TWI787784B - OPTICAL CALIBRATION TOOL FOR QPCR STYSTEM, FABRICATION METHOD OF OPTICAL CALIBRATION TOOL, AND CALIBRATION METHOD OF qPCR STYSTEM - Google Patents

Abstract

An optical calibration tool for a qPCR system includes a transparent substrate and fluorescent particles. The transparent substrate is solid, and the fluorescent particles are uniformly distributed in the transparent substrate.

Description

Optical correction tool for real-time quantitative polymerase chain reaction (qPCR) system, preparation method thereof, and correction method for qPCR system

The disclosure relates to an optical correction tool for a real-time quantitative polymerase chain reaction (qPCR) system, a preparation method thereof, and a correction method for the qPCR system.

In the optical calibration tools of existing real-time quantitative polymerase chain reaction (qPCR) systems, organic fluorescent dyes are often used as excited materials. However, organic fluorescent dyes often suffer from the problems that the fluorescence intensity decays too quickly, and the luminous intensity is affected by environmental light source and temperature and is unstable. Therefore, the optical correction tool of the existing qPCR system is not easy to be reused, the preparation method is complicated, and the accuracy is difficult to improve.

In view of this, how to provide an optical correction tool with high precision, reusability, and simple preparation method is still one of the goals that the industry needs to study urgently.

One technical aspect of the present disclosure is an optical calibration tool for a real-time quantitative polymerase chain reaction system.

In an embodiment of the present disclosure, the optical calibration tool of the RTQPCR system includes a transparent substrate and fluorescent particles. Transparent substrates are solid. Fluorescent particles are uniformly dispersed in the transparent substrate.

In an embodiment of the present disclosure, the optical calibration tool of the RTQPCR system further includes a container configured to accommodate the transparent substrate and the fluorescent particles.

In an embodiment of the present disclosure, the difference between the refractive index of the container and the refractive index of the transparent substrate falls within a range of about 0.2 to 0.3.

In an embodiment of the present disclosure, the fluorescent particles are configured to be excited by a light source, and the wavelength of the light source falls within a range of about 400 nm to 800 nm.

In an embodiment of the present disclosure, the fluorescent particles include one or any combination of quantum dots, fluorescent particles, silicon nanospheres, or nanodiamonds.

In an embodiment of the present disclosure, the average diameter of the fluorescent particles falls within a range of about 1 micron to 10 microns.

In an embodiment of the present disclosure, the concentration of fluorescent particles falls within a range of about 1 ppm to 10000 ppm.

In an embodiment of the present disclosure, the material of the transparent substrate includes polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate or resin.

In an embodiment of the present disclosure, the viscosity of the transparent substrate in a liquid state is less than 5500 centipoise (cP).

Another technical aspect of the present disclosure is a preparation method of an optical calibration tool for a real-time quantitative polymerase chain reaction system.

In an embodiment of the present disclosure, a method for preparing an optical calibration tool for a qPCR system includes: preparing a liquid transparent substrate; mixing the liquid transparent substrate and fluorescent particles to form a mixture; vacuumizing the mixture; and solidifying the mixture to form a solid state Transparent substrate, in which fluorescent particles are uniformly dispersed in a solid transparent substrate.

In an embodiment of the present disclosure, the step of mixing the liquid transparent substrate and the fluorescent particles is performed by centrifugal stirring, and the rotational speed of the centrifugal stirring is less than 100 rpm.

In an embodiment of the present disclosure, the preparation method of the optical calibration tool of the qPCR system further includes filling the mixture into a container before curing the mixture.

In an embodiment of the present disclosure, the preparation method of the optical calibration tool for the qPCR system further includes shaping the solid transparent substrate by diamond engraving.

In an embodiment of the present disclosure, the Shore hardness of the solid transparent substrate falls in the range of about 90D to 100D.

In one embodiment of the present disclosure, curing the mixture is performed by moisture curing, heat curing or light curing.

Another technical aspect of the present disclosure is a calibration method for a qPCR system.

In an embodiment of the present disclosure, the calibration method of the qPCR system includes placing an optical calibration tool in the qPCR system; turning on the light source of the qPCR system to excite the fluorescent particles in the optical calibration tool; comparing the emitted light of the fluorescent particles with the built-in data and generate comparison results; and adjust the settings of the qPCR system according to the comparison results.

In an embodiment of the present disclosure, the setting of the qPCR system includes the light intensity of the light source. In an embodiment of the present disclosure, the wavelength of the light source of the qPCR system corresponds to the wavelength of the excitation light of the fluorescent particles.

In the above embodiments, the luminous intensity of the fluorescent particles has high stability, so the optical correction tool disclosed in the present disclosure is reusable, not prone to photobleaching, and has high accuracy. In addition, the excitation light intensity of the fluorescent particles can have a high signal-to-noise ratio (S/N Ratio), which can improve the optical calibration performance of the qPCR system.

Several embodiments of the present invention will be disclosed in the following figures. For the sake of clarity, many practical details will be described together in the following description. It should be understood, however, that these practical details should not be used to limit the invention. That is, in some embodiments of the present invention, these practical details are unnecessary. In addition, for the sake of simplifying the drawings, some well-known structures and components will be shown in a simple and schematic manner in the drawings. Also, the thicknesses of layers and regions in the drawings may be exaggerated for clarity, and the same reference numerals denote the same elements in the description of the drawings.

FIG. 1 is a schematic diagram of an optical calibration tool 100 of a qPCR system according to an embodiment of the present disclosure. The optical calibration tool 100 includes a transparent substrate 110 , a plurality of fluorescent particles 120 and a container 130 . The transparent substrate 110 is solid. The fluorescent particles 120 are uniformly dispersed in the transparent substrate 110 . The container 130 is configured to accommodate the transparent substrate 110 and the fluorescent particles 120 . The optical calibration tool 100 is used to calibrate the optical quality of a qPCR system. The fluorescent particles 120 are configured to be excited by the light source emitted by the qPCR system. The container 130 is an optical grade container, such as a centrifuge tube. Therefore, the light source of the qPCR system can penetrate the container 130 to excite the fluorescent particles 120 in the transparent substrate 110, so that the fluorescent particles 120 emit light of a specific wavelength for performing calibration of the qPCR system.

The material of the transparent substrate 110 includes polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate or resin. For example, the refractive index of the transparent substrate 110 may be 1.40 to 1.7, and the viscosity of the transparent substrate 110 in a liquid state is less than 5500 centipoise. With this feature, bubbles can be avoided when the fluorescent particles 120 are mixed into the liquid transparent substrate 110 . In this way, air bubbles or impurities can be prevented from remaining in the transparent substrate 110 and reducing the optical calibration performance of the qPCR system.

The difference between the refractive index of the container 130 and the refractive index of the transparent substrate 110 falls in the range of about 0.2 to 0.3. In this way, the aberration problem caused by the difference in the refractive index of the medium can reduce the optical correction accuracy of the qPCR system.

2A to 2D are schematic diagrams of the excitation light and emission light spectra of the fluorescent particles 120 of the optical calibration tool of the qPCR system according to an embodiment of the present disclosure. The fluorescent particles 120 are configured to be excited by the light source emitted by the qPCR system, and the wavelength of the light source falls in the range of about 400 nm to 800 nm. In other words, the fluorescent particles 120 are configured to emit visible light, and the wavelength of the excitation light of the fluorescent particles 120 corresponds to the wavelength of the light source of the qPCR system.

For example, a part of the fluorescent particles 120 can be excited by blue light and emit blue-green light. As shown in FIG. 2A, when the qPCR system can be configured to emit light source EX1 (blue light) with a peak wavelength of about 470 nm, the fluorescent particles 120 can be excited to emit light with a peak wavelength of about 520 nm. EM1 (blue-green light). As shown in Figure 2B, another part of the fluorescent particles 120 can be excited by the light source EX2 (blue-green light) with a peak wavelength of about 520 nm to emit light EM2 (green light) with a peak wavelength of about 570 nm. ). As shown in FIG. 2C , another part of the fluorescent particles 120 can be excited by the light source EX3 (yellow light) with a peak wavelength of about 590 nm to emit light EM3 (yellow light) with a peak wavelength of about 590 nm. As shown in FIG. 2D , another part of the fluorescent particles 120 can be excited by the light source EX4 (red light) with a peak wavelength of about 630 nm to emit light EM4 (red light) with a peak wavelength of about 640 nm.

According to the above, by using the fluorescent particles 120 that can be excited by the light source of the qPCR system, the light emitted by the fluorescent particles 120 can be received for optical calibration of the qPCR system. The detailed calibration steps will be described in detail later. It should be understood that the above light source wavelengths are just examples, and those skilled in the art can select appropriate fluorescent particles 120 according to the light source wavelengths of the qPCR system.

The fluorescent particles 120 may include one of Quantum Dots, Fluorescent Particles, Silica Nanoparticles, or Nanodiamonds, or any combination thereof. The average diameter of the fluorescent particles 120 falls in the range of about 1 micron to 10 microns. The concentration of fluorescent particles 120 falls within a range of about 1 part per million (ppm) to 10000 ppm.

3A to 3C are graphs showing the relationship between the concentration of fluorescent particles and the emitted light intensity of the optical calibration tool of the qPCR system according to another embodiment of the present disclosure. FIG. 3A is a graph corresponding to the relationship between the concentration of fluorescent particles 120 excited by blue light and the intensity of emitted light shown in FIG. 2A. For example, as shown in FIG. 3A , the concentration of the fluorescent particles 120 in the range of 0 to 1000 ppm is proportional to the intensity of the fluorescent signal, and the correlation coefficient R1 of the linear regression analysis is about 1. FIG. 3B is a diagram corresponding to the relationship between the concentration of the fluorescent particles 120 excited by yellow light and the emitted light intensity (mV) shown in FIG. 2C. The concentration of the fluorescent particles 120 in the range of 0 to 1000 ppm is directly proportional to the intensity of the fluorescent signal, and the correlation coefficient R2 of the linear regression analysis is about 0.9853. FIG. 3C is a graph corresponding to the relationship between the concentration of fluorescent particles 120 excited by red light and the intensity of emitted light shown in FIG. 2D. The concentration of the fluorescent particles 120 in the range of 0 to 1000 ppm is directly proportional to the intensity of the fluorescent signal, and the correlation coefficient R3 of the linear regression analysis is about 0.997. Therefore, by Controlling the concentration of the fluorescent particles 120 in the range of 0 to 1000 ppm can precisely control the intensity of the excitation light of the fluorescent particles 120 .

FIG. 4 shows the emitted light intensity and signal-to-noise ratio of the fluorescent particles 120 of the optical calibration tool of the qPCR system according to another embodiment of the present disclosure. In FIG. 4 , different fluorescent particles 120 with a concentration of 1000 ppm are taken as an example. In the example shown in FIG. 4 , the excitation light intensity of the fluorescent particles 120 that can emit blue light, green light, yellow light and red light can all have a high signal-to-noise ratio, so as to improve the optical calibration performance of the qPCR system.

FIG. 5 is a schematic diagram of an optical calibration tool 200 of a qPCR system according to another embodiment of the present disclosure. The optical calibration tool 200 includes a transparent substrate 210 and fluorescent particles 120 . The optical calibration tool 200 does not have a container for accommodating the transparent substrate 210 and the fluorescent particles 120 . The transparent substrate 210 of the optical calibration tool 200 can be shaped by high-precision machining such as a diamond cutting machine, so that the outer surface of the transparent substrate 210 can meet the quality required for optical calibration. The transparent substrate 210 of the optical calibration tool 200 can have different shapes, such as a centrifuge tube shape similar to the optical calibration tool 100 shown in FIG. 1 , conical shape, cylindrical shape, square shape, flat shape, etc. In other words, as long as the shape of the optical calibration tool 200 is compatible with the space where the optical calibration tool 200 is placed in the qPCR system.

FIG. 6 is the thermal test data of the optical calibration tool of the qPCR system according to an embodiment of the present disclosure. The data in FIG. 6 is the experimental data obtained after placing the optical calibration tool 100 shown in FIG. 1 in a 65-degree environment for two days. It can be seen from the experimental data in Fig. 6 that in this embodiment, the variation of the emitted light intensity of the fluorescent particles 120 that emit yellow light and blue light is less than 10%, and the emission of fluorescent particles 120 with a concentration of 1000ppm The variation in light intensity is approximately less than 2%. Therefore, the fluorescent particles 120 of the optical calibration tool of the present disclosure can withstand high temperature without fading phenomenon of luminous intensity.

FIG. 7 is photostability data of an optical calibration tool of a qPCR system according to an embodiment of the present disclosure. The data in FIG. 7 are experimental data obtained by placing the optical calibration tool 100 shown in FIG. 1 under ambient light conditions for about five months. It can be seen from the experimental data in FIG. 7 that the emitted light intensity of the fluorescent particles 120 emitting red light and yellow light remains approximately stable. Therefore, the fluorescent particles 120 of the optical calibration tool of the present disclosure can be reused for a long time, so as to achieve the effect of reducing waste of resources.

To sum up, since the luminous intensity of the fluorescent particles 120 has high stability, the optical calibration tool of the present disclosure is reusable, less prone to photobleaching, and has high accuracy. In addition, the design of the container 130 or the shaped transparent substrate 110 allows users to use it conveniently and reduces the complexity of the optical calibration steps.

FIG. 8 is a flowchart of a method 300 for preparing an optical calibration tool of a qPCR system according to an embodiment of the present disclosure. Step S11 of the method 300 starts with preparing a liquid transparent substrate. Next, step S12 is to mix the liquid transparent substrate and a plurality of fluorescent particles to form a mixture. Next, step S13 is to vacuumize the mixture. Finally, step S14 is curing the mixture to form a solid transparent substrate, wherein the fluorescent particles are evenly dispersed in the solid transparent substrate.

9 to 10 are schematic diagrams of the intermediate steps of the method 300 for preparing the optical calibration tool of the qPCR system of FIG. 8 . As shown in FIG. 9 , in steps S11 to S12 , a liquid transparent substrate 110L is prepared, and fluorescent particles 120 are added into the liquid transparent substrate 110L. Next, the transparent substrate 110L and the fluorescent particles 120 are mixed. In this embodiment, the fluorescent particles 120 in the transparent substrate 110L are stirred by centrifugal force provided by the centrifuge 140 to form a mixture. The rotational speed of centrifugal stirring is less than 100 rpm. In other embodiments, the fluorescent particles 120 in the transparent substrate 110L may also be stirred by an electric mixer.

As shown in FIG. 10 , in step S13 , the mixture is vacuumed to remove air bubbles G in the transparent substrate 110L. Next, the mixture is filled into container 130 before curing the mixture. Finally, in step S14 , the mixture is cured by light L to form the optical calibration tool 100 . In other embodiments, curing the mixture can also be performed by moisture curing or thermal curing.

The Shore hardness of the solid transparent substrate 110 falls in the range of about 90D to 100D. In addition, in other embodiments, taking the optical calibration tool 200 as an example, after the mixture is solidified, the solid transparent substrate 110 can be shaped by diamond engraving without being placed in a container.

FIG. 11 is a flowchart of an optical calibration method 400 of a qPCR system according to an embodiment of the present disclosure. Step S21 of the method 400 starts with placing the optical calibration tool in the qPCR system. Next, step S22 is to turn on the light source of the qPCR system to excite the fluorescent particles in the optical calibration tool. Next, in step S23, the qPCR system receives the emitted light signal of the fluorescent particles, compares the emitted light intensity of the fluorescent particles with the built-in data, and generates a comparison result. For example, the built-in data includes the normal range of light intensity emitted by fluorescent particles corresponding to each wavelength light source of the qPCR system. Therefore, in step S24, it can be determined whether the intensity of the emitted light emitted by the fluorescent particles in the optical calibration tool that can be excited by excitation lights of different wavelengths is within a normal range.

If the comparison result obtained in step S24 shows that the emitted light intensity is within the normal range, proceed to step S25 to return the qPCR system to the default mode. Next, proceed to step S26, and end the calibration procedure of the qPCR system.

If the comparison result obtained in step S24 shows that the emitted light intensity is not within the normal range, adjust the settings of the qPCR system according to the comparison result. The settings of the qPCR system include the light intensity of the light source for each wavelength. For example, step S24 may proceed to step S27 to adjust the light source intensity of the qPCR system. Next, proceed to step S28 to determine whether the number of light source intensity adjustments of the qPCR system is less than 10. If the result of step S28 is yes, return to step S24 to compare the adjusted emitted light intensity with the built-in data. If the result of step S28 is negative, proceed to step S29 to execute the maintenance procedure of the qPCR system.

In summary, due to the high stability of the luminous intensity of the fluorescent particles, the optical calibration tool of the present disclosure can be used repeatedly with high accuracy. The design of the container or the shaped transparent substrate allows the user to use it conveniently and reduces the complexity of the optical calibration steps. By controlling the concentration of fluorescent particles in the range of 0 to 1000 ppm, the light intensity of the fluorescent particle excitation light can be precisely controlled. In addition, the excitation light intensity of the fluorescent particles can have a high signal-to-noise ratio, which can improve the optical calibration performance of the qPCR system. The viscosity of the transparent base material is less than 5500 centipoise, which can avoid air bubbles when mixing the fluorescent particles into the liquid transparent base material. The difference between the refractive index of the container and the transparent substrate falls in the range of about 0.2 to 0.3, which can reduce the optical correction accuracy of the qPCR system due to the aberration problem caused by the difference in the refractive index of the medium.

Although the present invention has been disclosed above in terms of implementation, it is not intended to limit the present invention. Anyone skilled in this art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection of the present invention The scope shall be defined by the appended patent application scope.

100,200: Optical correction tools

110, 110L, 210: transparent substrate

120: fluorescent particles

130: container

140: Centrifuge

300,400: method

EX1, EX2, EX3, EX4: excitation light

EM1, EM2, EM3, EM4: emit light

R1, R2, R3: correlation coefficient

G: Bubbles

S11~S14, S21~S29: steps

FIG. 1 is a schematic diagram of an optical calibration tool of a qPCR system according to an embodiment of the present disclosure. 2A to 2D are schematic diagrams of the excitation light and emission light spectra of the fluorescent particles of the optical calibration tool of the qPCR system according to an embodiment of the present disclosure. 3A to 3C are graphs showing the relationship between the concentration of fluorescent particles and the emitted light intensity of the optical calibration tool of the qPCR system according to another embodiment of the present disclosure. FIG. 4 shows the emitted light intensity and signal-to-noise ratio of the fluorescent particles of the optical calibration tool of the qPCR system according to another embodiment of the present disclosure. FIG. 5 is a schematic diagram of an optical calibration tool of a qPCR system according to another embodiment of the present disclosure. FIG. 6 is the thermal test data of the optical calibration tool of the qPCR system according to an embodiment of the present disclosure. FIG. 7 is photostability data of an optical calibration tool of a qPCR system according to an embodiment of the present disclosure. FIG. 8 is a flow chart of the preparation method of the optical calibration tool of the qPCR system according to an embodiment of the present disclosure. 9 to 10 are schematic diagrams of the intermediate steps of the preparation method of the optical calibration tool of the qPCR system in FIG. 8 . FIG. 11 is a flowchart of an optical calibration method of a qPCR system according to an embodiment of the present disclosure.

100: Optical Correction Tools 110: transparent substrate 120: fluorescent particles 130: container

Claims (18)

An optical correction tool for real-time quantitative polymerase chain reaction system, comprising: a transparent substrate, wherein the transparent substrate is solid; and a plurality of fluorescent particles uniformly dispersed in the transparent substrate. The optical calibration tool of the real-time quantitative polymerase chain reaction system according to Claim 1 further comprises: a container configured to accommodate the transparent substrate and the fluorescent particles. The optical calibration tool of the real-time quantitative polymerase chain reaction system according to claim 2, wherein the difference between a refractive index of the container and a refractive index of the transparent substrate falls in the range of about 0.2 to 0.3. The optical calibration tool of the real-time quantitative polymerase chain reaction system as described in claim 1, wherein the fluorescent particles are configured to be excited by a light source, and the wavelength of the light source falls in the range of about 400 nm to 800 nm . The optical calibration tool of the real-time quantitative polymerase chain reaction system as described in claim 1, wherein the fluorescent particles include one of quantum dots, fluorescent particles, nano silicon spheres or nano diamonds or any combination of the above . Real-time quantitative polymerase chain reaction as described in claim item 1 The optical correction tool of the system, wherein the average diameter of the fluorescent particles falls in the range of about 1 micron to 10 microns. The optical calibration tool of the real-time quantitative polymerase chain reaction system according to claim 1, wherein the concentration of the fluorescent particles falls in the range of about 1 ppm to 10000 ppm. The optical calibration tool of the real-time quantitative polymerase chain reaction system as described in claim 1, wherein the material of the transparent substrate comprises polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate ester or resin. The optical calibration tool of the real-time quantitative polymerase chain reaction system according to claim 1, wherein the viscosity of the transparent substrate in liquid state is less than 5500 centipoise. A method for preparing an optical correction tool for a real-time quantitative polymerase chain reaction (qPCR) system, comprising: preparing a liquid transparent substrate; mixing the liquid transparent substrate and a plurality of fluorescent particles to form a mixture; pumping the mixture vacuum; and solidifying the mixture to form a solid transparent substrate, wherein the fluorescent particles are uniformly dispersed in the solid transparent substrate. The preparation method of the optical calibration tool of the real-time quantitative polymerase chain reaction system as described in claim 10, wherein the step of mixing the liquid transparent substrate and the fluorescent particles is stirred by centrifugal force, and a rotational speed of the centrifugal force is stirred Less than 100rpm. The preparation method of the optical calibration tool of the real-time quantitative polymerase chain reaction system according to claim 10 further comprises: before solidifying the mixture, putting the mixture into a container. The preparation method of the optical correction tool of the real-time quantitative polymerase chain reaction system as described in Claim 10 further comprises: shaping the solid transparent substrate by diamond engraving. The preparation method of the optical calibration tool of the real-time quantitative polymerase chain reaction system according to claim 10, wherein the Shore hardness of the solid transparent substrate falls in the range of about 90D to 100D. The preparation method of the optical calibration tool of the real-time quantitative polymerase chain reaction system as described in claim 10, wherein curing the mixture is performed by moisture curing, heat curing or light curing. A calibration method for a real-time quantitative polymerase chain reaction (qPCR) system, comprising: placing an optical calibration tool in the qPCR system, wherein the optical calibration tool includes: a transparent substrate, wherein the transparent substrate is solid; and a plurality of fluorescent particles uniformly dispersed in the transparent substrate; opening the qPCR A light source of the system to excite the fluorescent particles in the optical calibration tool; compare the emitted light of the fluorescent particles with a built-in data and generate a comparison result; and adjust the qPCR according to the comparison result System settings. The calibration method of the real-time quantitative polymerase chain reaction system according to claim 16, wherein the setting of the qPCR system includes a light intensity of the light source. The calibration method of the real-time quantitative polymerase chain reaction system according to claim 16, wherein the wavelength of the light source of the qPCR system corresponds to the excitation light wavelength of the fluorescent particles.

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