KR20040048754A - Temperature controlled real time fluorescence detection apparatus - Google Patents

Abstract

PURPOSE: An apparatus for detecting fluorescence in real-time and capable of controlling a temperature is provided to detect a great number of biological samples by detecting fluorescence at a high speed and to sensitively detect fluorescence without loss of fluorescence by exchanging a filter at a high speed. CONSTITUTION: An apparatus for detecting fluorescence in real-time and capable of controlling a temperature includes a sample vessel(2), a light source(1), a detector(5), a fluorescence transport unit, a wavelength selection unit(3), and a control part. The light source is positioned such that the light source is incident on the sample vessel(2). The detector(5) detects fluorescence emitted from a sample. The fluorescence transport unit transports the emitted fluorescence to the detector(5).

Description

Real-time fluorescence detection device with temperature control {TEMPERATURE CONTROLLED REAL TIME FLUORESCENCE DETECTION APPARATUS}

The present invention relates to a device for monitoring and analyzing the condition and reaction of a large number of biological samples, in particular to search for fluorescence emitted after the light (Excitation) to the sample, or the temperature control ( The present invention relates to a device for searching and analyzing fluorescence in real time when a nucleic acid amplification reaction or a sequence of nucleic acid is added with a temperature controller.

More specifically, the present invention detects fluorescence of various wavelengths in hundreds to thousands of samples sensitively even at low and fast sample concentrations within seconds, while realizing the ability to search and analyze enzyme reactions in real time. A compact fluorescence retrieval apparatus portable to a.

In general, a device for searching and analyzing biological samples includes a spectrophotometer and a spectrofluorometer.

Spectrophotometers measure the conduction of light, while spectrofluorometers measure the emission fluorescence, which is usually measured in the vertical direction of the excitation light to eliminate the effects of direct excitation light. For fluorescence microscopy, epi-fluorescence is used.

Such devices typically consist of a light source, a sample compartment, a detector, a wavelength selector, and a lens.

As a light source, lamps, lasers or LEDs are usually used.

The lamps are advantageous because they are flexible in their wavelength range, but they require a separate monochrometer, are difficult to install, and costly to install the array required for fast retrieval of large samples. Lasers do not require monochromators, but they are unrealistic because of the large size of the unit, the fixed wavelength band, and the high cost of array installation.

On the other hand, LEDs eliminate the need for monochromatic devices, enable cost-effective array installation, and are extremely advantageous for making compact devices with fast sample retrieval.

Sample containers are usually cuvettes, microtubes, microplates or capillary tubes.

Cuvettes cannot be minimized to the appropriate size for the experiment, cannot be installed with the array required for fast retrieval of large samples, and the free replacement of the containers required for efficient experimentation is expensive and expensive. Capillary tubes can be arranged, but they have the disadvantage of being low in capacity and free to replace freely and costly.

On the other hand, tubes and microplates can be used as samples in small volumes, can be arranged, and can be freely replaced like disposable products, and are inexpensive, so they are used as a standard for fast fluorescent screening of large samples.

Detectors usually use CCD cameras, photodiodes, or photo-multiplier tubes (PMTs).

Although CCD cameras are inexpensive and focus using a lens, arrangement is unnecessary for each sample container, but it has a disadvantage in that sensitivity is low. Photodiodes have the advantage of being inexpensive, but they require arrangements and are moderately sensitive, while still having the disadvantage of relatively high noise signals.

PMT, on the other hand, requires an array and is expensive, but it can be the best method if you can develop a device that can use a minimum number of devices because it is very sensitive and has a low noise signal, which is optimal for detecting a small amount of fluorescence. . Currently, multi-channel products are also available.

As a wavelength selector, a monochromator or a filter is usually used.

Monochromatic devices are easy to select wavelengths, but have the disadvantage of being expensive and difficult to apply to large samples.

Filters, on the other hand, require the use of multiple filters for the search of multiple wavelengths, but are advantageous because they are inexpensive and easy to develop for array installation.

The lens is used only when focus is needed, and a large number of lenses are required to search for a lot of excitation light and emission light.

At present, there is a need for an economical device that is compact and portable while searching for the largest amount of samples simultaneously and quickly and precisely in various wavelength bands.

Therefore, in the types of basic components described above, it is required to select components that can meet these needs, but to develop additional technologies that can overcome the disadvantages. need.

First, a technique to be combined to scan / analyze fluorescence in a large amount of samples (sample vessels) quickly and rapidly includes a scanning method or an array method.

In the scanning method, (1) the excitation light and emission detectors scan fluorescence by moving from one end to the other end of a fixed mass of sample containers, or (2) the fixed excitation light and emission The detector searches for fluorescence by sequentially scanning a large number of rotating sample containers. This method is economical, but there is a limit to the simultaneous retrieval of a large number of samples because the usage can be limited by the speed and retrieval time of the scan. In addition, the method of rotating the sample container has a disadvantage that it is difficult to use an unmanned apparatus that automatically exchanges and automatically loads the microplate.

On the other hand, in the array method, the excitation light and the fluorescence detector are arranged for each sample container without moving parts to detect fluorescence.

The arrangement of excitation light can be accomplished by (1) using a bundle of fibers for one light source, such as by separating the laser beam into bundles of optical fibers and arranging them in individual sample containers, or (2) using multiple LEDs (or photodiodes). There is a method of arranging a plurality of light sources for each sample container, such as arranging the respective sample containers.

In addition, the arrangement of the fluorescence detector includes (1) partially arranging the multi-channel micro-PMT, (2) arranging the photodiodes per container, or (3) separating the entire image detected by one CCD camera for each sample. In order to produce an array effect, the CCD camera method is limited because of its low sensitivity. Among the above methods, PMT is most commonly used because it detects a small amount of fluorescence with low sensitivity and low sensitivity.

These arrangements allow simultaneous fluorescence searches in large sample containers, thus enabling rapid fluorescence searches without limiting the number of samples.

However, this approach requires high throughput, such as the need to arrange 96 or 384 light sources and detectors at most to simultaneously monitor large samples, for example 96 or 384 well PCR samples. More will have complex mechanical structures and can be expensive systems.

Therefore, there is a demand for developing a technology capable of achieving the same performance while arranging a minimum number of light sources and detectors.

In addition, when a large amount of samples are injected into a large number of exchangeable sample containers, and a fluorescence search must be performed while continuously changing, a problem in handling a plurality of 96-well or 384-well microplates is a manual operation. The possibility of errors and the inconvenience of labor-intensive work that requires loading a large number of plates sequentially.

Therefore, there is additionally a need for a well plate autoloading structure that can use a robotic device that not only automatically loads one plate into the device, but also sequentially loads multiple plates into the plate guide.

Devices that add scanning or arraying technology to detect fluorescence in large samples are commonly referred to as fluorescent microtiter plate detectors. Currently available devices include Gemini Microplate Detectors and Applied Biosystems from Molecular Devices. CytoFluor 4000 Fluorescence Multi-well Plate Detector, Tecan GENios, etc.

Molecular Devices' Gemini Microplate Detectors are designed to search from 6 to 384 wells of microplates up or down. Free and high sensitivity PMT is used as a detector. However, because the lamp is used as an excitation light source, it requires a lens and a monochromator and is large, and the arrangement method for retrieving a large amount of samples is unrealistic, which results in a 96-well search time of at least 15 seconds and more in the 384-well. It takes time.

CytoFluor 4000 Fluorescence Multi-well Plate Detector from Applied Biosystems is a structure that can search 6 to 384 well microplates below, and it is 320nm ~ 700nm using 6 types of Excitation / Emission filter pairs. It is possible to select wavelength within and use high sensitivity PMT as detector. As a scanning method using a lamp as an excitation light source and using a single PMT, a scan time of 96 wells is 45 seconds, and in the case of 384 wells, more time is required.

Tecan's GENios can be used not only for 6 to 384 well microplates, but also for tubes and cuvettes. Its wavelength scanning using lamps allows for free wavelength selection within 340 nm to 700 nm and provides high sensitivity PMT. There is a feature to use as a detector. However, because the lamp is used as an excitation light source, a lens and a monochromator are required and large, and the arrangement method for retrieving a large amount of samples is unrealistic, and the search time of 96 wells is 30 seconds. It takes a lot of time.

These microplate fluorescence detectors are ideally suited for the development of fluorescence-centric reactions and high-efficiency screening, as well as additional screening functions. Cell-surface receptor binding assays, cell-based Cell-based drug screening, Fluorescence-Linked Immunoabsorbent Assays (FLISA), protein-protein interactions, protein-ligand interactions, protein-DNA interactions, enzyme reactions (kinetics) Study) and apoptosis studies of cells.

However, devices currently on the market are still in search of tens of seconds for 96 wells, and the need for several seconds of high-speed search technology is needed for large sample retrieval.

In addition, another disadvantage of the above microplate fluorescence detection devices is the lack of temperature control, which makes it difficult to monitor true biological responses.

Therefore, adding a Temperature Controller to these devices results in a real-time PCR device that searches for PCR (Polymerase Chain Reaction) sensitive to temperature changes in real time. Nucleic Acid Amplification (PCR) and Rather than performing a separate fluorescence search, amplification of nucleic acids while real-time (fluorescence) can be quickly searched and monitored in real time (Real-Time) is currently in the spotlight because it is possible to search the nucleic acid sequence within 1-2 hours.

As described above, the "microplate search apparatus" includes a light source 1, a sample container 2, a wavelength selector 3, a fluorescence moving device 4, and a detector 5 as basic components, and the focus is Lens 6 may be added if necessary.

1 is a conceptual diagram of a general microplate search apparatus. The real-time PCR apparatus is a temperature controller (Temperature Controller) is attached to the well chamber block for inserting the sample container in order to repeatedly give a temperature change to the sample based on the apparatus shown in FIG.

Referring to the operation of the fluorescence detector based on a real-time PCR device as follows.

In the first step, DNA and reagents extracted from a sample (eg blood, saliva, air, cultured cells) to be analyzed are injected into a sample vessel (typically 96 well or 384 well microplates).

In the next step, the computer program specifies variables such as step temperature, number of cycles (= cycles), and data collection steps.

In the next step, depending on the parameters set in the program, amplification reactions are repeated by repeating 30-40 cycles with three cycles of temperature change (eg 95 ° C, 56 ° C, 72 ° C) as one cycle. .

In the next step, light 1 is irradiated onto the sample in the sample vessel at each step (selected by the program) at each cycle.

In the next step, if the wavelength selection device (i.e. filter) 3 between the sample container and the light source passes only light of a specific wavelength, fluorescence is emitted in all directions from the sample.

In the next step, a fluorescence emitted from the sample is transferred to a focusing lens 6 using a dichroic lens 4. The focus lens 6 collects fluorescence and focuses on the detector 5 (i.e., a PMT or CCD camera).

In the next step, the wavelength selector 3 between the focus lens 6 and the detector 5 passes only light of a specific wavelength.

Finally, in the PC program, the amount of fluorescence in each sample is displayed for each cycle.

Real-time monitoring of PCR amplification or dynamic PCR analysis allows the measurement of DNA replication on a cycle-by-cycle basis. This is made possible by real-time detection of fluorescence changes during PCR amplification reactions, including the appropriate amount of DNA-inserted fluorophores (eg, SYBR Green I) in the sample that are characterized by an increased fluorescence amount when bound to double-stranded DNA (dsDNA). Proved by

FIG. 2 is a result graph in which the X-axis is the cycle and the Y-axis is the intensity (Intensity), where each line is a value in each sample.

First, when the first cycle is completed, the result value is drawn for each entire sample (= well), and when the second cycle is completed, the second value is drawn to display in real time until the entire cycle is completed.

As shown in FIG. 2, an exponentially abruptly rising curve shows the sample's DNA sequence (eg, anthrax) as expected (expected with reagent injected with the sample). It can be determined that there is a bacteriostatic bacterium, and that the higher the sample is in the previous cycle, the greater the amount of bacteria.

Compared to other methods of quantitative analysis through PCR amplification, real-time PCR can suppress potential contamination by using "closed tubes" as sample containers, and can be used in real time without simple mechanical structure, high efficiency and post-reaction data analysis. It has advantages such as displaying data in time.

Moreover, monitoring of the progress cycle unit allows tracking of PCR amplification processes, which allows other methods to facilitate the quantification of sequences within many acceptable concentration ranges without the necessary sample dilution.

In general, in PCR amplification, the cycle-by-cycle DNA strand variation and primer binding are performed by temperature control that repeats the temperature change of the high and low temperatures in cycles according to programming. (annealing), DNA amplification (cloning) by the catalysis of the polymerase (Polymerase) is made. It can be seen that this reaction is a reaction experiment sensitive to the influence of temperature.

The temperature control device used here is of various kinds depending on the method of heating and cooling the well chamber block (metal material).

Conventionally, air circulation, electrical resistance, halogen vacuum tube, electric coil, light, cartridge heater (Cartridge Heater) are used for heating and air circulation through water or fan is used for cooling. Due to the temperature control feature, a method using a thermoelectric module using a Peltier device is mainly used.

The current need for technology development focuses on (1) how to accurately and rapidly increase or decrease the maximum capacity of the well chamber block by programming, and (2) how to control the entire well chamber block to a uniform temperature. It is becoming.

Current real-time PCR devices include Applied Biosystems' ABI Prism 7700 Sequence Detection System (SDS), Gene Amp 5700 SDS and ABI Prism 7700HT, Roche Molecular Biochemicals' LightCycler, Bio-Rad Instruments' iCycler iQ and Stratagene's Mx4000 Multiplex , Cepheid's Smart Cycler System, and Corbett Research's Rotor-Gene.

The ABI Prism 7700 Sequence Detection System (SDS) from Applied Biosystems (Forster City, Calif., USA) is the first commercially available real-time PCR device with temperature control. The laser induces fluorescence as a light source, and through multiple fluorescence wavelengths of 500 ~ 660nm detected, a multiplex PCR can be used to simultaneously use a plurality of fluorescent materials in a single reaction.

The device can be used for reactions such as hydrolysis probes, molecular beacons, and double-stranded DNA-binding dyes. You can run a 96-well experiment at a time, which takes about two hours. However, there is a disadvantage that the result can be analyzed only after amplification is completed, as in the conventional hybrid probe method.

Another device from the company is the Gene Amp 5700 SDS. Like the 7700 SDS device, real-time reverse transcription PCR (RT-PCR) using TaqMan, molecular beacons, and SYBR Green I that binds to DNA ) Can be performed. According to a related article, Legionella Pneumophila demonstrated the quantification of cytokine mRNA in lung infections using Gene Amp 5700 and SYBR Green I technology. The difference with the 7700 SDS is that it uses a halogen lamp as a light source instead of a laser, which is inexpensive, while only searching for a single wavelength.

Another device from the company is the ABI Prism 7900HT, which has the same specifications as the 7700 SDS, but can automatically prepare a large number of search plates by automating some processes for high efficiency applications. It can handle microtiter plates up to well. In addition, automated attachments can be optionally attached to automatically load 96-well or 384-well plates into the device, allowing full automatic handling 24 hours a day.

LightCycle from Roche Molecular Biochemicals (Mannheim, Germany) enables real-time RT-PCR with Borosilicate Glass Capillary, which can hold 32 samples. A small amount of glass capillary tube can be easily heated and cooled with air, and a rotary conveyor is used to retrieve 32 PCR samples in turn through the light source and detector to be retrieved once per cycle during PCR. The blue LED is used as the excitation light, and is fluorescence-detected by three silicon photodiodes with different wavelengths to enable multiplex PCR amplification.

The device is mainly SYBR Green I which binds to double stranded DNA (dsDNA). The advantage of the 7700 SDS is that it is inexpensive and highly efficient, allowing real-time data display during PCR amplification, fast temperature control with air, and 30-40 cycles in 20-30 minutes through capillary vessels. In addition, it is possible to analyze the specificity of the results through the melting curves (Melting Curves), so that SYBR Green I that binds to double-stranded DNA (dsDNA) can be used more stably.

However, it is not practical because it uses a capillary tube that is expensive and cannot be replaced freely, and only 32 wells can be searched simultaneously and only a limited number of samples can be used because some wells must be used for standard samples for quantitative analysis through standard curves. There is a crucial drawback to this.

This is a problem that is inconvenient for the user when performing a large-scale search that must deal with a large variety of samples in one experiment.

ICycler IQ from Bio-Rad Instruments (Hercules, Calif.) Is another device that can measure fluorescence during PCR amplification, and because it rotates the filter, it has multiplex functions using four fluorescent materials in the same tube. It is possible. You can also search for 96 samples at the same time.

However, there is a disadvantage that it is not suitable to detect a large number of samples sensitively because a complex lens is required and an unarranged lamp is used as an excitation light source and a low sensitivity CCD camera is used as a detector.

The Mx4000 Multiplex from Stratagene (La Jolla, Calif.) Can search for a variety of fluorescence, including TaqMan probes, hybrid probes, and molecular beacons, and a variety of sample vessels, including 96-well plates, eight tubes or individual tubes. The light source is a tungsten halogen lamp with multiple wavelengths from 350 to 750 nm, enabling multiplexing and data analysis during PCR amplification.

Cepheid (Sunnyvale, Calif.) Smart Cycler System can be used with molecular beacons, scorpions, hybrid probes, TaqMan probes or SYBR Green I. The advantage is that it has 16 individual modules that can be programmed differently and have a separate optical system that allows the use of four probes in one experiment, allowing for free use. The software also allows you to view data as you collect it.

However, because of the use of individual tubes, only a small number of samples can be analyzed for quantitative analysis.

Rotor-Gene from Corbett Research (Mortlake, Sydney, Australia) uses a "centrifugal" temperature control, similar to LightCycler, and uses four LEDs of different wavelengths as the light source and multiple filters to search for a variety of fluorescent materials. Can be.

However, up to 48 samples can be experimented at a time, and the scanning method using a relatively long search time is difficult, so a large amount of sample search is difficult.

As described above, the conventional fluorescence detection device still has a number of limitations such as searching over a few tens of seconds on a 96-well basis, and the concentration of the DNA sample should be at least several hundreds to thousands of copies in terms of sensitivity.

In addition, the conventional fluorescence retrieval device has a limitation that can be used only in the laboratory because it is expensive and portable.

In order to solve the above problems, the present invention improves the late detection speed of the prior art, which sequentially scans the entire well partly while the detector moves, so that a large number of biological samples can be simultaneously retrieved in a few seconds with a high-speed fluorescence search. An object of the present invention is to provide a compact device.

In addition, another object of the present invention is to increase the sensitivity to detect the concentration of several tens of copies by eliminating the low fraction of the prior art that the concentration of DNA or RNA samples should be at least hundreds to thousands of copies (copy).

In addition, another object of the present invention is not to increase the price exponentially as the number of samples (well number) more than hundreds as in the prior art, but the economical price of the fluorescence detection device of thousands of well plate capacity To make it searchable.

In addition, another object of the present invention is to miniaturize to a portable level so that it can be applied in the field that requires maneuverability, such as biological terrorism or infectious diseases, rather than a large device that can be used only in the laboratory as in the prior art.

In addition, another object of the present invention is to remotely load the sample-injected microplate as necessary to solve the difficulty of the fluorescence detection in the contaminated site caused by the conventional well chamber block portion and the detector and control unit together It is to provide a controllable remote control fluorescent search apparatus.

1 is a conceptual diagram of a conventional general microplate detection device (Microtiter Plate Detector).

2 is a graph of the results of cycle vs. fluorescence retrieved in conventional real time.

3A to 3C are front, side, and plan views of a Real-Time Fluorescence Microtiter Plate Detector according to one embodiment of the present invention.

4A to 4C are front, side, and plan views of a real-time PCR apparatus of one embodiment of the present invention.

5A and 5B are a front view and a plan view of an LED board of a real-time microplate fluorescence retrieval apparatus or a real-time PCR apparatus according to one embodiment of the present invention.

6A and 6B are a front view and a plan view of an excitation filter guide of a real-time microplate fluorescence retrieval device or a real-time PCR device according to one embodiment of the present invention.

7A to 7C are front, side and top views of a well chamber block thermoelectric module device of a real-time microplate fluorescence retrieval device or a real-time PCR device according to one embodiment of the present invention.

8A and 8B are a front view and a plan view of a microplate of a real-time microplate fluorescence retrieval apparatus or a real-time PCR apparatus according to one embodiment of the present invention.

9A and 9B are a front view and a plan view of a header device of a real-time microplate fluorescence retrieval device or a real-time PCR device according to one embodiment of the present invention.

10 is a front view of a PMT of a real-time microplate fluorescence retrieval device or a real-time PCR device according to one embodiment of the present invention.

11A and 11B are a front view and a plan view of a rotary automatic filter changer of a real-time microplate fluorescence retrieval apparatus or a real-time PCR apparatus, which is an embodiment of the present invention.

12 is a diagram illustrating a coupling process as a front view of each of the microplate, the well chamber block, the excitation filter guide, and the LED board according to one embodiment of the present invention;

13 is an explanatory diagram illustrating each LED array of the LED board according to an embodiment of the present invention.

14 is a front view showing a connection relationship between a header, a thermoelectric module device for a heating cover, and a PMT as a detector according to an embodiment of the present invention.

15 is a front view of a PMT and a plan view of a header, according to an embodiment of the present invention, illustrating interconnections;

FIG. 16 is a diagram showing an interconnection relationship between an LED board, an excitation filter guide, a thermoelectric module device for a heating cover, and a microplate according to another embodiment of the present invention. FIG.

FIG. 17 illustrates an interconnect relationship between a microplate, a well chamber block, a header, and a PMT according to another embodiment of the present invention. FIG.

In order to achieve the above object, the present invention is an apparatus for analyzing a sample by searching for a fluorescence emitted from the sample after irradiating a light source to a biological sample, a sample container, a light source positioned to irradiate the sample container, in the sample A fluorescence detection device comprising a detector for detecting emitted fluorescence, a fluorescence shifting device for moving the fluorescence emitted from the sample to the detector, a wavelength selection device, and a control unit.

An LED array in which a plurality of LEDs are sequentially emitted;

A well chamber block having a plurality of wells for inserting a sample container;

A multi-channel PMT for detecting fluorescence emitted from the sample by each LED emission of the LED array; And

It provides a temperature-controlled real-time fluorescence search device comprising a plurality of optical fibers for moving the fluorescence emitted from each sample individually to the multi-channel PMT.

In another aspect, the present invention provides a real-time fluorescence retrieval device capable of temperature control characterized in that it further comprises a thermoelectric module unit attached to the side surface of the well chamber block for controlling the temperature of the sample.

Referring to the characteristics of the present invention based on 96 wells of one embodiment of the present invention.

In the present invention, 96 LEDs are used under each 96 well as the light source 1, a microtube or microplate with the sample container 2, a filter with the wavelength selector 3, and 96 strands instead of the reflector 4. The optical fiber of was attached on each well, 16 channel PMT which is the largest channel capacity currently selected with the detector 5, and the focus lens 6 was stuck under each optical fiber.

In addition, in order to express the detected fluorescence by photon counting, two additional 8-channel photon counting boards, which are currently the largest channel capacity, have been added to represent a graph or a number on a computer.

As usual, if 96 LEDs simultaneously emit light and one strand of fiber is connected to one channel of the PMT, six 16-channel PMTs (6 * 16 = 96) and twelve eight-channel photon count boards (12 * 8 = 96), which are expensive and expensive because the PMT and photon count boards cost millions of dollars.

Therefore, in the present invention, in order to separate and designate all 96 wells into one 16-channel PMT, 6 fiber bundles of 6 fibers are bundled in one channel (6 * 16 = 96).

However, when fluorescence is simultaneously obtained from six optical fibers in one channel of the PMT, a problem may occur in that it is impossible to distinguish which fluorescent signal is from which well.

Therefore, in the present invention, the same structure as the channel of the PMT by separating the six LEDs into one channel, each LED is "A", "B", "C", "D", "E", "F" And sequentially emit light six times from the "A" LEDs to the "E" LEDs. That is, if the 16 channels of "A" LEDs emit light, one fluorescent signal is detected from each channel of the 16 channel PMT. Next, if the 16 channels of "B" LEDs emit light, each channel of the 16 channel PMT is detected. Luminescence and detection are performed sequentially, such as by detecting one fluorescence signal, thereby detecting fluorescence from a 96-well sample in six times.

At this time, LED shapes of "A", "B", "C", "D", "E", and "F" have four shapes of 1 * 6, 6 * 1, 2 * 3, and 3 * 2. Although possible, the configurations of 1 * 6 and 6 * 1 have a problem of severe cross talk of light as the LEDs are adjacent to each other in sequential light emission.

Therefore, in the present invention, one channel of the LED is configured in the form of 2 * 3 or 3 * 2.

According to the present invention, a small hole is formed in the bottom of each well of a well chamber block into which a microtube or microplate into which a sample is injected can be inserted, and an LED, which is an excitation light source, is arranged under each well. (Array), LEDs for multiple sample containers are used as one channel, and 16 LEDs are emitted at the same time in the same position of each channel, and six LEDs are emitted sequentially and six times in sequence (SequentialIllumination). (Multi-channel) This method searches fluorescence 6 times sequentially in PMT.

Also. The present invention uses a control board device including the LED and a firmware program for quickly and efficiently controlling temperature / voltage as a computer memory chip, and quickly detects fluorescence within one second instead of tens of seconds on a 96-well basis. Biological samples also provide devices that are simultaneously searchable in seconds.

Here, the number of LEDs in one channel is configured to be equal to the number of fiber optics planted in one channel of the multichannel PMT used as the detector.

In an embodiment of the present invention, six LEDs are configured as one channel, and the entire 96 well is divided into 16 channel units, and then only the first LED of each channel emits light first, and then only the second LED of each channel emits light. Equation 6 provides sequential illumination with only six sequential illuminations within 1 second, providing a device capable of emitting and searching all 16 * 6 = 96 wells.

Hereinafter, the configuration of the present invention in detail with reference to the drawings.

3 and 4 show a front view, a side view, and a plan view of a microplate reader device and a real-time PCR device, respectively, according to the present invention.

In an exemplary embodiment of the present invention, as shown in FIG. 3 or 4, an LED 1b, an Excitation light source, is arranged under each well 3a under the microplate 5a, which is a sample container. The LED board 1a is configured to be controlled by the control board 15. The LED board 1a controlled by the control board 15 simultaneously uses a plurality of LEDs 1b as one channel to simultaneously group the same positions of the respective channels. It is comprised so that light may be emitted.

As shown in the LED array of the LED board according to the present invention in Figure 13, a total of 96 LEDs are composed of 16 channels, 6 in 2 * 3 form as one channel to operate in 96 wells In addition, according to the command of the control board 15, sixteen LEDs 1b from the first to sixth in each channel are sequentially emitted six times.

Referring back to FIG. 3 or FIG. 4, the present invention includes an excitation filter 2b for allowing only the light of the required wavelength band to pass through in the light of the emitted LED 1b. Each LED 1b is inserted into a hole formed for each well 3a in the excitation filter guide 2a, and an excitation filter 2b is positioned on the LED 1b.

The present invention also includes a well chamber block 3b for inserting a microtube or microplate 5a into which a sample is injected. Holes are formed for each of the wells 3a so that light of the wavelength band passing through (2b) can be irradiated. The tube or microplate 5a has a lid 5b for each well 3a to prevent evaporation, bubble formation, and contamination of the sample during the experiment.

4 is a case in which a thermoelectric module 4 is attached to four sides of a well chamber block 3b to add a temperature controller device to implement a real-time PCR device. 7 is a front view, a side view, and a plan view of a well chamber block in which the thermoelectric module device 4 is attached to the well chamber block 3b.

The thermoelectric module device 4 has a plurality of P type and N type Thermoelectric Semiconductor elements 4b between two ceramic plates 4a, and a heat sink (Heat Sink) on the outer ceramic plate 4a. 4c) is attached.

The thermoelectric module device 4 uses a P type and an N type thermoelectric semiconductor elements 4b having both ends of the heat absorbing or dissipating effect by the Peltier effect when a DC current is provided. It serves as a heating device or a cooling device capable of temperature control by 15), and the heat control effect is transmitted to the sample in the microplate 5a via the side surface of the well chamber block 3b.

In another embodiment of the present invention, a cooling fan is attached to the heat sink 4c for faster temperature cooling.

The present invention includes a header 7a located on top of the microplate 5a, as shown in FIGS. 3 and 4.

As can be seen in the header device shown in Fig. 9, the header 7a includes a fiber optics 8 for detecting fluorescence in a sample, and a GRIN (Gradient Index) in the header for each well 3a. It is planted to the top and an epoxy-like adhesive 7b is applied on top so that the fiber optics 8 are fixed.

The header 7a is attached with a thermoelectric module 6a which serves as a heating cover of the microplate 5a when the cover 5b of the microplate 5a comes into close contact with the microplate 5a. Evaporation or condensation of the sample in the microplate 5a does not occur.

In the thermoelectric module 6a for the heating cover, holes 6b are formed for each well 3a so that fluorescence emitted from the sample can be transferred to the optical fiber bundle 8 in the header 7a.

The header 7a helps press the cover 5b of the microplate 5a so that the microplate 5a is inserted as closely as possible into the well chamber block 3b device.

In addition, the present invention automatically moves the well chamber block 3b device into and out of the device by adjusting the control board 15 according to a command in the MuenAnalyzer ^™ computer software, and moves the header 7a to the microplate 5a. In close contact, an autoloading device that allows a user or a robot to experiment by exchanging a plurality of microplates 5a in turn.

Referring to the operation of the autoloading device, first, the well chamber block 3b uses the power of the up-and-down step motor 16a to move outwards, so that the upper holder 16e and the lower holder 16e of the apparatus are respectively headers. (7a) The relevant portion and the well chamber block 3b are moved up and down of the first vertical movement guide 16b and the second vertical movement guide not shown, so that the header 7a and the well chamber block 3b are moved. To separate.

Next, by using the power of the up / down rotation step motor 16c, the well chamber block 3b moves to the outside according to the up / down rotation guide 16d.

10 is a front view of a PMT according to an embodiment of the present invention.

As shown in FIG. 10, the present invention provides a multi-channel micro photomultiplier tube (PMT) as a detector that receives fluorescence emitted from a sample through an optical fiber bundle 8 and then multiplies to a meaningful level. Wherein the PMT is located within a PMT guide 9e.

The invention includes an optical fiber bundle 8 that carries fluorescence emitted from a sample, wherein the optical fiber bundle 8 planted up to the GRIN lens 7c in the header 7a is a well defined as one channel in the LED board 1a. Those in charge of (3a) are bundled together and planted together in the Cathode (9b) position of each channel (9a) in the PMT (Photo Multiplier Tube) glass cover (9c).

Since parts other than Cathode 9b in PMT 9a are parts that cannot accept fluorescence as Anodes, it is important to plant them at the position of Cathode 9b.

There are four rows of Cathode (9b) in one channel (9a) of PMT, and four fibers (8) can be planted in one row of Cathode (9b), so that 16 fibers (8) in one channel (9a). Can be planted.

As an embodiment of the present invention in FIG. 10, six of each is bundled and three of them are two rows, which are precisely planted at the position of Cathode 9b in the PMT glass cover 9c.

As shown in FIG. 3 or 4, the PMT according to the present invention receives fluorescence from the optical fiber bundle 8 through the PMT glass cover 9c on one side and doubles the fluorescence according to the high voltage by the control board 15. After the connection, the photonic counting connection cable 14b connected to the opposite side is connected to the two 16-channel photon counting boards 14a via the connection passage 11. One embodiment of the present invention uses one 16 channel, which is currently the maximum capacity.

In another embodiment of the present invention, as shown in FIGS. 16 and 17, the positions of the LED 1b light source and the PMT 9a detector are changed with respect to the microplate 5a and the optical fiber bundle 8 is replaced with the microplate ( Closely adhere to the sample for each well of 5a) to detect minute fluorescence.

In addition, an embodiment of the present invention includes an automatic filter changer 10f to use fluorescent materials in various wavelength bands.

3 or 4 shows an automatic filter changer 10f, which is a sliding method in which five filters 10a are formed in a band shape, and in FIG. 11, another filter 10a of another embodiment of the present invention. Represents a rotary automatic filter changer 10b that is configured in a circular shape.

As shown in Fig. 11, the automatic filter changer is adapted to a step motor 10c which causes movement about the drive shaft 10d by adjustment of the control board 15 according to a user selection in the MuenAnalyzer ^™ computer software. It is driven by a gyroscope 10e which adjusts the position so that only the filter 10a moves accurately.

In the present invention, as shown in Fig. 3, Fig. 4, or Fig. 11, a thin gap is formed on three surfaces of the PMT guide 9e, and the automatic filter changing devices 10b and 10f are inserted therebetween, and the PMT glass cover is inserted. Close contact with both (9c) and PMT (9a) is made so that the filter can be exchanged precisely without loss of fluorescence.

In addition, in one embodiment of the present invention, as shown in Fig. 3 or 4, one communication board 13, two eights, which are plugged onto a base board 12, A Channel Photon Counting Board 14a, and one Control Board 15 and MuenAnalyzer ^™ computer software installed on the computer.

MuenAnalyzer ^TM computer software allows users to freely and conveniently control parameters such as LED emission time, temperature step and cycle times, high voltage of PMT and desired emission filter type through control board 15. In addition, the results of the sequential photon counts per well / cycle of the entire well 3a input through the photon count board 14a are displayed on a computer monitor in real time as graphs or data, and the analysis tool is also presented.

The communication board 13 is connected to the computer via a cable, allowing the device to send and receive control and results through the MuenAnalyzer ™ computer software.

The photon counting board 14a calculates the fluorescence amplified and input from the PMT 9a by photon counting and delivers the result to the computer in real time through the communication board 3 so that the MuenAnalyzer ^TM computer software sends the result Make it visible on the monitor.

The control board 15 includes a firmware program as a computer memory chip that actually controls related hardware according to various variables designated by a user through MuenAnalyzer ^TM computer software, and includes an LED board 1a and a well chamber block ( 3b) thermoelectric module device (4), thermoelectric module (6a) for heating cover, PMT (9a), step motor (10c) for automatic filter changing device, vertical motion step motor for automatic loading device, up / down for automatic loading device Connected to the rotary motion step motor, the light emitting time of the LED 1b, the temperature of the thermoelectric module devices 3b and 6a, the high voltage of the PMT 9a, the type of the filter 10a, and the up / down movement of the well chamber block 3b. , Variables of the photon count board 14a, and the like are controlled.

In another embodiment of the present invention, the photon counting function is integrated into the control board 15 without designing a commercially available photon counting board 14a, thereby designing a single board. Minimize the size of the device to make it simpler and more portable.

In another embodiment of the present invention, the PMT 9a portion, the base board 12, the communication board 13, the photon count board 14a, and the control board 15 are separated into separate devices, and then placed in another device. The optical fiber bundle 8 is connected to an optical switch, and the two devices are connected by an optical cable capable of long distance communication, so that the device having the well chamber block 3b can be controlled at a far distance.

Hereinafter, the operation of the real-time fluorescence retrieval apparatus capable of temperature control according to the present invention will be described.

The automatic loading device of the invention automatically controls the well chamber by the control of the control board 15 according to a command of the MuenAnalyzer ^TM computer software which the user designates to load the microplate 5a into which the sample is injected. The block 3b is moved outward.

When a command is issued by the MuenAnalyzer ^™ computer software, the up / down stepping motor 16c is driven by the control of the control board 15 so that the upper layer holder 16e of the device moves up and down the header 7a related part. 16b) up to the bottom and the lower support 16e raises the well chamber block 3b related part only up to the inlet position along the up-and-down motion guide 16b.

Next, the up / down stepping motor 16c is driven to rotate the well chamber block 3b according to the up / down / rotating motion guide 16d, and moves to the outside through the inlet.

When the user places the microplate 5a into which the sample is injected, on the well chamber block 3b and instructs automatic loading in the MuenAnalyzer ^™ computer software, the well chamber block (in accordance with the operation of the stepper motor 16c for up / down rotation) 3b) moves inward and the microplate 5a is pushed to the maximum as the relevant portion of the header 7a moves downward in accordance with the drive of the stepper motor 16c for vertical movement.

The LED 1b, which is an excitation light source of the present invention, is arranged below each position of the well 3a, and the LED board 1 is formed to bundle the plurality of channels into one channel so that the LEDs 1b at the same position in each channel are operated together. 1a) is designed. However, the problem of interference of light is solved by determining the shape of the LED of one channel so that the LEDs emitting together are not adjacent to each other.

The emission time of the LED 1b can be freely set by the user in the MuenAnalyzer ^™ computer software and controlled via the control board 15.

As shown in FIG. 13, in one embodiment of the present invention, six LEDs are formed in a 2 * 3 channel so that there is no interference problem of light, and each channel is positioned below 96 wells. In another embodiment of the present invention, Six are configured in the form of 3 * 2, and one channel is freely configured according to the number of wells 3a and the capacity of the PMT 9a within a maximum of 16.

Excitation filter (2b) of the present invention is inserted into the upper portion of the hole formed for each well (3a) in the excitation filter guide (2a) of the horizontal / vertical size of the well chamber block (3b) Under the hole, the LED 1b is inserted so as to pass only a desired wavelength band from the light of the emitted LED 1b.

The excitation filter guide 2a in close contact with the LED board 1a can be detached together, so that the user can easily replace the filter with a desired wavelength band.

As shown in FIG. 3, FIG. 4, or FIG. 6, an embodiment of the present invention uses an excitation filter guide 2a which is only a 96-well well chamber block 3b.

The well chamber block 3b into which the microtube 5a of the sample of the present invention is injected or the microplate 5a of the multi-tub structure can be inserted is closely attached to the excitation filter guide 2a and the LED 1b. The light passing through the excitation filter (2b) of the light is transmitted to the sample well without being dispersed.

In an embodiment of the present invention, the well chamber block 3b is capable of inserting a 96 well microplate 5a, and has a structure in which the bottom surface is grooved in a well shape.

In another embodiment of the present invention, a well chamber block having a structure filled with a flat bottom surface may be provided for rapid thermal change regardless of well capacity.

The thermoelectric module device 4 of the present invention attaches as closely as possible to the four sides of the well chamber block 3b for temperature control required by real-time PCR.

When a DC current is provided to a plurality of P type and N type thermoelectric semiconductor elements 4b between the two ceramic plates 4a through the control board 15, the well chamber block 3b according to the flow thereof. By heating or cooling the ceramic plate 4a attached to), it serves as a heating device or cooling device of the well chamber block 3b.

4 and 7, in one embodiment of the present invention, a heat sink 4c is attached to a ceramic plate on four surfaces of a well chamber block 3b of a 96 well.

In another embodiment of the present invention, a cooling fan is attached to each of the heat sinks 4c to increase the size of the device but for a faster cooling rate.

FIG. 7 shows a front view, a side view, and a plan view of a 96-well well chamber block 3b in which a thermoelectric module device 4 is in close contact with four sides for a real-time PCR device.

As can be seen in FIG. 9, which shows a header according to an embodiment of the present invention, the header 7a is as close to the microplate 5a as possible, and is irradiated with the LED light passing through the filter 2b. Detect fluorescence from each sample. Under the header 7a, heating is performed to maintain a constant high temperature in the lid 5b of the microplate 5a so that evaporation or condensation of the sample in the microplate 5a does not occur even during temperature control. A cover is attached and holes 6b are formed for each well 3a to move fluorescence. The temperature of the heating cover is controlled to be constant by the control board 15.

In the header 7a, the optical fiber bundle 8 is planted to the bottom of the header 7a for each well 3a so that the fluorescence can move, and the adhesive 7b is attached to the optical fiber bundle to fix the header 7a in the header 7a. 8) It is filled around.

In an embodiment of the present invention, the thermoelectric module 6a is used as a heating cover.

In another embodiment of the present invention, while the heating coils are evenly arranged, the upper surface uses a thin heating plate insulated with asbestos or the like.

The optical fiber bundle 8 of the present invention detects fluorescence in each well 3a at the bottom of the header 7a and moves to the multi-channel micro PMT 9a, which is a detector, in the LED 1b. By definition, the optical fiber bundles 8 in each channel are bundled and planted in the channels 9a in the glass 9c of the multichannel PMT, respectively.

When planting for each channel, the optical fiber bundle 8 should be planted only at the portion of the cathode 9b capable of receiving fluorescence, and the rest is an anode, which does not receive light. Up to 16 fiber bundles 8 are planted in one channel of the PMT.

As shown in FIG. 3, FIG. 4, or FIG. 9, in an embodiment of the present invention, six wells 3a are divided into 16 channels in a 96-well microplate 5a and are divided into sixteen channels. Of the optical fiber bundles 8 are planted at a position 9d in the cathode 9b of each channel 9a of the 16-channel PMT.

In another embodiment of the present invention, a bundle of optical fibers 8 may be bundled and planted in each channel 9a of the PMT so as to cover the entire well within 16 channels regardless of the well capacity.

The multi-channel micro PMT 9a of the present invention is a detector. The photon counting board 14a is multiplyed to a meaningful level by the fluorescence of each well 3a received through the optical fiber bundle 8. The high voltage is controlled by the control board 15 according to the instructions of the MuenAnalyzer ^™ computer software.

One channel 9a of the PMT can receive only one fluorescence. As shown in FIG. 9, after dividing 96 wells into 16 channels in one embodiment of the present invention, the bundles of optical fibers 8 are bundled by six channels for each channel. In each channel 9a of the 16-channel PMT, the LEDs of the same position in each of the 16 channels sequentially emit 6 times (Sequential Illuminating), so in the first LED light emission from the sample of the first well 3a in each channel. Fluorescence is detected sequentially, six times, including channel-specific detection in 16 channels of the PMT, detecting the entire 96 wells within one second.

Photon counting board (14a) of the invention by counting the fluorescence detected in the PMT (9a) into photons (Phonton) MuenAnalyzer ^TM juneunde deliver to the computer software, MuenAnalyzer ^TM variable is passed to the dictionary of the data acquisition time by computer software do.

In one embodiment of the present invention, the fluorescence doubled in the 16-channel PMT is photon counted using two 8-channel photon count boards 15.

MuenAnalyzer ^TM computer software of the present invention allows the user to set the emission time of LEDs, the temperature and repetition times of the steps, the high voltage of the PMT, the desired emission filter type, and the like. 15) The experiment is carried out while controlling, and the result is received from the photon coefficient board 14a and converted into graphs or data and displayed on a monitor for each well / cycle. It also provides a variety of tools to analyze the results.

FIG. 12 is a front view of the microplate 5a, the well chamber block 3b, the excitation filter guide 2a, and the LED board 1a, before being combined with each other. An excitation filter 2b is fitted in the upper hole of the excitation filter guide 2a.

To combine, first, the LED (1b) on the bottom LED board (1a) is inserted into each hole of the excitation filter guide (2a) to contact the excitation filter (2b) And the well chamber block 3b thereon.

Finally, the microplate 5a containing the sample and the reagent for each well is inserted into the well chamber block 3b. The light from the LED passes through the filter and reaches the sample.

FIG. 13 is a plan view of an LED board, in which an exemplary embodiment of the present invention is a case in which a total of 96 wells are configured as 16 channels by configuring 6 channels each in a 3 * 2 form.

Sequential emission means that only the first LEDs (16 total) of each channel emits light first, and then the second LEDs (16 total) emits light in total. Say that.

In addition, the present invention, by placing a separate optical fiber bundle 8 for each well (3a) and bundled in a multi-channel PMT (9a) which is a detector (Detector) by moving and detecting the fluorescence without loss, DNA In the case of RNA, the present invention provides a method and apparatus having a sensitivity capable of retrieving a sample concentration of 10 copies.

14 shows a front view of the PMT 9a and a heating cover thermoelectric module device 6a and a header on a 96-well microplate 5a so that the optical fiber bundle 8 in the header 7a is positioned above each well 3a. It is a front view of the state which connects the header 7a and the PMT 9a which is a detector, acting as a fluorescent device.

FIG. 15 is a front view of the PMT 9a and a plan view of the header 7a showing how the optical fiber bundles 8 are connected in one channel.

FIG. 16 is a front view of the LED board 1a, the excitation filter guide 2a, the heating cover thermoelectric module device 6a, and the microplate 5a as another embodiment of the present invention, before being combined with each other.

Hereinafter, a description of another embodiment of the present invention shown in FIG. 16 and FIG. 17 will be described with reference to FIG. 12 and FIG. 14 described above in detail. The description will be made on the basis of the description, and description of common parts in the present invention will be omitted.

Referring to FIG. 16, the LED light source emits light from above instead of below to reach the sample through the lid 5b of the microplate 5a, and as shown in FIG. 17 to be described later, the header 7a for detecting fluorescence. ) And PMT 9a are positioned under the microplate 5a.

The coupling method is that the LED 1b below the LED board 1a is inserted into each hole of the excitation filter guide 2a, and the thermoelectric module device 6a for the heating cover is attached below the excitation filter guide 2a. Each lid 5b of the microplate 5a is inserted into each well hole 6b.

FIG. 17 shows another embodiment of the present invention in which the header 7a and the PMT 9a are located below the microplate 5a, rather than above it, and show a state before joining.

At this time, the LED which is a light source is located on the microplate 5a, as shown in FIG.

The combining method is that the microplate 5a is inserted above the well chamber block 3b, the header 7a is inserted below the well chamber block 3b, and the optical fiber bundle 8 is separated for each channel of the PMT 9a. It is planted by In the coupling method, since the optical fiber is in close contact with the lower part of the sample, minute fluorescence can be detected more sensitively without further loss.

As shown in FIG. 16 or FIG. 17, the LED 1b is inserted into the hole 2c of the excitation filter guide 2a located at the lower portion, and below the excitation filter guide 2a. The microplate 5a is located, and the microplate 5a is inserted into each well 3a of the well chamber block 3b located below, and the optical fiber bundle 8 is located below the well chamber block 3b. Header portion 7a is fixed.

In addition, the embodiment of the present invention, the excitation filter guide (excitation filter guide; 2a) by pressing the microplate cover (5b) so that the microplate (5a) in close contact with the well chamber block (3b), And a thermoelectric module 6a for a heating cover to serve as a heating cover of the microplate 5a when the cover 5b of the microplate is in close contact.

In addition, the embodiment of the present invention provides a step motor 10c for separating the excitation filter guide 2a and the well chamber block 3b so that the microplates 5a may be exchanged and tested. Include.

In addition, the embodiment of the present invention is characterized in that the lower part of each well of the wall chamber block is opened so that the fluorescence emitted from the sample of the microplate is transmitted to each optical fiber 8 connected to the header portion 7a. It is done.

One optical fiber is connected to one channel of the PMT of a conventional fluorescent device, because when one channel receives light from several optical fibers at the same time, light interference occurs and cannot be distinguished.

However, in one embodiment of the present invention, since the LED emits light six times sequentially while emitting only the first ones (16 total) of each channel, fluorescence is emitted only from one optical fiber per channel from the PMT point of view. Fluorescence can be searched for samples across 96 wells with only one sequential emission / search.

That is, if only the first LEDs of each channel (16) emit light, fluorescence is generated only in the first sample of each channel, and simultaneously detected in 16 channels of PMT. Fluorescence occurs only in the second sample of and is detected simultaneously on 16 channels of the PMT. When the detected fluorescence is sequentially photon counted by the photon counting board 14a and handed over to the computer through the communication board 13, the computer program displays a graph for each well 3a.

In addition, the present invention uses a multi-channel micro PMT (8) as a detector (Detector), but not simply connecting one well (3a) to one channel (9a), the sample vessel in each channel The entire fiber bundle (8) above (3a) is precisely planted in four lines of cathodes (9b) per channel, the light-sensing part of the PMT, tied up to 16 channels in each channel 9a of the PMT. By adopting the planting method, the entire well 3a can be searched by sequential light emission by the number of LEDs 1b (= number of optical fibers) in one channel and sequential search by the PMT 9a. Depending on the number of channels and the array, the capacity of hundreds of thousands of well plates 5a can be established at a fast and economical price.

In the case of 16-channel PMT, fluorescence can be detected only in 16 wells. However, in the present invention, as described above, since 6 optical fibers are planted in one channel, sequential light emission / retrieval enables economic system construction.

Each channel of PMT has 4 rows of Cathode that can receive light, and up to 4 fibers can be planted in one row of Cathode in one channel.

Since the rest of the cathode cannot accept light as an anode, when several optical fibers are planted in one channel, as in the present invention, only a small amount of thinner fiber than the thickness of the cathode should be accurately planted in the cathode portion, resulting in slight fluorescence in the sample. Can be detected precisely.

In one embodiment of the present invention, each channel of the 16-channel microPMT is composed of 16 channels with six LEDs 1b as one channel in a 98 well, and bundles six optical fiber bundles 8 by channel. Plant in (9a).

In this case, each fluorescence is transmitted and searched for each channel 8 of the PMT while emitting only the first LEDs 1b of each channel, and then for each channel 9a of the PMT while emitting only the second LEDs 1b of each channel. Each fluorescence is delivered and searched in order, thereby providing a device capable of searching the 96 wells 3a within one second by sequential light emission and retrieval only six times.

In another embodiment of the present invention, one 16-channel PMT is used as a detector, and one LED is used as one channel in 16 wells, and two LEDs are used as one channel in 32 wells. 64Well provides a device for searching the entire well by configuring 4 LEDs in one channel and sequentially emitting / searching 1/2, 4/4.

According to another embodiment of the present invention, sixteen LEDs are used as one channel and 96 LED boards of 16 channels are moved four times with a step motor to sequentially emit light in 384 wells, and two 16-channel PMT detectors are used. Searching sequentially provides an apparatus that can search 384 wells in less than four seconds at an economical price.

In addition, 384 LED boards with 12 LEDs as one channel are used, and 16-channel PMT can be searched for 384 wells within 3 seconds by using 16 detectors with two detectors. Provide the device.

In addition, the present invention provides a space between the PMT glass (9c) and the PMT (9a) in which the optical fiber bundle (8) is planted in the PMT guide (9e) to form a close contact, and the various emission filters configured in the form of bands or circles. Insert the (10a) and move it in a sliding or rotating manner using a step motor 10c that moves according to the selection in the MuenAnalyzer ^™ computer software and a gyroscope 10e that controls the position. By auto-exchanging, it implements a multiplex function to quickly search for fluorescence of various wavelengths, but there is no loss of fluorescence so that sensitivity is not reduced.

In one embodiment of the present invention was configured using six radiation filters (10a, 10f).

In addition, the present invention develops a sequential illumination method by arranging the LED 1b, which does not require a focal length of the lens, to a light source for each sample, and uses the optical fiber bundle 8 for the shortest distance. The present invention provides a method and apparatus for making a portable device by developing a method of shifting fluorescent light.

In another embodiment of the present invention, the photon counting function is integrated into the control board 15 without designing a commercially available photon counting board 14a, thereby designing a single board. Minimize the size of the device to make it simpler and more portable.

Lenses require a fixed focal length and complex devices to move the fluorescence to the desired location, but since the optical fiber flexes flexibly with the shortest distance, it can be easily moved within the device to the desired location. have.

In addition, the present invention freely and conveniently controls not only variables such as LED 1b Excitation time / temperature / high voltage, but also the parameters of hardware components of the device, and the LED light source 1b and the optical fiber bundle 8 and multiplexing. Whole wells (3a) using MuenAnalyzer ^™ computer software, which can display the results of sequential photon counting on a computer monitor using a channel PMT (8) and a Photon Counting Board (14a), etc. ) Can be monitored and analyzed in real time by well / cycle.

In another embodiment of the present invention, the PMT 9a portion, the base board 12, the communication board 13, the photon count board 14a, and the control board 15 are separated into separate devices, and then placed in another device. The optical fiber bundle 8 is connected to an optical switch, and the two devices are connected by an optical cable capable of long distance communication, so that the device having the well chamber block 3b can be controlled at a far distance.

The present invention monitors the intensity, lifetime, and polarization of fluorescence simultaneously in real time on a large number of samples injected into a large number of wells 3a (1). ) Use for fluorescence enzyme assay, cell assay, or nucleic acid assay, or (2) drug screening using fluorescence, that is, cell-centered drug selection (cell) -based drug screening, (3) diagnostics using fluorescence, (4) nucleic acid sequences using fluorescence, or (5) polymerase chain reaction (PCR) or reverse transcription PCR. Experiments can be used to perform fluorescence analysis in real time (cycle-by-cycle).

The invention (1) "microplate search apparatus" for searching for fluorescence emitted from the sample after irradiating light to the sample; (2) A device comprising a "real-time PCR" device capable of performing nucleic acid (DNA or RNA) amplification experiments through repeated temperature changes by adding a temperature controller to the device, in particular to Applicable to the hourly search of sensitive biological reactions.

In addition, the real-time PCR device according to the present invention can also perform a biological experiment requiring a variety of temperature changes by controlling the temperature of the existing very limited thermal cycling control by various user settings through the control board. have.

The present invention is not limited to the above-described embodiments, and modifications may be made by those skilled in the art without departing from the spirit of the present invention. Therefore, the scope of claims in the present invention will not be defined within the scope of the detailed description, but rather by the claims below.

As described above, according to the present invention, there is an advantage that the number of PMTs and the cost of the device can be minimized through the complex use of bundled optical fiber bundles that are separately assigned to each channel of the LED and the multi-channel microPMT. .

In addition, the present invention is to search sequentially in the multi-channel PMT (9a), which is a detector (Detector) while emitting light simultaneously and sequentially in groups of the same position of each channel using a plurality of LED (1b) as a channel, 96-based reference Fast fluorescence searches in less than one second enable multiple biological samples to be searched simultaneously in seconds.

In addition, the present invention includes an automatic filter exchange device for forming thin gaps on the three sides of the PMT guide 9e and using fluorescent materials in various wavelengths therebetween, while replacing the filter 10a at high speed without loss of fluorescence. Make it searchable sensitively.

In addition, the present invention uses the multi-channel micro PMT 9a as a detector while the entire optical fiber bundle 8 above the well 3a in each channel is precisely applied to the cathode 9b of each channel 9a of the PMT. Through the bundling method, the entire sample container can be quickly and economically priced by sequential emission of the number of LEDs 1b (= number of optical fibers) and sequential search by the PMT 9a for only a few seconds. Make it searchable.

Claims (36)

A device for analyzing a sample by searching for a fluorescence emitted from the sample after irradiating a light source to a biological sample, comprising: a sample container, a light source positioned to irradiate the sample container, a detector for detecting the fluorescence emitted from the sample, in the sample A fluorescence retrieval apparatus including a fluorescence shifting device, a wavelength selection device, and a control unit for moving the emitted fluorescence to the detector, An LED array in which a plurality of LEDs are sequentially emitted; A well chamber block having a plurality of wells for inserting a sample container; A multi-channel PMT for detecting fluorescence emitted from the sample by each LED emission of the LED array; And And a plurality of optical fibers for individually moving the fluorescence emitted from each sample to the multi-channel PMT. The method of claim 1, And a photon count board for digitizing the fluorescence amplified by the multi-channel PMT with photons and displaying the number or graph in a number or graph. The method of claim 1, And a thermoelectric module attached to an inclined side surface of the well chamber block to control the temperature of the sample. The method of claim 3, wherein The thermoelectric module includes two ceramic plates and a heat sink, one of the ceramic plates is connected to the well chamber block, and the other of the ceramic plates is connected to a heat sink. Search device. The method of claim 4, wherein The thermoelectric module further includes a plurality of P type and N type thermoelectric semiconductor elements connecting between the two ceramic plates. The method of claim 3, wherein And the thermoelectric module further attaches a cooling fan to the heat sink. The method of claim 3, wherein The lower structure of the well chamber block is a real-time fluorescence detection device with temperature control, characterized in that the lower surface is filled with parallel structure for rapid thermal change. The method of claim 1, And the plurality of optical fibers are separated and bundled into a plurality of channels of each of the multi-channel PMTs. The method of claim 1, The LED array is a plurality of channels, wherein each channel is configured to include a plurality of phased LEDs, the temperature control characterized in that each of the LEDs of the same phase belonging to each channel is controlled to emit light sequentially at the same time Real-time fluorescence detection device available. The method of claim 9, And a control board for controlling sequential emission and emission time of the plurality of LEDs. The method of claim 1, When the LEDs corresponding to the same phase of each channel among the LED arrays emit light at the same time, the LEDs of the same phase belonging to each channel are arranged to be spaced apart from each other to prevent interference between LEDs belonging to adjacent channels. A real-time fluorescence detection device capable of temperature control. The method of claim 11, When the LED array is divided into 16 channels as 96 LED arrays, six LEDs belonging to each channel are arranged in 2 * 3 matrixes or 3 * 2 matrixes. Device. The method of claim 1, And an excitation filter guide having a hole into which each LED of the LED array is inserted. The method of claim 13, It is disposed to contact the LED inserted into each hole of the excitation filter guide (excitation filter guide), further comprising an excitation filter (excitation filter) for passing only the light of the required wavelength band of the emitted LED wavelength Real-time fluorescence detection device capable of temperature control, characterized in that. The method of claim 1, And the sample vessel is a microtube. The method of claim 1, And said sample container is a microplate having a plurality of sample receiving portions. The method of claim 16, The microplate further includes a sample cover for preventing contamination and evaporation of each injected sample and removing bubble formation. The method of claim 1, And a header portion to which one end of the optical fiber for detecting fluorescence from the sample is bonded and fixed. The method of claim 18, And the other end of the optical fiber in which one end is fixed to the header part is connected in a cathode region of each channel of the multi-channel PMT. The method according to any one of claims 1 to 19, A measurement unit including the LED array, the excitation filter, the well chamber block, the header part, and an optical fiber bundle; Separating the control unit including the radiation filter, the PMT, the photon counting board, the control board, and the communication board into separate devices on a distance; And a fiber bundle in the separated device is connected to an optical switch. The method of claim 20, Communication between the two separated devices is a real-time fluorescence detection device capable of temperature control, characterized in that consisting of an optical cable capable of long-distance communication. The method according to any one of claims 1 to 19, The LED array is inserted into a hole of an excitation filter guide located at an upper portion, a wall chamber block is positioned at an upper portion of the excitation filter guide, and the microplate is disposed at an upper portion of the well chamber block. A real-time fluorescence retrieval apparatus capable of temperature control, which is inserted into each well of a chamber block and has a structure in which a header portion for fixing an optical fiber bundle is coupled to the microplate. The method of claim 22, The header portion is pressed by the microplate cover to allow the microplate to be in close contact with the well chamber block, and further comprises a thermoelectric module for a heating cover to serve as a heating cover of the microplate when the cover of the microplate is closely attached A real-time fluorescence detection device capable of temperature control. The method of claim 22, And a step motor for separating the header portion and the well chamber block so as to test the plurality of microplates in turn. The method of claim 22, A lower part of each well of the wall chamber block is opened such that a light source of each LED passing through the excitation filter of the excitation filter guide is irradiated to a microplate inserted into each well of the well chamber block. Real-time fluorescence detection device capable of temperature control. The method according to any one of claims 1 to 19, The LED array is inserted into the hole of the excitation filter guide located at the bottom, a microplate is located below the excitation filter guide, the microplate is inserted into each well of the well chamber block located at the bottom, And a header portion to which the optical fiber bundle is fixed is coupled to a lower portion of the well chamber block. The method of claim 26, The excitation filter guide (excitation filter guide) to press the microplate cover to ensure that the microplate is in close contact with the well chamber block, the heating cover for serving as a heating cover of the microplate when the cover of the microplate is in close The temperature control real-time fluorescence detection device further comprises a thermoelectric module. The method of claim 26, And a step motor for separating the excitation filter guide and the well chamber block so as to test a plurality of microplates in turn. The method of claim 26, And the lower part of each well of the wall chamber block is opened so that the fluorescence emitted from the sample of the microplate is transferred to each optical fiber connected to the header part. The method of claim 1, And a plurality of optical fiber bundles are separated and connected to a plurality of channels of the multi-channel PMT through PMT guides, and the multi-channel PMTs are located in the PMT guides. The method of claim 30, And the multi-channel PMT receives fluorescence from the optical fiber bundle that is in close contact with the PMT glass cover. The method of claim 30, Further comprising an automatic filter changer for automatically replacing the radiation filter for using a fluorescent material of various wavelengths, the automatic filter changer is characterized in that the thin gap formed on the three sides of the PMT guide and disposed therebetween Real-time fluorescence detection device capable of temperature control. The method of claim 32, And the automatic filter changer is in close contact with both the PMT glass cover and the multi-channel PMT such that there is no loss of the received fluorescence. The method of claim 32, The automatic filter change device is a step motor for generating a movement around the drive shaft, and A temperature controlled real-time fluorescence detection device comprising a gyroscope for adjusting the position so that only one filter moves accurately. The method according to claim 2 or 10, The control board is a real-time fluorescence retrieval device capable of temperature control, characterized in that performed by integrating the function of the photon coefficient board The method according to claim 1 or 10, The control board is a real-time fluorescence detection device capable of temperature control, characterized in that the firmware program for controlling the hardware in accordance with a user-designated variable is built into the computer chip.