WO2012157979A2 - Module optique portable d'imagerie hypermulticolore et système de criblage d'un médicament inhibiteur de la kinase basés sur un codage de couleurs de microbilles - Google Patents

Module optique portable d'imagerie hypermulticolore et système de criblage d'un médicament inhibiteur de la kinase basés sur un codage de couleurs de microbilles Download PDF

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Publication number
WO2012157979A2
WO2012157979A2 PCT/KR2012/003904 KR2012003904W WO2012157979A2 WO 2012157979 A2 WO2012157979 A2 WO 2012157979A2 KR 2012003904 W KR2012003904 W KR 2012003904W WO 2012157979 A2 WO2012157979 A2 WO 2012157979A2
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microbeads
drug
microbead
image
kinase
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Korean (ko)
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WO2012157979A3 (fr
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송준명
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SNU R&DB Foundation
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SNU R&DB Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility

Definitions

  • Portable kinase inhibitor drug screening system based on hypermulticolor imaging optics and microbead colorcoding
  • 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.
  • HCS high-content screening
  • 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.
  • HCS high-content screening
  • 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.
  • 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.
  • hepatotoxicity evaluation of drugs mediated by mitochondrial dysfunction was performed using microbead technology.
  • 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.
  • 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.
  • HCS high-content screening
  • 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.
  • microbead 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • red (630-740 nm) orange (585-620 nm)
  • yellow 570-580 nm
  • green 520-570 nm
  • blue 440-490 nm
  • A0TF is very advantageous for this microbead based color coding.
  • AOTF-based microbead imaging systems can be used to obtain transmission images at each wavelength of microbeads with 1 nm resolution.
  • 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.
  • 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
  • De-addressing can be enabled.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇
  • 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
  • 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.
  • FIG. 1 is a schematic diagram of a color coding process of a microbead using a portable HMI.
  • 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.
  • 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.
  • the portable HMI optical models 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
  • 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.
  • 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.
  • 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.
  • 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 '
  • 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.
  • 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
  • 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.
  • the HMI system is free to substitute A0TF one wavelength per second and transmit wavelength interval (Article 26). (1 nm-several tens nm- 1
  • FIG. 4 is a schematic diagram of an HMI 1 system composed of a c-mount lens, an AOTF, and a CCD
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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)
  • 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.
  • 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.
  • the variable filter 40 is preferably an acoustic optical wavelength variable filter 40 (acousto-optic tunable filter 40 (acousto-optic tunable filter, A0TF)).
  • the image sensor 60 is preferably a two-dimensional image sensor 60 including a charge coupled device camera and a photodiode array.
  • 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.
  • the case 10 is opaque to block external light from entering
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • the intensities of the CCD at a specific location of the pixel at each wavelength can be obtained simultaneously.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • Air-cooling or water-cooling system is installed to prevent heat.
  • 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.
  • 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.
  • a microbead sample of a specific color is placed on the microscope sample platform.
  • 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.
  • 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
  • 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.
  • One color polystyrene dye with kinase was added to indicate the kinase.
  • 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.
  • 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 °].
  • kinase was added to a mixture of ATP, DTT, kinase buffer, and kinase substrate, and the reaction was carried out.
  • Kinases replaced by drugs (Article 26 of the Rule) If it becomes inhibition ⁇
  • the following figure is the result of measuring the inhibition of Kinase GSK3b by Methanesulfonate Inhibitor using HMI-fluorescence microscopy system.
  • 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.
  • the kinase inhibition by each drug can be determined through the fluorescence image of the microbeads through the microscope -HMI system.
  • 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.
  • 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.
  • Example 3 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.
  • 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.
  • microbeads were reacted overnight with Kinase GSK3b.
  • GSK3b Kinase GSK3b
  • all Kinases can be applied to this essay.
  • 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.
  • 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.
  • the microbeads's mixed solution as a drug reservoir containing each different drug is placed on the microscope sample stage and repeated.
  • 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.
  • luciferin reacts with oxygen to oxidize to oxyluciferin, producing light (luciferin + 3 ⁇ 4 ⁇ oxyluciferin + light).
  • 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.
  • 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.
  • 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.
  • HepG2 8xl0 6 cell / mL liver cell
  • 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
  • Permeabi lithium transit ion 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- ⁇ 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.
  • 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
  • Air cooling member 20 First lens member
  • Condenser lens 24 Pan collimator or beam expander
  • Lens support member 40 Variable filter
  • 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.
  • bioimaging research can be spread by installing a modeled optical system.
  • 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.
  • 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.
  • the drug target protein reservoir such as Kinase is made of microbeads and the combinations thereof.
  • the present invention 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.
  • the present invention is a new era of drug screening platform without pipetting because it can indicate ion each drug by encapsulating different dyes or drug targets by color coding method and encapsulat ion Can be opened.
  • 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. .

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Abstract

La présente invention concerne le développement d'un module optique portable d'imagerie hypermulticolore (HMI) et d'un nouveau système de criblage d'un médicament basés sur un codage de couleurs de microbilles. Le module optique HMI est un nouveau système d'imagerie portable à longueurs d'ondes multiples qui remplace un dispositif de criblage à haute teneur (HCS) actuellement utilisé à titre de dispositif important pour le criblage d'un médicament. Il s'agit également d'un système HMI portable qui est fixé sur le côté d'un microscope optique classique et qui exécute un criblage d'un médicament à un niveau cellulaire et à un niveau moléculaire. De plus, pour palier les limites du criblage de médicament robotisé qui est utilisé pour cribler l'action d'un médicament et la toxicité cellulaire de composés d'une bibliothèque à grande échelle par l'intermédiaire d'une plate-forme d'échantillons de type à cuvettes, un système de criblage de composés d'une bibliothèque à grande échelle sans pince à tube et utilisant des microbilles est présenté à titre de nouveau stockage de cible pharmacologique et de médicament. L'utilisation d'un module optique HMI portable fixé à un microscope optique et de microbilles stockant un médicament ou une cible pharmacologique permet un criblage d'un médicament rapide et plus économique. Son application permet un criblage de la toxicité d'un médicament, qui déclenche un effet de blocage de la kinase, et de l'hépatotoxicité d'un médicament.
PCT/KR2012/003904 2011-05-19 2012-05-17 Module optique portable d'imagerie hypermulticolore et système de criblage d'un médicament inhibiteur de la kinase basés sur un codage de couleurs de microbilles Ceased WO2012157979A2 (fr)

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