TW202217303A - Photoactivation device and method of controlling the same - Google Patents

Abstract

A photoactivation device for viability PCR includes a light source, a filer, a diffusing optics, a reflective structure, and a cooling device. The light source provides a light beam with a specific wavelength to illuminate a bio sample for photoactivation. The filter reflects infrared light from the light source to reduce thermal radiation. The diffusing optics homogenizes the light beam transmitted through the filter. The reflective structure surrounds the bio sample, and reflects the light beam transmitted through the diffusing optics to enhance uniform illumination. The cooling device is disposed adjacent the light source and dissipates heat from the photoactivation device. Thereby, the light beam provided by the light source first transmits through the filter then the diffusing optics, and is reflected by the reflective structure, so that the bio sample gets uniform illumination to improve photoactivation efficiency, and the thermal radiation is blocked by the filter and the reflective structure to avoid damaging the bio sample.

Description

Photoactivation device and control method thereof

This case is about a photoactivation device and its control method, especially a photoactivation device used for viability PCR (viability polymerase chain reaction (viability PCR), also known as v-PCR or vPCR) and its control method.

Viability PCR (viability PCR, also known as v-PCR or vPCR) is an evolutionary technology of polymerase chain reaction (PCR). Viability PCR uses specific intercalating photo-reactive reagents (intercalating photo-reactive reagents), or Photoactive dyes are used for simple pretreatment of samples. Since photoreactive reagents can selectively enter dead cells with damaged cell membranes and covalently cross-link with nucleic acids after irradiation with visible light, they inhibit nucleic acid amplification. Only nucleic acids from living cells can be detected by PCR. However, the viability PCR currently on the market has the following problems.

The first problem is uneven lighting. The optical structure of the prior art cannot provide uniform illumination for each biological sample, resulting in uneven photoactivation of the biological sample in the tube. Uneven illumination can also lead to poor photoactivation efficiency of some biological samples. High-power light sources can cause localized thermal transfer of biological samples, which may reduce the photoactivation of viability PCR.

The second problem is the long exposure time. The shortest exposure time for existing systems on the market today is 10-30 minutes. Longer photoactivation times may damage biological samples due to the heat accumulated by the light source.

Therefore, there is a need to provide a special photoactivation system for viability PCR to solve the above-mentioned problems encountered with the prior art.

The purpose of the embodiment of this case is to provide a photoactivation device for viability PCR, so that the biological sample can be illuminated uniformly and the illumination time can be reduced.

Another object of the embodiments of the present invention is to provide a photoactivation device for viability PCR, which can isolate thermal convection or thermal radiation to avoid damage to biological samples.

In order to achieve the above purpose, the embodiment of the present application provides a photoactivation device for viability PCR, which includes a light source, a filter, a diffusing optical element, a reflective structure, and a heat dissipation device. The light source structure is configured to provide a light beam with a specific wavelength for irradiating the biological sample for photoactivation. The filter is disposed downstream of the light path of the light source, and is configured to reflect infrared light from the light source to reduce thermal radiation. The diffusing optical element is disposed downstream of the optical path of the optical filter, and is constructed to homogenize the light beam passing through the optical filter. The reflective structure is arranged around the biological sample, and is constructed to reflect the light beam passing through the diffusing optical element to enhance the uniform light effect. The heat dissipation device is disposed adjacent to the light source, and is configured to dissipate heat to the light activation device. In this way, the light beam provided by the light source first passes through the filter, then passes through the diffusing optical element, and is reflected by the reflective structure, so that the biological sample is illuminated uniformly, so as to improve the photoactivation efficiency. Block thermal radiation to avoid damage to biological samples.

In one embodiment, the light source includes a light emitting diode circuit board, a halogen lamp, a diode laser, or a through hole light emitting diode.

In one embodiment, the filter comprises a hot mirror or a low pass filter.

In one embodiment, the diffusing optical element comprises a diffusing optical film, a light shaping diffuser, a diffusing plate, ground glass, a dynamic diffuser, or a liquid or liquid crystal speckle reducer.

In one embodiment, the off angle of the diffusing optical element is greater than 60° full width at half maximum.

In one embodiment, the reflective structure is a casing surrounding the biological sample, and a high-reflection material is attached to the inner wall of the casing, and the high-reflection material includes at least one of a reflective film, a highly polished mirror, and a reflective coating or its combination.

In one embodiment, the photoactivation device further includes a sample tray on which a plurality of sample tubes for accommodating biological samples are carried, wherein the reflective structure is correspondingly disposed below the sample tray to enclose the plurality of sample tubes therein.

In one embodiment, a highly reflective material is attached to the bottom surface of the sample tray, and the highly reflective material includes at least one of a reflective film, a highly polished mirror surface, and a reflective coating or a combination thereof.

In one embodiment, the heat dissipation device includes an active heat dissipation device and a passive heat dissipation device, the active heat dissipation device includes a cooling fan, and the passive heat dissipation device includes a heat dissipation fin.

In one embodiment, the photoactivation device further includes a power supply configured to supply power to the light source and the heat dissipation device.

In one embodiment, the photoactivation device further includes a control module configured to control the intensity of the light source, the exposure time of the light source, and the temperature of the photoactivation device.

In order to achieve the above purpose, the embodiment of the present application further provides a control method for the aforementioned photoactivation device, which includes the steps of: distributing the biological sample pre-mixed with the photoactivatable dye into a sample tube, and placing the sample tube in a sample tray of the photoactivation device middle; closing the upper cover of the photoactivation device, and using a timer to set the time required for photoactivation; and turning on the photoactivation device to activate the light source and the heat sink to start the photoactivation process.

Some embodiments embodying the features and advantages of the present case will be described in detail in the following description. It should be understood that this case can have various changes in different aspects, all of which do not depart from the scope of this case, and the descriptions and drawings therein are essentially for illustrative purposes rather than limiting this case.

The embodiment of this case provides a photoactivation system for viability PCR. Since viability PCR uses photoactivatable dyes to enter dead cells with damaged cell membranes, and covalently cross-links with nucleic acids after irradiating visible light to inhibit nucleic acid amplification, only nucleic acids from living cells can be detected by PCR. In the device, the photoactivation reaction is carried out first, and then amplification and detection are carried out in the PCR instrument.

Figure 1 shows a photoactivation system for viability PCR, the system includes a manually adjustable power source 1, a high power resistor assembly 2, and a photoactivation device 3, wherein the manually adjustable power source 1 and the high power resistor assembly 2 can provide the power required by the photoactivation device 3 . In one embodiment, the light activation device 3 is provided with a touch panel 4, and a timer and a current controller can be configured. In addition, ventilation holes 5 are provided on the side and bottom surfaces of the housing of the light activation device 3 for heat dissipation.

In another embodiment, the power supply and related control modules can also be integrated into the photoactivation device 3, so that the photoactivation device 3 can be an independent device.

Figure 2 shows the photoactivation device with the top cover removed to illustrate the manner in which the sample is contained in the photoactivation device. As shown in FIG. 2, the photoactivation device 3 has a detachable sample tray 6, and the sample tray 6 is provided with a plurality of mounting holes for a plurality of sample tubes (PCR tubes) 7 for accommodating biological samples to be carried thereon, The biological sample can be illuminated uniformly in the photoactivation device 3 for photoactivation. In one embodiment, the mounting holes are arranged in at least one row, for example, two rows are shown in FIG. 2 , but it is not limited to this, and can also be arranged in other ways. In one embodiment, the diameter of the mounting hole is slightly smaller than the diameter of the cover of the sample tube 7 , so that the sample tube 7 can be inserted into the mounting hole and carried on the sample tray 6 . In addition, the number of the mounting hole can be marked on the sample tray 6 to facilitate sample identification and process control.

According to the concept of the embodiment of the present application, the main effect of the photoactivation device for viability PCR is that the biological sample can be illuminated uniformly and the illumination time can be reduced, and thermal convection or thermal radiation can be isolated to avoid damage to the biological sample. Therefore, the biological sample of the embodiment of the present invention is placed in a thermally insulated casing, so that the temperature of the biological sample will not be affected by the convection of hot air and the thermal radiation of the high-power light source.

According to the conception of the embodiment of the present case, the photoactivation device for viability PCR mainly includes a light source, an optical component, a cooling module and a control module, wherein the light source provides light of a specific wavelength to illuminate the biological sample for photoactivation, and the optical component is used to activate the biological sample. The beam is uniform to achieve uniform illumination and to isolate thermal radiation. The cooling module includes a heat sink to maintain the temperature of the biological sample and prevent degradation. The control module is used to control the light intensity, light source exposure time, and equipment temperature.

Exemplary embodiments of the photoactivation device of the present application will be described below with reference to Figures 3 to 5, wherein Figure 3 shows a perspective view of the photoactivation device, Figure 4 shows a cross-sectional view of the photoactivation device in section A-A of Figure 3, Figure 5 shows a schematic diagram of part of the internal structure of the photoactivation device. As shown in the figure, the photoactivation device 3 mainly includes a light source 31 , a filter 32 , a diffusing optics 33 , a reflection structure 34 , and a heat dissipation device 35 .

The light source 31 is configured to provide a light beam with a specific wavelength for irradiating the biological sample for photoactivation. In one embodiment, the light source 31 includes a configuration of at least one light emitting diode (LED), such as a light emitting diode circuit board (LED PCB), which is a high power surface mount light emitting diode array (SMD LED). array), with a wavelength range of 460 to 490 nm, reactive with photoactivatable dyes for viability PCR. The configuration of the LEDs can be a rectangular array conforming to the position of the biological sample, wherein the LEDs are arranged in a plurality of rows and columns, but not limited thereto. Of course, the light source in this case is not limited to LED PCBs, and other types of light sources whose spectral range includes the desired visible light spectrum can also be used, such as halogen lamps, diode lasers, through-hole LEDs, or other surface light sources.

The filter 32 is disposed downstream of the light path of the light source 31, and is configured to reflect the infrared light from the light source 31 to reduce thermal radiation. More specifically, in one embodiment, the filter 32 selectively allows specific light beams from the light source 31 with wavelengths in the range of 400 to 800 nm to pass therethrough, and blocks and reflects infrared light, so as to reduce light from high Thermal radiation of the power light source to avoid damage to biological samples. In one embodiment, the filter 32 comprises a hot mirror or a low pass filter.

The diffusing optical element 33 is disposed downstream of the optical path of the optical filter 32 , and is configured to homogenize the light beam passing through the optical filter 32 , that is, the light beam emitted from the light source 31 and passing through the optical filter 32 is evenly distributed. In one embodiment, the diffusing optical element 33 includes a diffusing optical film, which has random microscopic features to make the light beam uniform. Of course, the diffusing optical elements in this case are not limited to diffusing optical films, and other types of diffusing optical elements can also be used, such as light shaping diffusers, diffuser plates, ground glass ), a dynamic diffuser, or a liquid or liquid crystal speckle reducer. A preferred deviation angle for the diffusing optical element is circular and greater than 60° full width at half maximum (FWHM).

The reflective structure 34 is disposed around the biological sample, and is constructed to reflect the light beam passing through the diffusing optical element 33 to enhance the uniform light effect. More specifically, the reflective structure 34 is a casing surrounding the biological sample, and a highly reflective material is attached to the inner wall of the reflective structure 34 to enhance optical scattering, so as to further homogenize the light beam during the photoactivation process, so that the biological sample can be further homogenized. The scattered light inside the reflection structure 34 can be fully utilized to improve the photoactivation efficiency. In one embodiment, the highly reflective material includes a reflective film, but not limited thereto, other types of materials can also be used, such as a highly polished mirror surface or a reflective coating, or a combination of the aforementioned highly reflective materials .

In one embodiment, the reflective structure 34 is a shell formed by four side walls, and is correspondingly disposed under the sample tray 6 to enclose the sample tube 7 containing the biological sample therein, so that the light beam is in the reflective structure 34 . Reflection to achieve uniform illumination of biological samples. In one embodiment, the bottom surface of the sample tray 6 is also attached with a highly reflective material, such as at least one of a reflective film, a highly polished mirror surface, and a reflective coating, or a combination thereof. In other words, the bottom of the sample tube 7 is the light emitting surface, and the four surrounding surfaces and the top surface of the sample tube 7 are all reflective surfaces, so that the uniform light effect can be enhanced. In addition, the reflective structure 34 can also isolate external thermal convection or thermal radiation, so as to avoid damage to the internal biological samples.

In the embodiment shown in FIGS. 4 and 5 , there is still a gap between the bottom of the reflective structure 34 and the diffusing optical element 33 . In another embodiment, as shown in FIG. 6 , the bottom of the reflective structure 34 can directly abut against the optical structure formed by the diffusing optical element 33 and the filter 32 , that is, the reflective structure 34 and the sample tray 6 . The bottom surface and the optical structure formed by the diffusing optical element 33 and the filter 32 together define a closed casing, so that the biological sample is accommodated in the closed casing for the photoactivation process, and is isolated from external thermal convection or thermal radiation .

The heat dissipation device 35 is disposed adjacent to the light source 31. For example, as shown in FIG. 4 and FIG. 5, the heat dissipation device 35 is disposed below the light source 31 and is configured to dissipate heat to the photoactivation device, especially the cooling is generated by the high-power light source. heat to further reduce the temperature of the photoactivated device and avoid damage to biological samples. The heat dissipation device 35 includes an active heat dissipation device 351 and a passive heat dissipation device 352. For example, the passive heat dissipation device 352 is disposed under the light source 31, and the active heat dissipation device 351 is disposed under the passive heat dissipation device 352. This configuration can improve the heat dissipation efficiency. Allows the photoactivation device to cool down evenly.

In one embodiment, the active heat dissipation device 351 includes a cooling fan, and the passive heat dissipation device 352 includes heat dissipation fins, but not limited thereto, other types of temperature control devices, such as thermoelectric coolers, may also be used Or a thermal pipe, etc.

Therefore, according to the configuration of the components in the photoactivation device 3 , the light beam with a specific wavelength provided by the light source 31 will first pass through the filter 32 and then pass through the diffusing optical element 33 to enter the reflection structure 34 , and pass through the reflection structure 34 . Reflection, so that different biological samples in the reflective structure 34 can be illuminated uniformly, so as to improve the photoactivation efficiency, and the filter 32 and the reflective structure 34 block thermal radiation, and supplemented by the cooling effect of the heat sink 35, it can be Reduce the temperature of the photoactivation device 3 to avoid damage to the biological sample.

In one embodiment, the light activation device 3 further includes a power supply 36 configured to supply power to the light source 31 and the active heat dissipation device 351 . In another embodiment, the photoactivation device 3 further includes a control module 37 configured to control the intensity of the light source, the exposure time of the light source, and the temperature of the photoactivation device.

Figures 7A and 7B show the beam path within the photoactivation device. Taking 12 sample tubes arranged in 6 x 2 as an example, Figure 7A shows the six sample tubes on the long side receiving light, and Figure 7B shows the two sample tubes on the short side receiving light. As shown in the figure, the light beam with a specific wavelength emitted from the light source 31 first passes through the filter 32, then passes through the diffusing optical element 33, and then enters the interior of the housing of the reflective structure 34, so as to detect the biological sample in the sample tube 7. photoactivation. The reflective film on the inner wall surface of the reflective structure 34 and the bottom surface of the sample tray 6 will collect and reflect scattered light to increase the optical efficiency of photoactivation.

Figure 8 shows the absorbance of the six sample tubes in the photoactivation device. It can be seen from Fig. 8 that the light absorption of the six sample tubes is quite similar, indicating that the photoactivation device of this embodiment can uniformly illuminate each biological sample (Δt) and different sample heights in each tube during the photoactivation process (Δh), that is, the photoactivation device of the embodiment of the present application can uniformly illuminate the biological samples at different positions, so as to effectively perform photoactivation of each biological sample.

Figure 9 shows the optical power in different sample tubes in the photoactivation device. As can be seen from Fig. 9, the maximum deviation of the optical power measured by the sample tubes at different positions is about 13%. Compared with the deviation of up to 200% of the commercially available device, the photoactivation of one embodiment of the present case The device does provide fairly uniform illumination to each biological sample, allowing efficient photoactivation of each biological sample.

Figure 10 shows the temperature of the biological sample in the photoactivation device, which is monitored by a thermal sensor. It can be seen from Fig. 10 that during the photoactivation process, the temperature of the biological sample can still be kept below 37°C for 30 minutes, which shows that the photoactivation device of the embodiment of this case can effectively dissipate heat and prevent the biological sample from being subjected to high temperature. Influenced by the thermal radiation of the power light source.

Figure 11 shows the analysis of viability PCR, in which the virus sample was photoactivated for 2 minutes in the photoactivation device of one embodiment of the present application, and then amplified in the qPCR system. As can be seen from the amplification curves in Figure 11, the amplification of the photoactivated samples is delayed (the Cq value is larger) compared to the non-photoactivated samples, indicating that effective photoactivation can be clearly seen from the biological samples Differentiate between live and dead cells. In addition, 2 minutes of illumination in the photoactivation device of the embodiment of the present application is sufficient for effective photoactivation.

Figure 12 shows another analysis of viability PCR, in which the virus sample is HCoV-229E, and the photoactivation device of one embodiment of the present case is also photoactivated for 2 minutes, and then amplified in the qPCR system. As can be seen from the amplification curve in Figure 12, when the live (infectious) virus is inactivated by heating (eg, to 75°C), most cells die, so after dye treatment and photoactivation, the resulting Amplification delay. Normal amplification was obtained in inactive 229E purified controls, infectious 229E purified controls, and inactive 229E treated with dye but not photoactivated. Therefore, after effective photoactivation by the photoactivation device of the embodiment of the present invention, it is helpful for the viability PCR to distinguish between live cells and dead cells, thereby detecting the infectivity of the virus.

On the other hand, the embodiment of the present application further provides a control method of a photoactivation device. First, dispense the biological sample premixed with the photoactivated dye into a sample tube and place the sample tube in the mounting hole of the sample tray. The top cover of the photoactivation device was then closed, and a timer was used to set the time required for photoactivation. Then, the photoactivation device is turned on to activate the light source and the heat dissipation device to start the photoactivation process. After the timer expires, the light source will be turned off and the heat sink will continue to operate until the internal temperature of the photoactivation device reaches room temperature. During the photoactivation process, if the internal temperature of the photoactivation device exceeds an acceptable temperature, the photoactivation device will issue a warning signal and terminate the photoactivation process.

To sum up, the embodiments of the present invention provide a photoactivation device and a control method thereof, which utilize novel designs in the fields of optics, heat, electricity, and control to improve photoactivation efficiency. The photoactivation device can uniformly illuminate each location of each biological sample during photoactivation. The specific spacing and arrangement of the light sources, as well as the use of filters and diffusing optics, further contribute to uniform illumination, which not only reduces power consumption, but also avoids localized temperature increases that could damage biological samples. Compared with the prior art, the photoactivation device of the embodiment of the present application uses less light sources, improves the optical performance, and reduces the photoactivation time, so it has better efficiency. The light-activated device helps eliminate false-positive effects caused by nucleic acids from dead cells, thereby assisting in accurate diagnosis of qPCR systems. The independent photoactivation device also includes a timer and temperature control and current control of the light source. The user also does not need to remember the complicated operation process to perform photoactivation, so it is very user-friendly. In addition, the design of this case considers many aspects, including the design and material properties of optical components, the design of heat dissipation structure, the mechanism layout of the light source, and the circuit control of the device, thus improving the optimal performance of the photoactivation device in this case.

Even though the present invention has been described in detail by the above-mentioned embodiments, various modifications can be made by those skilled in the art, but they are all within the scope of the appended claims.

1: Manually adjustable power supply 2: High Power Resistor Assembly 3: Photoactivation device 31: Light source 32: Filter 33: Diffuse optics 34: Reflective Structure 35: Cooling device 351: Active cooling device 352: Passive cooling device 36: Power Supply 37: Control Module 4: Touch panel 5: Ventilation hole 6: Sample Tray 7: Sample tube

Figure 1 shows the photoactivated system for viability PCR. Figure 2 shows the photoactivation device with the cover removed. Figure 3 shows a perspective view of the photoactivation device. FIG. 4 shows a cross-sectional view of the photoactivation device of FIG. 3 taken at section A-A. Figure 5 shows a schematic diagram of part of the internal structure of the photoactivation device. FIG. 6 shows a cross-sectional view of a photoactivation device according to another embodiment. Figures 7A and 7B show the beam path within the photoactivation device. Figure 8 shows the absorbance of the six sample tubes in the photoactivation device. Figure 9 shows the optical power in different sample tubes in the photoactivation device. Figure 10 shows the temperature of the biological sample in the photoactivated device. Figure 11 shows the analysis of viability PCR. Figure 12 shows another analysis of viability PCR.

31: Light source

32: Filter

33: Diffuse optics

34: Reflective Structure

35: Cooling device

351: Active cooling device

352: Passive cooling device

36: Power Supply

37: Control Module

Claims (16)

A light-activated device for viability PCR, comprising: a light source configured to provide a light beam with a specific wavelength for irradiating a biological sample for photoactivation; a filter, disposed downstream of the light path of the light source, and configured to reflect infrared light from the light source to reduce thermal radiation; a diffusing optical element disposed downstream of the optical path of the optical filter and configured to homogenize the light beam passing through the optical filter; a reflective structure, disposed around the biological sample, configured to reflect the light beam passing through the diffusing optical element to enhance the uniform light effect; and a heat dissipation device, disposed adjacent to the light source, and configured to dissipate heat from the light activation device; Thereby, the light beam provided by the light source first passes through the filter, then passes through the diffusing optical element, and is reflected by the reflective structure, so that the biological sample is illuminated uniformly, so as to improve the photoactivation efficiency, and by The filter and the reflective structure block thermal radiation to avoid damage to the biological sample. The photoactivation device for viability PCR according to claim 1, wherein the light source comprises a light-emitting diode circuit board, a halogen lamp, a diode laser, or a through-hole light-emitting diode. The light-activated device for viability PCR of claim 1, wherein the filter comprises a thermal mirror or a low-pass filter. The photoactivation device for vitality PCR according to claim 1, wherein the diffusing optical element comprises a diffusing optical film, a light shaping diffuser, a diffusing plate, a frosted glass, a dynamic diffuser, Or a liquid or liquid crystal speckle reducer. The photoactivation device for viability PCR according to claim 1, wherein an off-angle of the diffusing optical element is greater than 60° full width at half maximum. The photoactivation device for viability PCR according to claim 1, wherein the reflective structure is a casing surrounding the biological sample, and a highly reflective material is attached to the inner wall of the casing. The light-activated device for viability PCR according to claim 6, wherein the highly reflective material comprises at least one of a reflective film, a highly polished mirror surface, and a reflective coating, or a combination thereof. The photoactivation device for viability PCR according to claim 6, further comprising a sample tray on which a plurality of sample tubes for accommodating the biological sample are supported, wherein the reflective structure is correspondingly disposed below the sample tray , so as to enclose the plurality of sample tubes therein. The photoactivation device for viability PCR according to claim 8, wherein a bottom surface of the sample tray is attached with a highly reflective material. The photoactivation device for viability PCR according to claim 9, wherein the highly reflective material comprises at least one of a reflective film, a highly polished mirror surface, and a reflective coating, or a combination thereof. The photoactivation device for active PCR according to claim 1, wherein the heat dissipation device includes an active heat dissipation device and a passive heat dissipation device. The light-activated device for active PCR of claim 11, wherein the active heat dissipation device includes a cooling fan. The photoactivation device for active PCR according to claim 11, wherein the passive heat dissipation device comprises a heat dissipation fin. The light activation device for active PCR according to claim 1, further comprising a power supply configured to supply power to the light source and the heat dissipation device. The photoactivation device for vitality PCR according to claim 1, further comprising a control module configured to control the intensity of the light source, the exposure time of the light source, and the temperature of the photoactivation device. A method for controlling a light-activated device for vitality PCR as claimed in claim 1, comprising the steps of: Dispensing the biological sample premixed with a photoactivatable dye into a sample tube and placing the sample tube in a sample tray of the photoactivation device; closing a top cover of the photoactivation device, and using a timer to set the time required for photoactivation; and The photoactivation device is turned on to activate the light source and the heat dissipation device to start the photoactivation process.

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