TWI498562B - Apparatus and method for detecting biochemical reaction - Google Patents

Description

Biochemical reaction detecting device and method thereof

The present invention relates to a detection technique for nucleic acid amplification reaction, and more particularly to a detection device for polymerase chain reaction (PCR), which uses a light source and a light guide plate as a fluorescent reaction substance. The excitation source illuminates the light evenly, and the fluorescence performance of the polymerase chain reaction is improved.

When performing genetic research such as genetics or molecular biology or detection of animal and plant diseases, a small amount of nucleic acid samples should be replicated in a short time by means of amplifying nucleic acids (eg, polymerase chain reaction, PCR). Increase to the amount that can be detected. The nucleic acid amplification product may further bind a target nucleic acid fragment to a nucleic acid probe having a fluorescent, radioactive or coloring enzyme via hybridization to generate a fluorescent, radiographic or color reaction. . Fluorescent dyes are currently used as markers on many biochips, mainly because fluorescent dyes provide better analytical results. Fluorescent dyes can provide up to about 1000 compared to conventional color formers. Up to 500,000 times sensitivity.

The fluorescent reaction of the above fluorescent dye can be observed, detected and analyzed by using a fluorescence microscope (Fluorescence Microscopy), a fluorescence analyzer, a flow cytometer or an image capturing device to capture fluorescent images; The technology involves the selection of an excitation light source. A specific fluorescent dye needs to use light of a specific wavelength range as an excitation light source. When a light of a suitable wavelength illuminates a molecule having a fluorescent property, the molecule absorbs the energy of the light and is excited. In the high energy state, and returning to the low energy state in a very short time, and releasing the excess energy in the form of light emission, it can be seen that the excitation light source is the key to affect the excitation effect of the fluorescent dye.

The detection method of the fluorescent reaction of the fluorescent dye is to place the test tube between the excitation light source and the image capturing device, wherein the excitation light source mostly adopts an LED lamp, and an excitation filter is arranged between the excitation light source and the test tube. The excitation light source is used to filter the incident light source of a specific wavelength to stimulate the fluorescent dye in the test tube to generate a fluorescent reaction, and the image of the fluorescent reaction signal is captured by the image capturing device for detection and analysis; When the excitation light source is projected on the excitation filter, the illumination angle of the LED lamp itself and the angle of the reflected light after the excitation filter are generated, so that the excitation light source will inevitably have uneven illumination, so that the fluorescent dye generation cannot be ensured. The best stimulating effect.

On the other hand, when performing the above-mentioned detection operation, the detection and analysis are performed on the plurality of test tubes at the same time, and in order for the image capture device to simultaneously capture the fluorescent reaction in each of the test tubes, the image capture device and the test tube must have a certain relationship. The distance can be used to capture all the tubes, so the volume of the whole device can not be effectively reduced; although the above problem can be achieved by setting a lens between the image capturing device and the test tube to focus the fluorescent reaction signal by refraction In order to shorten the distance required between the image capturing device and the test tube, but the refractive angle is not easy, and the fluorescent signal is likely to be attenuated after the refraction.

In general, the disadvantages of the prior art are generally included in the above-mentioned detection method of the fluorescent reaction of the conventional fluorescent dye, which hinders the illumination angle of the LED lamp itself and the angle of the reflected light after the excitation filter, so that the excitation light source will inevitably have In the case of uneven illuminance, in order to enable the image capturing device to simultaneously capture the fluorescent reaction in the plurality of test tubes, the image capturing device must have a certain distance from the test tube, so the volume of the overall device cannot be effectively reduced. However, if a lens is disposed between the image capturing device and the test tube, the refractive angle is not easy, and the fluorescent signal is likely to be attenuated after the refraction; and in view of solving the above disadvantages, the present invention provides a biochemical reaction detecting device. And its method.

The invention relates to a biochemical reaction detecting device, comprising: a positioning seat having at least one accommodating space for receiving a test tube, the test tube being filled with at least one biochemical reaction buffer and at least one fluorescent reaction substance The positioning seat is provided with at least one observation hole for observing the accommodating space on one side, and an excitation light source module is disposed on one side of the positioning seat to correspond to the end of the test tube, the excitation light source The module includes at least one light source that can project light toward the accommodating space, and a light guide plate is disposed between the light source and the accommodating space, and the light guide plate can uniformly diffuse the light projected by the light source into the accommodating space for exciting The fluorescent reaction in the test tube and the reaction of the buffer can be detected from the observation well.

The above detection method comprises the steps of: a) filling at least one biochemical reaction buffer in a test tube; b) contacting and heating the near end of the test tube with a heat source to make the buffer at both ends of the test tube The temperature forms a temperature difference, and a thermal convection phenomenon occurs, and the DNA in the buffer can perform a PCR proliferative reaction in a cycle of heating and cooling; c) using at least one light source to project light onto a light guide plate to borrow light from the light guide plate. Producing a homogenizing effect, and uniformly projecting into the test tube to excite the fluorescent reaction substance in the test tube to generate a fluorescent reaction signal; d) detecting the fluorescent reaction signal image of the buffer through a light sensor and transmitting through the optical fiber Transfer to an image capture device to capture its image.

One of the objectives of the present invention is to enable the light guide plate to guide the light direction through the excitation light source module composed of the plurality of light sources and the light guide plate, and uniformly diffuse the reflected light toward the accommodating space for exciting the fluorescent light in the test tube. Reaction, thereby effectively improving the homogenization of the excitation light source and avoiding generation The illuminance distribution is uneven, so as to effectively improve the fluorescence performance of the polymerase chain reaction.

The second object of the present invention is that a display device can be configured to transmit a specific wavelength of the fluorescent reaction signal image to the image capturing device through the optical fiber for image capturing, thereby greatly reducing the image capturing device and the test tube. The distance between the two can further reduce the volume of the overall structure, so as to improve the practicality and convenience of use.

The third object of the present invention is that the combination of the plurality of light sources and the light guide plate enables the light source to pass through other light sources so as not to affect the generation of the excitation light source even if one of the light sources fails to function.

The fourth object of the present invention is that the excitation light source module can have a plurality of light sources capable of generating different wavelengths, and is disposed on the side of the light guide plate to be projected on the light guide plate through the side light entrance mode, thereby exciting When the light source is used, the light source of the corresponding wavelength can be selected to be irradiated according to the light source of the specific wavelength required.

For the convenience of the description, the central idea expressed by the present invention in the column of the above summary of the invention is expressed by the specific embodiments. Various items in the embodiments are depicted in terms of ratios, dimensions, amounts of deformation, or displacements that are suitable for illustration, and are not drawn to the proportions of actual elements, as set forth above. In the following description, like elements are denoted by the same reference numerals.

As shown in the first to third figures, the present invention is a biochemical reaction detecting device, which mainly includes a positioning base 10, a heating device 20, an excitation light source module 30, and a display module 40.

The positioning base 10 has a plurality of accommodating spaces 11 arranged in a row for accommodating a negative tube 50 for performing nucleic acid extraction, and the inside thereof is filled with at least one polymerase chain reaction buffer and at least one firefly. Photoreactive material, the buffer and the fluorescent reaction substance can be quantitatively analyzed by various methods The method may be different, for example, DNA dye (DNA Intercalate, which may be ErBr, SyBr Green, etc.), turbidity reaction (Turbidity, which may be magnesium Pyrophosphate), fluorescent staining method ( Fluorescent dye, which may be FAM, Cy3, Cy5), visible light detection or coloring reagent, etc.; and to provide convenience for the test tube 50, the positioning seat 10 is provided with a detachable carrier 12, When the plurality of test tubes 50 are carried, the test tubes 50 are respectively accommodated in the accommodating spaces 11 when the detachable carrier 12 is mounted on the positioning base 10; The observation holes 13 of the respective accommodation spaces 11 are observed.

The heating device 20 is disposed on one side of the positioning base 10, and the heating device 20 can make the buffer in the test tube 50 exhibit the required temperature of the nucleic acid constant temperature amplification reaction; the heating device 20 includes a heating seat 21 and a heat conduction. The plate 22 and a driving device 23 are disposed in the heating base 21 and provide a heat conduction effect by a heat source. The heating seat 21 is provided with a receiving passage 211 and a heating passage 212. The accommodating passage 211 is permeable to the end of the test tube 50, and the heating passage 212 is configured to traverse the heat conducting plate 22 to contact the proximal end of the test tube 50. The driving device 23 can be used to control the heat conducting plate 22 to contact or separate from the test tube. 50; However, the heating device 20 can be a heating device used in the conventional biochemical reaction, and is not limited to the type shown in the legend, and can be used as the heating device 20 only for the same purpose of heating the test tube 50. .

The excitation light source module 30 is disposed in a fixing base 60. The fixing base 60 is fixed on a base 70 so as to be located on one side of the heating base 21. The excitation light source module 30 includes a plurality of The illuminating space 11 is configured to illuminate the light source 31 to correspond to the end of each of the adjacent tubes 50, and the light sources 31 can generate different wavelengths. A light guide plate 32 is disposed between the light source 31 and the accommodating space 11. The material of the light plate 32 is not particularly limited, and may be, for example, plastic, acrylic, polystyrene (PS), etc.; each light source 31 may be a light emitting diode, a laser, a halogen lamp, a xenon lamp or a xenon gas. Lights, and In order to cause the light source 31 to project light of a specific wavelength, a light filter plate 33 is disposed between the light source 31 and the accommodating space 11 for light of a specific wavelength, wherein the filter plate 33 can be disposed on the light source 31 and the light guide plate. Between 32, it can also be disposed between the light guide plate 32 and the accommodating space 11.

The display device 40 can be a photo sensor such as a photosensitive coupling element (CCD), a complementary metal oxide semiconductor (CMOS), a photodiode, a phototransistor, a photomultiplier tube (PMT), a photoresistor, and a spectrophotometer. Or a spectrometer; for example, the filter 41 can be electrically connected to a fiber connector 411, and the fiber connector 411 is connected to the observation hole 13; the filter 41 can be connected to the observation hole 13; The housing 42 is disposed in a housing 42. The housing 42 can be provided with an image capturing device (not shown). The image capturing device can be coupled to a display.

After the above structure is clarified, the following is a detailed description of the operation and principle of the present invention: as shown in the fourth and fifth figures, the test tubes 50 filled with the biochemical reaction buffer and the fluorescent reaction material are placed. The detachable carrier 12 is mounted on the locating base 10 for the nucleic acid amplifying operation. At this time, the heat conducting plate 22 is controlled by the driving device 23 to contact the end of the test tube 50. The heating is performed such that the temperature of the buffer at both ends of the tube 50 forms a temperature difference to generate a thermal convection phenomenon, so that the DNA in the buffer can be subjected to a PCR proliferative reaction in a cycle of temperature rise and temperature decrease.

As shown in the sixth and seventh figures, after the PCR amplification reaction is completed, the detection operation can be performed. At this time, the light source 31 of the excitation light source module 30 is projected on the light guide plate 32 with light of a specific wavelength to utilize The dense and different size diffusion dot pattern on the light guide plate 32 is designed to make the light uniform effect and uniformly project on the test tube 50 to excite the fluorescent reaction in the test tube 50, and to transmit a specific wavelength of the fluorescent reaction signal through the optical fiber. The image capturing device is transmitted to capture an image thereof and displayed on a display for analyzing the reaction condition of the detection buffer.

The light guide plate 32 is guided by the excitation light source module 30 composed of the plurality of light sources 31 and the light guide plate 32 to guide the light guide direction to effectively improve the uniformity of the light emitted by the excitation light source, thereby avoiding uneven illumination distribution; The display device 40 can be configured to transmit a specific wavelength of the fluorescent reaction signal image to the image capturing device through the optical fiber for image capturing, thereby greatly reducing the distance between the image capturing device and the test tube, thereby reducing the overall structure. Volume to improve the practicality and convenience of use.

Furthermore, by the cooperation of the plurality of light sources 31 and the light guide plate 32, even if one of the light sources 31 fails to function, the other light sources 31 can be transmitted so as not to affect the generation of the excitation light source; The excitation light source is used to excite the fluorescent reaction in the test tube 50, which can effectively improve the fluorescence performance accuracy of the polymerase chain reaction.

In another embodiment of the present invention, the excitation light source module 30 can have a plurality of light sources 31 that can generate different wavelengths, and are disposed on at least one side of the light guide plate 32, for example, disposed on each side of the light guide plate 32 to transmit The side light incident mode is projected on the light guide plate 32. When the excitation light source is performed, the light source 31 of the corresponding wavelength can be selected according to the light source of the specific wavelength required. In this case, the filter plate 33 is not required. To filter specific wavelengths.

While the invention has been described in terms of the preferred embodiments of the present invention, various modifications of the various embodiments can be made without departing from the spirit and scope of the invention. The above embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention. All modifications and variations that are made without departing from the spirit of the invention are the scope of the invention.

10‧‧‧ Positioning Block

11‧‧‧ accommodating space

12‧‧‧Removable carrier

13‧‧‧ observation holes

20‧‧‧ heating device

21‧‧‧heating seat

211‧‧‧ accommodating channel

212‧‧‧heating channel

22‧‧‧heat conducting plate

23‧‧‧ drive

30‧‧‧Excitation light source module

31‧‧‧Light source

32‧‧‧Light guide plate

33‧‧‧Filter

40‧‧‧ display device

41‧‧‧Filter

411‧‧‧Fiber Optic Connector

42‧‧‧Shell

50‧‧‧test tube

60‧‧‧ fixed seat

70‧‧‧ machine base

The first figure is a three-dimensional combination diagram of the present invention.

The second figure is a perspective exploded view of the present invention.

The third figure is a schematic cross-sectional view of the present invention.

The fourth figure is a schematic view showing the state of use of the heating device of the present invention before heating.

The fifth figure is a schematic view showing the state of use of the heating device of the present invention in heating.

The sixth figure is a schematic diagram of the action of the excitation light source module to stimulate the fluorescence reaction according to the present invention.

The seventh figure is a schematic view showing the state of use of the display device of the present invention.

10. . . Positioning seat

12. . . Detachable carrier

13. . . Observation hole

20. . . heating equipment

twenty one. . . Heating seat

40. . . Display device

411. . . Fiber optic connector

42. . . case

50. . . test tube

60. . . Fixed seat

70. . . Machine base

Claims (11)

A method for detecting a biochemical reaction, comprising the steps of: a) filling at least one biochemical reaction buffer in a test tube; b) contacting the proximal end of the test tube with a heating source and heating the test tube to make the test tube The temperature of the buffer at the inner ends forms a temperature difference, which generates a thermal convection phenomenon, and the DNA in the buffer can perform a PCR proliferative reaction in a cycle of heating and cooling; c) using at least one light source to project light onto a light guide plate, The light absorbing effect is generated by the light of the light guide plate, and is uniformly projected on the test tube for exciting the fluorescent reaction material in the test tube to generate a fluorescent reaction signal; d) detecting the buffer solution by using a light sensor The fluorescent response signal image is transmitted through an optical fiber to an image capturing device to capture the image. The method for detecting a biochemical reaction according to the first aspect of the patent application, wherein the biochemical reaction may be a biochemical reaction using a fluorescent dye such as a nucleic acid amplification reaction, a fluorescent antibody calibration, or a fluorescent dye staining. The method of detecting a biochemical reaction according to the first aspect of the invention, wherein the light source generates light by using one of a light emitting diode, a laser, a halogen lamp, a xenon lamp or a xenon lamp. The method for detecting a biochemical reaction according to claim 1, wherein the light source is a plurality of light sources of different wavelengths, such as blue light, green light, and red light, for exciting the fluorescent reaction material in the test tube. An apparatus for applying the biochemical reaction detecting method according to the first application of the patent scope includes: a positioning base having at least one accommodating space for receiving a test tube, wherein the test tube is filled with at least one biochemical reaction buffer And at least one fluorescent reaction substance, the positioning seat is on one side At least one observation hole for observing the accommodating space is provided; a heating device is disposed on one side of the positioning seat, the heating device has a heating seat and a driving device, wherein the heating seat is provided with a heat conducting plate. The driving device is configured to control the heat conducting plate to contact or separate from the test tube, so that the heat conducting plate can be movably contacted or separated from the end of the tube, and the tube can be heated when contacting; an excitation light source module, The excitation light source module includes at least one light source that can project light toward the accommodating space, and the light source and the accommodating space are disposed between the light source and the accommodating space. a light guide plate for uniformly diffusing light projected by the light source into the accommodating space for exciting a fluorescent reaction in the test tube; and a light sensor having a fiber connector for connecting to the light guide The optical sensor further has an image capturing device for causing the photo sensor to capture the reaction condition detected from the observation hole through the optical fiber. The apparatus for detecting biochemical reactions according to claim 5, wherein a filter plate is disposed between the light source and the accommodating space for light of a specific wavelength to pass. The apparatus for detecting a biochemical reaction according to claim 5, wherein the light source is disposed on at least one side of the light guide plate. The apparatus for detecting a biochemical reaction according to claim 5, wherein the positioning seat is provided with a detachable carrier for carrying the test tube. The apparatus for detecting a biochemical reaction according to claim 5, wherein the heating seat is provided with a receiving passage and a heating passage, and the receiving passage is provided for the end of the test tube. A heating channel is provided for the heat conducting plate to be actively disposed therein. The apparatus for detecting a biochemical reaction according to claim 5, wherein the photosensor A filter is electrically connected to the fiber connector. The apparatus for detecting a biochemical reaction according to claim 5, wherein the image capturing device is connected to a display.