calcein-AM accumulates in the mitochondrial membrane, when the MPT changes, that is, the mitochondrial membrane is damaged, Calcein-AM accumulated in the mitochondrial membrane is diffused into the cytoplasm and calcein-AM. The brightness of the fluorescence by the overall decrease in the cell.

And Caspase-3 can be detected using Caspase-3 substrate. Caspase-3 substrate generates fluorescent material when it meets Caspase-3, and can detect that Caspase-3 is generated through fluorescence detection.

In order to observe 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 Calcein-AM and Caspase-3 substrates for a sufficient time. Fluorescent markers are introduced into the hepatocytes present in the microbeads, and then the drug (eg nefazodone) is treated, and after a certain time, the fluorescence intensity of each marker is measured by HMI-microscope system to determine the increase and decrease.

When the drug has hepatotoxicity, the increase in the calcium marker fluorescence, the decrease of Calcein-AM fluorescence according to the mitochondrial MPT, and the increase in fluorescence due to the phosphor generated in the Caspase-3 substrate with the increase of Caspase-3 can be confirmed. Monitoring the changes in the three fluorescence, when the above fluorescence changes are observed it can be confirmed that the cytotoxicity caused by the drug is due to mitochondrial dysfunction.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is to be understood that various changes and modifications may be made without departing from the scope of the appended claims.

10 case 12: opening and closing door
14 air cooling member 20 first lens member
22 condenser lens 24 beam collimator or beam expander
30 lens support member 40 variable filter
50: long pass filter or band pass filter 60: image sensor
80: support bar 90: rail member

Claims (18)

Modular case that one side can be fastened 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 fluorescent 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 fluorescent beam including a wavelength undiffracted from the fluorescent beam passing through the variable filter;
A long pass filter or a band pass filter disposed in the case to remove light from 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 long pass filter or the band pass filter; And
And a second lens member disposed at the front side of the image sensor and configured to obtain a high quality image, and is capable of hypermulticolor imaging (HMI),
A portable hypermulticolor imaging optical module, characterized in that it is configured to be detachably detachable to the microscope as portable.
Modular case that one side can be fastened to the microscope;
A first lens member installed in an opening of one side of the case and having a beam collimator or a beam expander for reducing the size of the fluorescent 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 fluorescent beam including a wavelength undiffracted from the fluorescent beam passing through the variable filter;
A long pass filter or a band pass filter disposed in the case to remove light from the excitation light source from the fluorescent beam passing through the variable filter; And
An image sensor installed in the case and detecting a fluorescent beam passing through the mechanical plate and the long pass filter or the band pass filter; And
And a second lens member disposed at the front side of the image sensor and configured to obtain a high quality image, and is capable of hypermulticolor imaging (HMI),
A portable hypermulticolor imaging optical module, characterized in that it is configured to be detachably detachable to the microscope as portable.
The method according to claim 1 or 2,
The variable filter, the mechanical plate, the long pass filter or the band pass filter, and a support bar for fixing the image sensor, respectively, is provided,
It is installed on the lower surface of the case, the lower end portion of the support bar in the longitudinal direction, so that the variable filter, the mechanical plate, the long pass filter or the band pass filter, and the image sensor can move in a straight direction from the first lens member The rail member is fastened to be movable; further comprising,
And an optical module configured to be focused.
The method according to claim 1 or 2,
In this case,
An opening / closing door formed on one surface of the first lens member, a variable filter, a mechanical plate, a long pass filter or a band pass filter, and an image sensor disposed therein so as to replace and repair the first lens member;
Optical module comprising a.
The method of claim 1,
The condensing lens is made of at least one,
In order to attach and detach the condensing lens to the case opening, a 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 module characterized in that the.
The method according to claim 1 or 2,
A lens support member configured to be supported by the first lens member in an opening of one side of the case;
Optical module comprising a further.
The method according to claim 1 or 2,
The variable filter is an optical-optic tunable filter (AOTF) characterized in that the optical module.
The method according to claim 1 or 2,
The image sensor,
An optical module comprising a two-dimensional image sensor including a charge coupled device camera and a photodiode array.
The method according to claim 1 or 2,
In this case,
An air-cooling member or a water-cooling member by cooling water configured to ventilate the coolant for cooling the variable filter or the image sensor;
Optical module characterized in that it further comprises.
The method according to claim 1 or 2,
In this case,
Optical module, characterized in that made of an opaque material to block the light from the outside.
After placing the microbeads with pigments on the microscope sample stage, a beam of white light is irradiated onto the microbeads, and an acoustic-optic tunable filter (AOTF) is applied at a resolution of 1 nm in the wavelength range from 400 nm to 800 nm. Scanning 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 of each wavelength obtained in step 1 through the metamorph software (Metamorph software) to the signal strengths of the transmission images of each wavelength obtained in step 2 (step 3);
In the wavelength-specific transmission image obtained in step 3, wavelengths corresponding to red (630-740 nm), orange (585-620 nm), yellow (570-580 nm), green (520-570 nm), and blue (440-490 nm) Using the Metamorph software to merge images of respective wavelengths corresponding to the wavelength ranges of each color to obtain a transmission image (step 4); And
Completion of the microbead color coding by inputting the color image obtained in the step 4 through the execution of the "Overlay" function of the software for each wavelength to obtain a microbead image of the color corresponding to the pigment into the microbead (step) 5);
Microbead determination step based on the microbead color coding comprising a.
The method of claim 11,
In the step 1,
The microbeads, alginate, poly lactide (PLA), photocurable photopolymer polymer (polyethylene glycol diacrylate, PEG-DA), polypropylene fumarate (Poly propylene fumarate, PPF), poly propylene fumarate (PPF) / diethyl fumarate (DEF), pentaerythritol triacrylate, or trimethylolpropane triacrylate ( Trimethylolpropane triacrylate) polymer,
The pigment includes a polystyrene microsphere bead polymer pigment, an organic pigment, an inorganic pigment quantum dot, a pigment-doped silica nanoparticles, and gold,
The software is applicable to software related to Metamorph, Metaview, or ImagePro image analysis, and has the same function as the step-by-step execution function executed in claim 11 among the functions of the software. And performing microbead color coding based on microbead color coding.
The method of claim 11,
A solution of a mixture of individual microbeads containing one specific pigment and one specific kinase is placed on a microscope sample stage and one specific drug to be screened enters the microbeads and reacts with each kinase. Incubating for a sufficient time to allow (step 1);
After washing the microbead with distilled water three times after step 1, adenosine triphosphate (ATP), dithiothreitol (DTT), kinase buffer (Kinase buffer), and kinase substrate (Kinase substrate) Placing in the mixed solution and reacting for a sufficient time at a specific temperature between 10-45 ° C. (step 2);
After step 2, the microbeads were washed with distilled water, placed on a microscope sample stage, and then irradiated with a beam of white light to the microbeads, an acoustic-optic tunable filter with a resolution of 1 nm in a wavelength range of 400 nm to 800 nm. scanning, filter (AOTF) to obtain a transmission image of white light corresponding to the pigment of the microbead at each wavelength (step 3);
After the step 3, the microscope was changed to the fluorescent mode, and then the phosphor formed in each microbead was excited through the microscope-HMI system, and then the wavelength of the acoustic-optic tunable filter (AOTF) was transmitted to the maximum emission wavelength of the phosphor. Fixing the obtained 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 the step 5, the microscope mode was changed to the brightfield mode, and white light was irradiated to the background solution to obtain an absorbance 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 images 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
Performing steps 3 and 5 of claim 3 to complete a kinase inhibitory drug efficacy screening in microbeads (step 9);
Microbead color coding based kinase (Kinase) inhibitor drug screening method comprising a.
The method of claim 13,
In the step 1,
The microbeads, alginate, poly lactide (PLA), photocurable photopolymer polymer (polyethylene glycol diacrylate, PEG-DA), polypropylene fumarate (Poly propylene fumarate, PPF), poly propylene fumarate (PPF) / diethyl fumarate (DEF), pentaerythritol triacrylate, or trimethylolpropane triacrylate ( Trimethylolpropane triacrylate) polymer,
In step 2 above,
The kinase substrate (Kinase substrate), in addition to Sox microbead color coding based kinase (Kinase) characterized in that it comprises any organic or inorganic kinase substrate (Kinase substrate) that can react with the kinase in the microbeads to generate a phosphor A) inhibitory drug screening method.
An aqueous solution of a mixture of microbeads, each containing one specific pigment and one specific drug, is placed on a microscope sample stage so that one specific kinase can enter the microbeads and react with the drug. Incubating for a sufficient time (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) Placing in the mixed solution and reacting for a sufficient time at a specific temperature between 10-45 ° 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 beam of white light to the microbeads, with an acoustic optical wavelength tunable filter at 1 nm resolution in the wavelength range of 400 nm to 800 nm. scanning, filter (AOTF) to obtain a transmission image of white light corresponding 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 (AOTF) is applied to the maximum emission wavelength of the phosphor. Fixing the transmission wavelength and then 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 the step 5, the microscope mode was changed to the brightfield mode, and white light was irradiated to the background solution to obtain an absorbance 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 images 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
Performing steps 3 and 5 of claim 3 to complete a kinase inhibitory drug efficacy screening in microbeads (step 9);
Kinase inhibitor drug screening method based on drug-containing microbead color coding comprising a.
16. The method of claim 15,
In the step 1,
The microbeads, alginate, poly lactide (PLA), photocurable photopolymer polymer (polyethylene glycol diacrylate, PEG-DA), polypropylene fumarate (Poly propylene fumarate, PPF), poly propylene fumarate (PPF) / diethyl fumarate (DEF), pentaerythritol triacrylate, or trimethylolpropane triacrylate ( A kinase inhibitor drug screening method based on drug-containing microbead color coding, which is made of a trimethylolpropane triacrylate polymer.
An aqueous solution of a mixture of microbeads containing one specific pigment or a mixture of specific pigments and one specific kinase is placed on a microscope sample stage, and then the specific drug is placed in the solution and incubated for a sufficient time to incubate the drug. Entering the microbeads to react with the drug (step 1);
After washing the microbeads after step 1, a kinase substrate to be phosphorylated by phosphorylation of adenosine triphosphate (ATP) and drug target kinase (Kinase) was added to the microbead solution, followed by sufficient reaction and washing with water. (Step 2);
After step 2, luciferin and luciferase were put in a microbead solution and reacted for a sufficient time, and measuring chemiluminescence generated in the microbeads through the optical module of claim 1 (step 3);
After the step 3, a beam of white light is irradiated to the microbeads, and an optical-optic tunable filter (AOTF) is scanned at a wavelength of 1 nm in a wavelength range of 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 the step 5, the microscope mode was changed to the brightfield mode, and white light was irradiated to the background solution to obtain an absorbance image (step 6);
Obtaining a chemiluminescent 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 images of the microbeads obtained in step 3 (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 4 for each wavelength (step 8); And
Performing steps 4 and 5 of claim 3 to complete a kinase inhibitory drug efficacy screening in microbeads (step 9);
Kinase inhibitor drug screening method using luciferin-luciferase assay based on drug-containing microbead color coding comprising a.
18. The method of claim 17,
The microbead of claim 8, wherein the microbead is an alginate, poly lactide, PLA, a photocurable photopolymer polymer, polyethylene glycol diacrylate, PEG-DA), Poly propylene fumarate (PPF), Poly propylene fumarate (PPF) / Diethyl fumarate (DEF), Pentaerythritol triacrylate Or Kinase inhibitor drug screening method using a luciferin-luciferase assay based on drug-containing microbead color coding, which is made of a trimethylolpropane triacrylate polymer.

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