TWI797783B - 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 for 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, simple pretreatment of samples, because photoreactive reagents will selectively enter dead cells with damaged cell membranes, and covalently cross-link with nucleic acids after irradiation with visible light to 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, so that the photoactivation of the biological samples in the tube is not uniform. Uneven illumination can also lead to poor photoactivation efficiency for some biological samples. High-power light sources can cause localized heat transfer to biological samples, which may reduce photoactivation for viability PCR.

The second problem is the long exposure time. Existing systems currently on the market have a minimum exposure time of 10-30 minutes. Longer photoactivation times may damage biological samples due to the heat accumulated by the light source.

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

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

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

To achieve the above purpose, the embodiment of the present case provides a photoactivation device for viability PCR, including a light source, a filter, a diffuse optical element, a reflective structure, and a heat dissipation device. The light source is structured to provide light beams with specific wavelengths for irradiating biological samples for photoactivation. The optical filter is arranged downstream of the light path of the light source, and is structured to reflect infrared light from the light source to reduce heat radiation. The diffuse optical element is arranged downstream of the optical path of the optical filter, and is structured to homogenize the light beam passing through the optical filter. The reflective structure is arranged around the biological sample, and is structured to reflect the light beam passing through the diffuse optical element to enhance the uniform light effect. The heat dissipation device is adjacent to the light source and is configured to dissipate heat from the photoactivation device. In this way, the light beam provided by the light source first passes through the filter and then through the diffuse optical element, and is reflected by the reflective structure, so that the biological sample can be uniformly illuminated to improve the photoactivation efficiency, and the optical filter and reflective structure Block thermal radiation to avoid damage to biological samples.

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

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

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

In one embodiment, the deflection angle of the diffuse optical element is greater than 60° FWHM.

In one embodiment, the reflective structure is a shell surrounding the biological sample, and a highly reflective material is attached to the inner wall of the shell, and the high reflective 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 containing biological samples are carried, wherein the reflective structure is correspondingly disposed under the sample tray to surround the plurality of sample tubes.

In one embodiment, the bottom surface of the sample tray is attached with a high reflective material, and the high reflective material includes at least one of a reflective film, a highly polished mirror, 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 heat dissipation fins.

In one embodiment, the photoactivation device further includes a power supply configured to supply power to the light source and the cooling 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 this case further provides a control method of the aforementioned photoactivation device, including the steps of: distributing the biological sample pre-mixed with the photoactivation dye into the sample tube, and placing the sample tube in the sample tray of the photoactivation device middle; close the upper cover of the photoactivation device, and use the timer to set the required time for photoactivation; and turn on the photoactivation device to start the photoactivation process by starting the light source and the cooling device.

Some embodiments embodying the features and advantages of the present application will be described in detail in the description in the following paragraphs. It should be understood that the present case can have various changes in different aspects without departing from the scope of the present case, and the descriptions and drawings therein are used for illustration in nature rather than limiting the present case.

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

Figure 1 shows a photoactivation system for vitality PCR, this system includes a manually adjustable power supply 1, a high-power resistor assembly 2, and a photoactivation device 3, wherein the manually adjustable power supply 1 and high-power resistor assembly 2 can provide the power required by the photoactivation device 3 . In one embodiment, the photoactivation device 3 is provided with a touch panel 4, and a timer and a current controller can be configured. In addition, vent holes 5 are provided on the side and bottom of the housing of the photoactivation 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 becomes a device capable of independent operation.

Figure 2 shows the photoactivation device with the top cover removed to illustrate how the sample is accommodated in the photoactivation device. As shown in Figure 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 carrying a plurality of sample tubes (PCR tubes) 7 containing biological samples on it. The biological sample can be uniformly irradiated with light in the photoactivation device 3 for photoactivation. In one embodiment, the mounting holes are arranged in at least one row, for example, two rows as shown in FIG. 2 , but not limited thereto, and can also be configured 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 holes can be marked on the sample tray 6 to facilitate sample identification and process control.

According to the idea of the embodiment of this case, the main function of the photoactivation device used for the activity PCR is to uniformly illuminate the biological samples and reduce the illumination time, and to isolate the thermal convection or thermal radiation to avoid damage to the biological samples. Therefore, the biological sample in this embodiment is placed in a heat-insulated casing, so that the temperature of the biological sample will not be affected by hot air convection and heat radiation from a high-power light source.

According to the idea of the embodiment of this case, the photoactivation device for vitality 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 irradiate the biological sample for photoactivation, and the optical component is used to use The beam is uniform to achieve uniform illumination and thermal radiation isolation. The cooling module includes heat dissipation devices to maintain the temperature of biological samples and prevent degradation. The control module is used to control light intensity, light source exposure time, and equipment temperature.

The exemplary embodiment of the photoactivation device of this case will be described below in conjunction with Figs. 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 reflective structure 34 , and a heat sink 35 .

The light source 31 is configured to provide light beams with specific wavelengths for irradiating biological samples for photoactivation. In one embodiment, the light source 31 includes an arrangement 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, reacts 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 PCB, and other types of light sources whose spectral range includes the required 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 infrared light from the light source 31 to reduce heat radiation. More specifically, in one embodiment, the optical filter 32 selectively allows specific light beams from the light source 31 with a wavelength range of 400 to 800 nm to pass through, and blocks and reflects infrared light to reduce light from high Thermal radiation of the power source avoids damage to biological samples. In one embodiment, the filter 32 includes a hot mirror or a low pass filter.

The diffuse optical element 33 is disposed downstream of the optical path of the optical filter 32 , and is structured to homogenize the light beam passing through the optical filter 32 , that is, evenly distribute the light beam emitted from the light source 31 and passing through the optical filter 32 . In one embodiment, the diffusing optical element 33 includes a diffusing optical film having random microscopic features to make the light beam uniform. Of course, the diffuse optical element in this case is not limited to the diffuse optical film, other types of diffuse optical elements, such as light shaping diffuser, diffuser plates, ground glass ), a dynamic diffuser, or a liquid or liquid crystal speckle reducer. The preferred deviation angle of the diffuse 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 structured to reflect the light beam passing through the diffuse optical element 33 to enhance the uniform light effect. More specifically, the reflective structure 34 is a shell that surrounds the biological sample, and a highly reflective material is attached to the inner wall of the reflective structure 34, which can enhance optical scattering, so as to further homogenize the light beam during the photoactivation process, so that the biological sample Scattered light inside the reflective structure 34 can be fully utilized to improve photoactivation efficiency. In one embodiment, the highly reflective material includes a reflective film, but it is not limited thereto. Other types of materials can also be used, such as highly polished mirror surfaces or reflective coatings, or a combination of the aforementioned highly reflective materials. .

In one embodiment, the reflective structure 34 is a shell formed by four side walls, which are correspondingly arranged under the sample tray 6 to surround the sample tube 7 containing the biological sample, so that the light beam is inside the reflective structure 34 reflection for 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, 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 surrounding four surfaces and the top surface of the sample tube 7 are all five reflective surfaces, so that the uniform light effect can be enhanced. In addition, the reflective structure 34 can also isolate external heat convection or heat radiation, so as to prevent internal biological samples from being damaged.

In the embodiments shown in FIG. 4 and FIG. 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 diffuse optical element 33 and the optical filter 32, that is, the reflective structure 34 and the sample tray 6 The bottom surface of the bottom surface and the optical structure formed by the diffuse optical element 33 and the filter 32 jointly define a closed shell, so that the biological sample is contained in the closed shell for the photoactivation process, and the external heat convection or heat radiation is isolated .

The heat dissipation device 35 is adjacent to the light source 31. For example, as shown in Figures 4 and 5, the heat dissipation device 35 is arranged below the light source 31, and is structured to dissipate heat from the light activation device, especially the cooling produced by the high-power light source. Heat to further reduce the temperature of the photoactivation device and avoid damage to biological samples. The cooling device 35 includes an active cooling device 351 and a passive cooling device 352. For example, the passive cooling device 352 is arranged under the light source 31, and the active cooling device 351 is arranged under the passive cooling device 352. Such a configuration can improve heat dissipation efficiency. Allow the photoactivation device to cool down evenly.

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

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

In one embodiment, the photoactivation device 3 further includes a power supply 36 configured to supply power to the light source 31 and the active cooling 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 paths within the photoactivation device. Taking 12 sample tubes arranged in 6 x 2 as an example, Figure 7A shows the situation of six sample tubes on the long side receiving light, and Figure 7B shows the situation of two sample tubes on the short side receiving light. As shown in the figure, the light beam of a specific wavelength emitted from the light source 31 first passes through the optical filter 32, then passes through the diffuse optical element 33, and then enters the inside of the housing of the reflection structure 34 to detect the biological sample in the sample tube 7. Perform photoactivation. The reflective film on the inner wall surface of the reflective structure 34 and the bottom surface of the sample tray 6 can collect and reflect scattered light to increase the optical efficiency of photoactivation.

Figure 8 shows the absorbance of six sample tubes in the photoactivation device. It can be seen from Figure 8 that the light absorption of the six sample tubes is quite similar, showing that the photoactivation device of the embodiment of this case can uniformly irradiate 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 irradiate the biological samples at different positions, so as to effectively photoactivate each biological sample.

Figure 9 shows the optical power in different sample tubes in the photoactivation setup. It can be seen from Figure 9 that the maximum deviation of the optical power measured by the sample tubes at different positions is about 13%. The device does provide fairly uniform illumination of each biological sample, allowing for efficient photoactivation of each biological sample.

Figure 10 shows the temperature of a biological sample in a photoactivation device, which is monitored by a thermal sensor. It can be seen from Figure 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 exposed to high temperature. Affected by the thermal radiation of the power source.

Figure 11 shows the analysis of activity PCR, wherein the virus sample is amplified in the qPCR system after being photoactivated for 2 minutes by the photoactivation device of one embodiment of the present case. From the amplification curve in Figure 11, it can be seen that the amplification of the photoactivated sample is delayed (larger Cq value) compared to the non-photoactivated sample, showing that effective photoactivation can be clearly obtained from biological samples. Differentiate between live and dead cells. In addition, 2 minutes of light irradiation in the photoactivation device of the embodiment of the present case is sufficient for effective photoactivation.

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

On the other hand, the embodiment of the present application further provides a control method of the photoactivation device. First, biological samples premixed with photoactivatable dyes are dispensed into sample tubes and the sample tubes are placed in the mounting holes of the sample tray. Then close the upper cover of the photoactivation device, and use a timer to set the required time for photoactivation. Subsequently, the photoactivation device is turned on to activate the light source and the cooling device to start the photoactivation process. After the timer expires, the light source will be turned off, and the cooling device 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 embodiment of this case provides a photoactivation device and its control method, which uses novel designs in the fields of optics, heat, electricity, and control to improve photoactivation efficiency. The photoactivation device can uniformly irradiate every position of every biological sample during the photoactivation process. The specific spacing and arrangement of light sources, as well as the application of filters and diffuse optics, further contribute to uniform illumination, reducing power consumption and avoiding localized temperature rises that could damage biological samples. Compared with the conventional technology, 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 photoactivation device helps to eliminate the false positive effect caused by dead cell nucleic acid, thereby assisting the accurate diagnosis of the qPCR system. The independent photoactivation device also includes temperature control and current control of the timer and light source. The user does not need to remember the complicated operation process to perform photoactivation, so it is very user-friendly. In addition, many aspects were considered in the design of this case, including the design of optical components and material properties, heat dissipation structure design, mechanism layout of the light source, and circuit control of the device, thus improving the best performance of the photoactivation device of this case.

Even though the present invention has been described in detail by the above-mentioned embodiments, it can be modified in various ways by those skilled in the art, all of which are within the scope of the attached patent application.

1: Manually adjustable power supply 2: High Power Resistor Assembly 3: Photoactivation device 31: light source 32: Optical 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 photoactivation system used for viability PCR. Figure 2 shows the photoactivated device with the top cover removed. Figure 3 shows a perspective view of the photoactivation device. Fig. 4 shows a cross-sectional view of the photoactivation device in Fig. 3 at section A-A. FIG. 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 of another embodiment. Figures 7A and 7B show the beam paths within the photoactivation device. Figure 8 shows the absorbance of six sample tubes in the photoactivation device. Figure 9 shows the optical power in different sample tubes in the photoactivation setup. Figure 10 shows the temperature of the biological sample in the photoactivation device. Figure 11 shows the analysis of the viability PCR. Figure 12 shows another analysis of the viability PCR.

31: light source

32: Optical 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 (15)

A photoactivation device for vitality PCR, comprising: a light source, structured to provide a light beam with a specific wavelength for irradiating a biological sample for photoactivation; a filter, arranged downstream of the light path of the light source, Structured to reflect infrared light from the light source to reduce heat radiation; a diffuse optical element, arranged downstream of the light path of the optical filter, structured to homogenize the light beam passing through the optical filter; a sample tray, structured A plurality of sample tubes accommodating the biological sample are carried on it; a reflective structure is arranged around the biological sample, and is structured to reflect the light beam passing through the diffuse optical element to enhance the uniform light effect, wherein the The reflective structure is correspondingly arranged under the sample tray, so as to surround the plurality of sample tubes; and a heat dissipation device is adjacent to the light source, and is configured to dissipate heat from the photoactivation device; thereby, the light source provides The light beam first passes through the optical filter and then passes through the diffuse optical element, and is reflected by the reflective structure, so that the biological sample is illuminated uniformly to improve the photoactivation efficiency, and the optical filter and the reflective The structure blocks thermal radiation to avoid damage to the biological sample. The photoactivation device for activity PCR according to claim 1, wherein the light source includes a light emitting diode circuit board, a halogen lamp, a diode laser, or a through hole light emitting diode. The photoactivation device for viability PCR as claimed in claim 1, wherein the optical filter comprises a thermal mirror or a low-pass filter. The photoactivation device for active PCR as described in claim 1, wherein the diffuse optical element includes a diffuse optical film, a light shaping diffuser, a diffuser plate, a ground glass, a dynamic diffuser, or a liquid or liquid crystal speckle reducer. The photoactivation device for activity PCR as claimed in claim 1, wherein an off angle of the diffusing optical element is greater than 60° half maximum width. The photoactivation device for live PCR according to claim 1, wherein the reflective structure is a shell surrounding the biological sample, and a high reflective material is attached to the inner wall of the shell. The photoactivation device for active PCR according to claim 6, wherein the highly reflective material comprises at least one of a reflective film, a highly polished mirror, and a reflective coating or a combination thereof. The photoactivation device for activity PCR according to claim 1, wherein a high reflective material is pasted on a bottom surface of the sample tray. The photoactivation device for active PCR according to claim 8, wherein the highly reflective material comprises at least one of a reflective film, a highly polished mirror, and a reflective coating or a combination thereof. The photoactivation device for vitality PCR according to claim 1, wherein the cooling device includes an active cooling device and a passive cooling device. The photoactivation device for vitality PCR according to claim 10, wherein the active cooling device includes a cooling fan. The photoactivation device for activity PCR according to claim 10, wherein the passive cooling device includes a cooling fin. The photoactivation device for vitality PCR as described in Claim 1 further includes a power supply configured to supply power to the light source and the cooling device. The photoactivation device for activity PCR as described in claim 1 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. A method for controlling a photoactivation device for viability PCR as described in claim 1, comprising the steps of distributing the biological sample premixed with a photoactivation dye into the sample tube, and placing the sample tube in the In the sample tray of the photoactivation device; close an upper cover of the photoactivation device, and use a timer to set the time required for photoactivation; and open the photoactivation device to start the light source and the cooling device to start the photoactivation process.

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