TWI636248B - Fluorescence detection device - Google Patents

Abstract

A fluorescent detecting device comprises a light emitting module and a detecting module. The light emitting module includes at least one light source and at least one first color filter, wherein the light source provides a wide-band illumination, and the first light filter is combined with the first color filter to allow a first light beam of a first specific bandwidth to pass along a first optical axis. To stimulate a target fluorescent probe stored in a fluorescent reaction mixture in a test tube and generate a fluorescent light. The detection module includes at least one second filter and at least one photodetector, wherein the second filter receives the fluorescent light and allows a second specific wavelength of the second light beam to pass along a second optical axis, the optical detection The detector receives the second beam of the second specific bandwidth and converts the second beam of the second specific bandwidth into an electronic signal, wherein the first optical axis is inclined at an angle from the second optical axis, ranging from 4.5 degrees to 9.5 degrees .

Description

Fluorescent detection device

The present invention relates to a fluorescent detecting device, and more particularly to a fluorescent detecting device with a high signal to noise ratio.

In recent years, the need to efficiently acquire a large number of DNA-specific fragments for different purposes is booming. Among all existing DNA sequencing technologies, Polymerase Chain Reaction (PCR) is one of the economical and rapid technologies that can obtain billions of copies of specific DNA fragments in a short period of time. PCR technology can be applied in a variety of fields, such as selective DNA isolation for genetic identification, forensic analysis of ancient DNA in archaeology, medical applications for genetic testing and tissue classification, special diagnosis of infectious diseases in hospitals and research institutions, and food safety environments. Monitoring of hazards and investigation of genetic fingerprinting of offenders. For PCR technology, only a small amount of DNA sample in blood or tissue is required. By adding the fluorescent dye to the nucleic acid solution, the amplified DNA fragment can be detected by the assistance of the fluorescent molecule.

In order to simultaneously detect and analyze the presence of target nucleic acids in a batch of biological samples, fluorescent dye detection techniques are commonly employed. After the light source of a particular wavelength illuminates the target nucleic acid, the DNA binding dye or luciferin-binding probe of the nucleic acid will react and emit a fluorescent signal. The fluorescent signal represents the presence of a target nucleic acid. This technology has been applied to new PCR techniques, called real time quantitative PCR or quantitative PCR (qPCR). Compared to traditional PCR technology, which is called end-point PCR detection, qPCR is early-phase PCR detection with higher sensitivity and better accuracy. Therefore, optical devices are indispensable tools for qPCR detection technology to detect fluorescence emitted by specific nucleic acid fragments. The optical device must provide a light source to excite the fluorescent probe at a specific wavelength, and simultaneously detect Measure the fluorescent signal from the probe.

Fluorescence detection systems have matured in many fields, such as the use of fluorescence spectroscopy and fluorescence microscopy. Monochromatic source arrays can be easily applied to specific fluorescent probes with a set of filters and optics. However, the development of a fluorescence detection device for a portable qPCR system is still difficult and has not yet been solved in the market, owing to the cost of achieving a high signal-to-noise ratio (SNR). Cost and size considerations.

In view of the foregoing needs and problems, it is necessary to provide a fluorescent detection device with high signal to noise ratio for qPCR applications.

The purpose of the present invention is to provide a fluorescent detection device with a high signal to noise ratio, which can reduce the size and weight of the device, and still provide a portable qPCR system with superior performance at a lower cost.

In order to achieve the above object, a broader aspect of the present invention provides a fluorescent detecting device comprising a light emitting module and a detecting module. The light emitting module includes at least one light source and at least one first color filter, wherein the light source provides a wide-band illumination, and the first light filter is combined with the first color filter to allow a first light beam of a first specific bandwidth to pass along a first optical axis. To stimulate a target fluorescent probe stored in a fluorescent reaction mixture in a test tube and generate a fluorescent light. The detection module includes at least one second filter and at least one photodetector, wherein the second filter receives the fluorescent light and allows a second specific wavelength of the second light beam to pass along a second optical axis, the optical detection The detector receives the second beam of the second specific bandwidth and converts the second beam of the second specific bandwidth into an electronic signal, wherein the first optical axis is inclined at an angle from the second optical axis, ranging from 4.5 degrees to 9.5 degrees .

In one embodiment, the light source is one of a group selected from the group consisting of a monochromatic LED, a laser diode, a mercury lamp, and a halogen bulb.

In an embodiment, the first filter and the second filter are a single band pass filter.

In one embodiment, the first filter and the second filter are respectively an excitation filter and an emission filter.

In one embodiment, the fluorescence detecting device further includes a heating module disposed between the light emitting module and the detecting module, wherein the heating module includes at least one heating slot for accommodating and storing the firefly The photoreaction mixture and the test tube of the target fluorescent probe.

In an embodiment, the heating module further includes a heater connected to the heating tank.

In one embodiment, the heater is a thermoelectric heater for thermal cycling control.

In one embodiment, the heating module further includes at least one first optical through hole and at least one second optical through hole, the first optical through hole is located on the first optical axis, and the second optical through hole is located on the second optical axis And the first optical through hole communicates with the second optical through hole through the heating groove.

In one embodiment, the first optical through hole has a diameter ranging from 1.8 mm to 2.2 mm, and the second optical through hole has a diameter ranging from 1.5 mm to 2.5 mm.

In one embodiment, the light emitting module further includes at least one pinhole disposed between the first filter and the first optical through hole on the first optical axis, and the diameter of the pinhole ranges from 1.3 mm to 1.8 mm.

In one embodiment, the second optical through hole of the heating module and the detecting module are combined to form a divergence half angle ranging from 18 degrees to 22 degrees.

In one embodiment, the detection module further includes at least one converging lens disposed between the heating slot and the second filter.

In one embodiment, the converging lens has a plane and a convex surface, the plane facing the second optical through hole, and the convex surface facing the second filter.

In one embodiment, the detection module further includes at least one imaging lens disposed between the second filter and the photodetector.

In one embodiment, the imaging lens has a plane and a convex surface, the plane facing the photodetector and the convex surface facing the second filter.

In one embodiment, the detecting module further comprises at least two optical lenses symmetrically disposed on opposite sides of the second filter at the same distance, wherein the convex surface of each optical lens faces the second filter.

In one embodiment, the fluorescence detecting device further includes a casing, wherein the light emitting module and the detecting module are jointly constructed on a casing.

In one embodiment, the detection module further includes a photodiode amplifier connected to the photodetector.

In one embodiment, the detection module further includes an electromagnetic interference mask and a ground structure to encapsulate the photodetector.

In one embodiment, the photodetector is one of a group consisting of a germanium photodiode, a photomultiplier tube, a photosensitive coupling element, and a complementary metal oxide semiconductor.

Some exemplary embodiments embodying the features and advantages of the present invention are described in detail in the following description. It is to be understood that the present invention is capable of various modifications in the various aspects of the present invention, and the description and illustration are in the nature of

The present invention provides a fluorescence detecting device which is an optical module that can sequentially illuminate a plurality of fluorescent samples arranged in a linear position. During qPCR amplification, the fluorescence detection device stimulates a fluorescent probe in a nucleic acid sample by providing a single light source, and sequentially detects a specific fluorescent signal emitted from the fluorescent probe.

Please refer to FIG. 1 to FIG. 4 , wherein FIG. 1 is a schematic view showing the three-dimensional structure of the fluorescent detecting device of the preferred embodiment of the present invention, and FIG. 2 and FIG. 3 respectively showing the fluorescent detecting device of FIG. 1 at different viewing angles. The exploded view, and Fig. 4 shows a cross-sectional view of the fluorescent detecting device of Fig. 1. As shown in FIG. 1 to FIG. 4 , the present disclosure provides a fluorescent detection device 1 including a light emitting module 10 and a detecting module 30 . The light emitting module 10 includes at least one light source 11 and at least one first color filter, for example, a first single band pass filter 12, wherein the light source 11 provides a broadband illumination, and the first filter The light sheet 12 is configured to allow a first light beam of a first specific bandwidth to pass along a first optical axis A1 to excite a target fluorescent probe stored in a fluorescent reaction mixture 40 of a test tube 41, and generate a fluorescent light. Light. The detection module 30 includes at least one second filter, such as a second single pass pass filter 32, and at least one photodetector 34, wherein the second filter 32 receives the firefly Light and allowing a second specific bandwidth of the second light beam to pass along a second optical axis A2, the light detector 34 receives the second specific bandwidth of the second light beam and converts the second specific bandwidth of the second light beam into an electronic signal The first optical axis A1 is inclined at an angle from the second optical axis A2, and the angle ranges from 4.5 degrees to 9.5 degrees. The fluorescent detection device 1 further includes a heating module 20 disposed between the illumination module 10 and the detection module 30 for accommodating the test tube 41 storing the fluorescent reaction mixture 40 and the target fluorescent probe. Maintain its thermal cycle control. In other words, the fluorescent detection device 1 mainly includes a light emitting module 10, a heating module 20, and a detecting module 30. The light emitting module 10 is disposed at a front end of the heating module 20, and the detecting module 30 is disposed at a rear end of the heating module 20. The illumination module 10, the heating module 20, and the detection module 30 are more commonly constructed on a housing 50.

In this embodiment, the light emitting module 10 includes at least one light source 11 and at least one first filter, such as a first single band pass filter 12. Wherein the light source 11 provides a broadband illumination, and the wavelength of the light source 11 includes an excitation bandwidth for exciting the target fluorescent probe stored in the fluorescent reaction mixture (also referred to as PCR mixture) 40 of the PCR tube 41. . The illumination module 10 is disposed on a first optical axis A1 and is designed to be inclined by a specific angle θ of 4.5 degrees to 9.5 degrees from the second optical axis A2 where the detection module 30 is located. In this embodiment, a light emitting diode (LED) can be selected as the light source 11, but other light sources such as a laser diode and a halogen bulb can also be applied, and the invention is not limited thereto. The first single band pass filter 12 applied to the light emitting module 10 is an excitation filter and allows only a light beam falling within the excitation bandwidth to pass through and reach the PCR mixture 40. In other words, the first single band pass filter 12 provides an excitation light signal to the target fluorescent probe in the PCR mixture 40. The first single band pass filter 12 has a passband width ranging from 20 nm to 30 nm.

Furthermore, in the present embodiment, as shown in FIG. 1 to FIG. 4, the light-emitting module 10 includes three channels arranged in a linear configuration and constructed in the housing 50, but the number of channels can be expanded to meet batch processing. high demand. Each channel has its own source 11 and a pinhole 13 between 1.3 mm and 1.8 mm in diameter to avoid large angle scattering of the excitation beam, one of the sources of background noise. The material of the casing 50 is a black ABS (acrylonitrile butadiene styrene) resin, which has the advantages of low thermal conductivity, high thermal resistance, and reduced internal light scattering. Of course, other low reflectance black plastic materials, or aluminum having a low reflectance and high absorptivity coated with a black anodized layer, can also be applied as the material of the casing 50.

Figure 5 shows the source spectrum of the preferred embodiment of the present invention. In this embodiment, the light emitted by the light source 11 is in a specific wavelength range of the visible light wavelength range, for example, a full-width at half maximum (FWHM) is between 450.34 nm and 481.26 nm to cover the target excitation. wavelength. More preferably, the light source 11 is more preferably a monochromatic LED light source that provides 7000 mcd optical power at 20 mA, and its viewing angle at the FWHM is 20 degrees. In addition, any light source 11 having a spectral energy including an excitation spectrum of a fluorescent dye, such as a laser, a mercury lamp, a halogen lamp, or the like, can be used, and can be applied to the present case.

On the other hand, Fig. 6 shows the pass band of the first single band pass filter of the preferred embodiment of the present invention, and Fig. 7 shows the spectrum of the light source of the preferred embodiment of the present invention and the first single band pass filter. In the present embodiment, the first single band pass filter 12 is an optical element capable of filtering a specific wavelength from the light source 11 for excitation, and can block other bands considered as noise. In other words, the light source 11 is combined with the first single band pass filter 12 to allow a first specific wavelength of the first light beam to pass along the first optical axis A1 to excite one of the fluorescent reaction mixtures 40 stored in the PCR tube 41. Target the fluorescent probe and produce a fluorescent light. In this embodiment, the first single band pass filter 12 has a passband width of about 27 nm and a passable wavelength range of 460.5 nm to 487.5 nm. Thus, the first single band pass filter 12 helps to increase the signal to noise ratio (SNR) of the target excited fluorescent dye. In the present embodiment, the size of the first single band pass filter 12 is 23 mm. 6.5 mm 2 mm to cover three channels. In another embodiment, the size of the first single band pass filter 12 can be disassembled to 5 mm for each channel. 5 mm 2 mm to further reduce costs.

Figure 8 is a cross-sectional view showing the heating module of Figure 1. As shown in FIGS. 1 to 4 and 8 , the heating module 20 includes a heating tank 21 , a heater 22 , a first optical through hole 23 , and a second optical through hole 24 . The heating module 20 can accommodate multiple channels laterally to achieve multi-path sample detection. In the present embodiment, the constituent material of the heating tank 21 may be selected from copper because it has thermal conductivity and can uniformly transfer heat in a few minutes to meet the demand for rapid thermal cycling. Of course, the heating tank 21 can also be constructed using other thermally conductive materials, such as aluminum. In the present embodiment, the heater 22 can be, for example but not limited to, a thermoelectric cooling (TEC) heater, and the heating tank 21 is disposed on the top of the heater 22. In addition, the first optical through hole 23 and the second optical through hole 24 are respectively constructed at the front end and the rear end of the heating groove 21, so that effective optical signal transmission can be input from the light source 11 and emitted from the PCR mixture 40. The heater 22 is cycled to change the temperature with a change in the input current.

Figure 9 is a view showing the optical path of the fluorescent detecting device of the preferred embodiment of the present invention on a single channel. In this example, biological samples are prepared and stored in PCR tubes 41 for PCR amplification and detection. As shown in FIGS. 4 and 9, the first optical through hole 23 and the second optical through hole 24 are respectively constructed at the front end and the rear end of the PCR heating tank 21. In other words, the first optical through hole 23 communicates with the second optical through hole 24 through the heating groove 21. Wherein, the diameter of the first optical through hole 23 ranges from 1.8 mm to 2.2 mm, and the first optical hole 23 is located on the first optical axis A1, and the first optical axis A1 is inclined clockwise from the second optical axis A2. 4.5 degrees to 9.5 degrees to detect the maximum fluorescent emission signal. In addition, the second optical through hole 24 has a diameter ranging from 1.5 mm to 2.5 mm, and the second optical through hole 24 is located on the second optical axis A2 where the detecting module 30 is located.

When the excitation beam passes through the fluorescent reaction mixture 40 having the target fluorescent probe stored in the PCR tube 41, it is affected by the shape of the PCR tube 41 and the refractive index of the test tube material (range 1.46 to 1.49), and The fluorescent reaction mixture 40 having the target fluorescent probe has a higher refractive index than air, and the incident angle of the light beam is designed to provide maximum excitation intensity.

Further, as shown in Figs. 1 to 4, in order to support and heat the PCR tube 41, the heating tank 21 may be made of copper having excellent thermal conductivity, and of course other heat conductive materials such as aluminum may be applied. Each heating tank 21 is linearly arranged for batch processing. Further, the heating tank 21 may be made of metal and other materials having high thermal conductivity, and may be fabricated by a method of computer numerical control machining (CNC machining), casting, laser cutting, 3D printing, and the like. The volume inside the PCR tube 41 is between 30 μL and 40 μL. In the present embodiment, the heater 22 is a thermoelectric cooling (TEC) heater. The temperature of the heater 22 is controlled to a fractional point to meet the needs of cyclic PCR amplification. The compactness of the TEC heater contributes to the miniaturization of the system compared to other thermal cyclers. In addition, TEC heaters have a long life and are easy to maintain. Of course, in addition to TEC technology, the traditional convection thermal cycling method by air or liquid is also applicable to this case.

On the other hand, in the embodiment, the fluorescence detecting device 1 further includes a detecting module 30 for detecting a specific fluorescent signal emitted from the fluorescent reaction mixture 40 having the target fluorescent probe. The sandwich detection module 30 includes a converging lens 31 and a second filter, such as a second single band pass filter 32, an imaging lens 33, and a photodetector 34. The second single band pass filter 32 can be an emission filter, and the photo detector 34 can be a photodiode. The number of detection channels of the detection module 30 is the same as the number of the light sources 11. Each channel of the illumination module 10 is mapped to a channel of the detection module 30.

As shown in FIG. 4, the detecting module 30 has a sandwich sandwich structure and includes a set of converging lenses 31, at least one second single band pass filter 32, a set of imaging lenses 33, and at least one light detecting. 34. The condenser lens 31 is located at the rear end of the second optical through hole 24 of the heating module 20. Converging lens 31 more collects the fluorescent emission signals from PCR mixture 40 and ensures that the concentrated beam of light can enter second single band pass filter 32. The second single band pass filter 32 is disposed at the rear end of the condenser lens 31 and allows only the fluorescent emission signals falling within the pass band thereof to be penetrated. The second single band pass filter 32 has a bandwidth between 20 nm and 30 nm. In the present embodiment, the imaging lens 33 focuses the filtered emission signal on the photodetector 34 and provides sufficient fluorescent emission signals for analysis. The photodetector 34 converts the fluorescent emission signal into an electronic signal for further analysis. In the present embodiment, a photodiode can be selected as the photodetector 34, but other types of detectors such as CCD, PMT and CMOS can also be applied to the fluorescent detecting device 1. The converging lens 31 and the imaging lens 33 used in the present case are made of optical grade glass, but injection molded optical plastics such as acrylic (also known as PMMA), polycarbonate (PC), polyphenylene. Polystyrene or polyolefin can also be used in this case.

In the present embodiment, the converging lens 31 located at the rear end of the heating tank 21 is for collecting the fluorescence emitted from the fluorescent reaction mixture 40 having the target fluorescent probe stored in the PCR tube 41. 10A and 10B are respectively a front view and a cross-sectional view showing a condenser lens of the preferred embodiment of the present invention. The condenser lens 31 has a convex surface S1 and a flat surface S2. The condenser lens 31 has a radius of curvature of 5.89 mm. The plane S2 faces the heating groove 21, and the convex surface S1 faces the second single band pass filter 32. The condenser lens 31 is for collecting the fluorescent light and converting the fluorescent light into a collimated light beam to uniformly illuminate the second single band pass filter 32. Second single bandpass filter 32 (23 mm 6.2 mm 2mm) covers all three channels. However, the size of the second single band pass filter 32 can also be reduced to 5 mm. 5 mm 2 mm to further reduce costs. Further, the material of the condenser lens 31 is N-SF11 glass. Of course, other materials, such as BK7 glass, or injection molded optical grade plastics, such as acrylic (PMMA), polycarbonate (PC), polystyrene (PS) or polyolefin (polyolefin) Can be applied to this case. The numerical aperture (NA) of the condenser lens 31 ranges from 0.37 to 0.42.

It is worth noting that each channel requires a first filter as an excitation filter and a second filter as an emission filter. In this embodiment, each channel includes a first single band pass filter 12 and a second single band pass filter 32. Figure 11 shows the pass band of the second single band pass filter of the preferred embodiment of the present invention. The emission wavelength of the target fluorescent probe is always longer than its excitation wavelength, so different filters are required. Similar to the first single band pass filter 12, the band pass coating of the second single band pass filter 32 allows only a second beam of a particular wavelength to pass through and blocks the rest of the light. The second single band pass filter 32 plays an important role in preventing interference of the noise signal from the light source 11 and the noise astigmatism from the surrounding environment. As shown in Fig. 11, the second single band pass filter 32 has a passband width of about 24 nm for filtering out noise signals that fall outside the passband. The second single band pass filter 32 has a filtered wavelength range between 512 nm and 536 nm.

Figure 12 shows the optical path of the detection module of the preferred embodiment of the present invention. In the present embodiment, the imaging lens 33 is made of N-SF11 glass and has the same structure as the condenser lens 31. The material of the imaging lens 33 is N-SF11. Of course, other materials, such as BK7 glass, or injection molded optical grade plastics, such as acrylic (PMMA), polycarbonate (PC), polystyrene (PS) or polyolefin (polyolefin) Can be applied to this case. The numerical aperture (NA) of the imaging lens 33 ranges from 0.37 to 0.42. The imaging lens 33 is disposed at a rear end of the second single band pass filter 32, and the distance from the second single band pass filter 32 is the same as the distance between the condenser lens 31 and the second single band pass filter 32. The filtered fluorescent signal is imaged on the imaging plane, the detection surface of the so-called photodetector 34. The convex surface of the imaging lens 33 faces the second single band pass filter 32. The converging lens 31 is disposed symmetrically with the corresponding imaging lens 33 such that the convex surfaces thereof face each other, which contributes to reducing wavefront aberration. The noise reflected inside the channel of the housing 50 (shown in Figure 4) is due to the fact that the scattered beam of light at a large angle of incidence may pass through the bandpass coating of the second single bandpass filter 32. The imaging lens 33 condenses the filtered emitted fluorescent light uniformly over a large area and focuses it on the area of the detection surface of the photodetector 34 which is much smaller than the fluorescent light source (1.1 mm). 1.1 mm). For the fluorescent signal of the PCR mixture 40, the imaging transparency 33 is received through the second optical through-hole 24 of the heating bath 21 in a divergent half angle of 18 to 22 degrees.

In this embodiment, the photodetector 34 can be a photoelectric photodiode for converting the optical signal into a current, and due to its high sensitivity, a small amount of photons in the filtered fluorescent light can still be detected. Measured in the wavelength range from 320 nm to 1100 nm. Figure 13 shows the response spectrum of the erbium photodiode. Of course, other types of photodetectors, such as photomultiplier tubes (PMTs), charged-coupled devices (CCDs), and complementary metal-oxide semiconductors (CMOS), can be used. Applicable to this case.

In one embodiment, the detection module 30 further includes a photodiode amplifier (not shown) that converts the current of the nanoamperes outputted by the photodetector 34 into volts. The photodiode amplifier amplifies the signal for further data analysis and utilization. The distance between the detection surface of the photodetector 34 and the rear end surface of the imaging lens 33 is about 7 mm.

In one embodiment, the detection module 30 further includes an electromagnetic interference (EMI) mask and a grounding structure (not shown) that surrounds the photodetector 34. The photodetector 34 is easily imaged by noise of the surrounding environment due to its high sensitivity. The EMI mask and ground structure can be integrated into the housing 50. The material of the housing 50 of the accommodating detection module 30 can be made of black ABS (acrylonitrile butadiene styrene) resin to avoid internal light reflection and light scattering. Of course, black machinable materials such as polylactide (PLA), polycarbonate (PC), polyetheretherketone (PEEK), polyphenylene ether (PPE) or black anodized layer The aluminum of the cloth can be used in this case. The outer surface of the housing 50 can be coated with a metallization coating to isolate EMI noise from the photodetector 34. Further, the case 50 may be made of an aluminum material coated with a black anodized layer. The black anodized coating not only avoids the short circuit between the positive and negative leads of the photodetector 33, but also reduces the noise generated by internal light scattering in the optical channel, which is another source of noise.

In this embodiment, the fluorescent dye FAM or FITC is used in this case. The PCR tube 41 on each channel is filled with nucleic acid that binds to the target fluorescent probe. Fluorescent dyes are commercially available fluorescent dyes. Figure 14A shows the excitation spectrum of the fluorescent dye FAM. Figure 14B shows the emission spectrum of the fluorescent dye FAM. Figure 15A shows the excitation spectrum of the fluorescent dye FITC. Figure 15B shows the emission spectrum of the fluorescent dye FITC. Although the preferred embodiment of the present invention is exemplified by these dyes, the system of the present invention is not limited to these two types of fluorescent dyes.

It is worth noting that a large number of instruments are known in the art to be able to image fluorescent signals. However, one of the main problems with such devices is the excitation of the emitted light relative to the fluorescent light emitted by the fluorescent probe. In order to overcome this problem, the fluorescent detecting device 1 of the present invention configures the light emitting module 10 and the detecting module 30 on opposite sides of a PCR tube 41 storing a fluorescent reaction mixture 40 having a target fluorescent probe, wherein The tilting optical axis (ie, the first optical axis A1) of the light-emitting module 10 is further inclined downward by 4.5 degrees to 9.5 degrees by the horizontal optical axis (ie, the second optical axis) where the detecting module 30 is located. . Therefore, the fluorescence detecting device 1 of the present invention can have a high signal to noise ratio (SNR).

Figure 16 shows a top view of an experimental setup for analyzing the distribution of the fluorescent emission signals. Figure 17 shows the intensity distribution of the fluorescent emission signal of the fluorescent dye FITC in different directions. Figure 18 shows the signal-to-noise ratio (SNR) plot of 20 nM FITC at different detection angles.

As shown in Figs. 16 to 18, the tested FITC fluorescent dye samples are stored in the cylindrical bottle 6 and excited by the light source 11, and the photodetector 34 detects the fluorescent emission signals in different directions. The maximum FITC fluorescence emission signal occurs in the forward direction (0 degrees). When the photodetector 34 deviates by ±2.5 degrees, the signal-to-noise ratio (SNR) drops by 25% from a peak of 0 degrees to 3 to 2.17. Although the maximum signal-to-noise ratio (SNR) at all deviation angles occurs at 50 degrees, the intensity of the detected fluorescent signal at 50 degrees is 22.8 mV, which is much lower than the 61.8 mV detected at 0 degrees.

Based on the experimental results and the nature of the materials such as the nature of the excitation source 11, the material properties of the PCR tube 41, the nature of the PCR reaction mixture 40, the nature of the emitted light, the optical properties of the PCR tube support, and the refraction and refraction in the test tubes and liquids. Scattering behavior must be considered in the design of this case. Therefore, in the present invention, the acceptable detection angle between the position of the light source 11 of the illumination module 10 relative to the position of the photodetector 34 of the detection module 30 falls within a specific angle θ of 4.5 degrees to 9.5 degrees. To achieve optimal operational performance.

In addition, according to the article by N. Lindlein - "Geometry and Technical Optics", paraxial ray can be propagated through a tilted refractive plane. 3 matrix form representation. The height and angle of the output paraxial ray are denoted as y' and α', respectively, and the matrix M _S is the propagation matrix of the optical system.

The transfer matrix describes the change in the height of the paraxial ray that travels within the distance d of the same medium. It can be expressed as follows:

A refraction matrix describes the deviation of the paraxial ray angle propagating from the medium 1 to the medium 2. The medium 1 has a refractive index n and the medium 2 has a refractive index n'. α is the angle of the plane.

Figure 19 shows a paraxial ray diagram similar to the optical system of the fluorescence detecting device. The light propagation matrix of the system shown in Figure 19 can be expressed as follows: Therefore, the relationship between the paraxial output and the height and angle of the input beam can be simplified as follows:

Accordingly, the angle relationship between the input beam and the output beam of the fluorescence detecting device 1 of the present invention can be known, and the results are shown in Table 1. It can be seen from Table 1 that when the input light source position is 7±2.5 degrees, the output angle of the excitation beam changes within 0±2.5 degrees, which can optimize the operation performance. Table 1

Figure 20 shows the intensity of the detection signal for different concentrations of FITC when the optical axis of the input source is set at 7 degrees tilt. Table 2 also shows the relative signal-to-noise ratio (SNR) of the detected signal strength at different concentrations. As shown in Fig. 20 and Table 2, as the concentration increases, the intensity of the detection signal and signal-to-noise ratio (SNR) increases. In Table 2, it is further shown that the fluorescent light detecting device 1 in which the input light source 11 is tilted at 7 degrees can provide a desired performance with a signal-to-noise ratio (SNR) of 111. Table 2

In summary, the present invention provides a fluorescent detection device including a light emitting module and a detecting module. The light emitting module includes at least one light source and at least one first color filter, wherein the light source provides a wide-band illumination, and the first light filter is combined with the first color filter to allow a first light beam of a first specific bandwidth to pass along a first optical axis. To stimulate a target fluorescent probe stored in a fluorescent reaction mixture in a test tube and generate a fluorescent light. The detection module includes at least one second filter and at least one photodetector, wherein the second filter receives the fluorescent light and allows a second specific wavelength of the second light beam to pass along a second optical axis, the optical detection The detector receives the second beam of the second specific bandwidth and converts the second beam of the second specific bandwidth into an electronic signal, wherein the first optical axis is tilted by a specific angle from the second optical axis, and the specific angle ranges from 4.5 degrees Up to 9.5 degrees and integrated with the heating module for thermal cycling control of PCR. The well-designed optical structure of this case minimizes the size and cost of the light-emitting module and the detection module, but still provides convincing performance with a signal-to-noise ratio (SNR) of 111. The overall size of the system is approximately 80 mm 35 mm 20 mm. The components and structure of the fluorescent detection device achieve a simplification of the optical system. The illumination module is configured to provide the most efficient excitation source for the target fluorescent probe in the PCR fluorescent reaction mixture. The sandwich sandwich arrangement of the condenser lens and the imaging lens also ensures miniaturization of the optical system because the spacing between the condenser lens, the emission filter and the imaging lens is well designed. This case avoids noise interference caused by scattering and reflection of the light source.

In addition, the light path of the case adopts a transmissive design, and according to the experimental result, the optical axis of the incident angle of the light source is inclined downward from the horizontal axis by a specific angle of 4.5 degrees to 10.5 degrees, so as to achieve the maximum light intensity applied to the low firefly. Optical signal detection system. From experimental and simulation results, when the fluorescent sample is stored in a cylindrical container, the peak intensity of the fluorescent emission signal occurs at zero degrees, also known as forward scatter. However, in order to match the conical shape of a commercial PCR tube, the angle between the light source and the photodetector falling between 4.5 and 9.5 degrees is an optimum condition. The signal-to-noise ratio (SNR) is also within the optimal performance range. The optimal angle of incidence can significantly reduce internal and multiple reflections in the PCR mixture. In addition, the optical through holes provided at the front and rear ends of the heating module ensure sufficient light signals for excitation and emission.

Furthermore, the design of the first and second single band pass filters as excitation and emission filters facilitates the simplification of the qPCR system. In addition, the filter bank can reduce noise interference, so that the excitation beam and the emission of fluorescence can be fully utilized, so that the fluorescence detection device can provide a high signal-to-noise ratio (SNR).

The present invention has been described in detail by the above-described embodiments, and is intended to be modified by those skilled in the art.

1‧‧‧Fluorescent detection device

10‧‧‧Lighting module

11‧‧‧Light source

12‧‧‧First Single Bandpass Filter

13‧‧‧ pinhole

20‧‧‧heating module

21‧‧‧heating tank

22‧‧‧heater

23‧‧‧First optical through hole

24‧‧‧Second optical through hole

30‧‧‧Detection module

31‧‧‧ Converging lens

32‧‧‧Second single bandpass filter

33‧‧‧ imaging lens

34‧‧‧Photodetector

40‧‧‧Fluorescent reaction mixture (also known as PCR mixture)

41‧‧‧test tube

50‧‧‧shell

6‧‧‧Cylinder bottle

A1‧‧‧first optical axis

A2‧‧‧second optical axis

d ₁ , d ₂ , d ₃ , d ₄ ‧‧‧ distance

n ₀ , n ₂ , n ₃ ‧‧ ‧ refractive index

Radius of curvature of R ₁ , R ₂ , R ₃ , R ₄ ‧‧

T ₁ , T ₂ , T ₃ , T ₄ , T ₅ ‧‧‧ transformation matrix

α, θ, φ‧‧‧ angle

Y‧‧‧ Height

FIG. 1 is a schematic perspective view showing the structure of a fluorescent detecting device according to a preferred embodiment of the present invention.

Fig. 2 is a view showing an exploded view of the fluorescent detecting device of Fig. 1.

Fig. 3 is a view showing an explosion view of the fluorescence detecting device of Fig. 1 from another angle of view.

Fig. 4 is a cross-sectional view showing the fluorescent detecting device of Fig. 1.

Figure 5 shows the spectrum of the source of light in the preferred embodiment of the present invention.

Figure 6 shows the pass band of the first single band pass filter of the preferred embodiment of the present invention.

Figure 7 shows the spectrum of the light source of the preferred embodiment of the present invention and the first single band pass filter.

Figure 8 is a cross-sectional view showing the heating module of Figure 1.

Figure 9 shows the optical path of the fluorescent detection device of the preferred embodiment of the present invention on a single channel.

10A and 10B are respectively a front view and a cross-sectional view showing a condenser lens of the preferred embodiment of the present invention.

Figure 11 shows the pass band of the second single band pass filter of the preferred embodiment of the present invention.

Figure 12 shows the optical path of the detection module of the preferred embodiment.

Figure 13 shows the response spectrum of the erbium photodiode.

Figure 14A shows the excitation spectrum of the fluorescent dye FAM.

Figure 14B shows the emission spectrum of the fluorescent dye FAM.

Figure 15A shows the excitation spectrum of the fluorescent dye FITC.

Figure 15B shows the emission spectrum of the fluorescent dye FITC.

Figure 16 shows a top view of an experimental setup for analyzing the distribution of the fluorescent emission signals.

Figure 17 shows the intensity distribution of the fluorescent emission signal of the fluorescent dye FITC in different directions.

Figure 18 shows the signal-to-noise ratio (SNR) plot of 20 nM FITC at different detection angles.

Figure 19 shows a paraxial ray diagram similar to the optical system of the fluorescence detecting device.

Figure 20 shows the intensity of the detection signal for different concentrations of FITC when the optical axis of the input source is set at 7 degrees tilt.

Claims (20)

A fluorescent detecting device comprising: a light emitting module comprising at least one light source and at least one first color filter, wherein the light source provides a broadband illumination and is combined with the first color filter to allow a first specific a first light beam of the bandwidth passes along a first optical axis to excite a target fluorescent probe stored in a fluorescent reaction mixture of a test tube and generates a fluorescent light; and a detecting module comprising at least one a second filter and at least one photodetector, wherein the second filter receives the fluorescent light and allows a second light beam of a second specific bandwidth to pass along a second optical axis, the photodetector receiving The second beam of the second specific bandwidth and converting the second beam of the second specific bandwidth into an electronic signal, wherein the first optical axis is inclined at an angle from the second optical axis, and the range is 4.5 degrees To 9.5 degrees. The fluorescent detecting device according to claim 1, wherein the light source is one selected from the group consisting of a monochrome LED, a laser diode, a mercury lamp, and a halogen bulb. The fluorescence detecting device of claim 1, wherein the first filter and the second filter are a single band pass filter. The fluorescence detecting device of claim 1, wherein the first filter and the second filter are an excitation filter and an emission filter, respectively. The illuminating device of claim 1 includes a heating module disposed between the illuminating module and the detecting module, wherein the heating module includes at least one heating slot for accommodating storage The test tube having the fluorescent reaction mixture and the target fluorescent probe. The fluorescent detecting device of claim 5, wherein the heating module further comprises a heater connected to the heating tank. The fluorescent detecting device of claim 6, wherein the heater is a thermoelectric cooling heater for thermal cycle control. The fluorescent detection device of claim 5, wherein the heating module further comprises at least one first optical through hole and at least one second optical through hole, the first optical through hole being located on the first optical axis, The second optical through hole is located on the second optical axis, and the first optical through hole communicates with the second optical through hole through the heating groove. The fluorescence detecting device of claim 8, wherein the first optical through hole has a diameter ranging from 1.8 mm to 2.2 mm, and the second optical through hole has a diameter ranging from 1.5 mm to 2.5 mm. The fluorescence detecting device of claim 9, wherein the second optical through hole of the heating module and the detecting module are combined to form a divergence half angle ranging from 18 degrees to 22 degrees. The luminescence detecting device of claim 8, wherein the illuminating module further comprises at least one pinhole disposed between the first optical filter and the first optical through hole on the first optical axis And the pinhole has a diameter ranging from 1.3 mm to 1.8 mm. The fluorescence detecting device of claim 5, wherein the detecting module further comprises at least one converging lens disposed between the heating slot and the second filter. The fluorescence detecting device of claim 12, wherein the converging lens has a plane and a convex surface, the plane facing the second optical through hole, the convex surface facing the second filter. The fluorescence detecting device of claim 1, wherein the detecting module further comprises at least one imaging lens disposed between the second filter and the photodetector. The fluorescence detecting device of claim 14, wherein the imaging lens has a plane and a convex surface, the plane facing the photodetector, the convex surface facing the second filter. The fluorescence detecting device of claim 1, wherein the detecting module further comprises at least two optical lenses symmetrically disposed on opposite sides of the second filter at the same distance, wherein each of the optical lenses There is a convex surface facing the second filter. The luminescence detection device of claim 1, wherein the illumination module and the detection module are jointly constructed on a housing. The fluorescence detecting device of claim 1, wherein the detecting module further comprises a photodiode amplifier connected to the photodetector. The fluorescence detecting device of claim 1, wherein the detecting module further comprises an electromagnetic interference mask and a grounding structure for covering the photodetector. The fluorescence detecting device of claim 1, wherein the photodetector is one of a group selected from the group consisting of a germanium photodiode, a photomultiplier tube, a photosensitive coupling element, and a complementary metal oxide semiconductor.