CN115216522A

CN115216522A - Optical correction tool of real-time quantitative polymerase chain reaction system and preparation method thereof

Info

Publication number: CN115216522A
Application number: CN202110400114.5A
Authority: CN
Inventors: 游杰颖; 马勃; 李浚荣; 颜翊安
Original assignee: Delta Electronics Inc
Current assignee: Delta Electronics Inc
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2022-10-21

Abstract

The optical calibration tool of the real-time quantitative polymerase chain reaction system comprises a transparent substrate and fluorescent particles. The transparent substrate is a solid. The fluorescent particles are uniformly dispersed in the transparent base material.

Description

Optical correction tool of real-time quantitative polymerase chain reaction system and preparation method thereof

Technical Field

The present disclosure relates to an optical calibration tool for a quantitative polymerase chain reaction (qPCR) system, a method for preparing the same, and a method for calibrating the qPCR system.

Background

In existing optical correction tools for quantitative polymerase chain reaction (qPCR) systems in real time, organic fluorescent dyes are often used as the excited material. However, the organic fluorescent dyes often suffer from problems of fast fluorescence intensity decay and unstable luminous intensity due to the influence of environmental light source and temperature. Therefore, the optical calibration tool of the existing qPCR system is not easily reused, the preparation method is complicated, and the accuracy is difficult to improve.

In view of the above, it is still one of the objectives of the present invention to provide an optical calibration tool with high accuracy, reusability and simple preparation method.

Disclosure of Invention

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

In one embodiment of the present disclosure, an optical calibration tool of a real-time quantitative polymerase chain reaction system includes a transparent substrate and fluorescent particles. The transparent substrate is a solid. The fluorescent particles are uniformly dispersed in the transparent base material.

In an embodiment of the present disclosure, the optical calibration tool of the real-time quantitative polymerase chain reaction system further comprises 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 one 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 nanometers to 800 nanometers.

In an embodiment of the present disclosure, the fluorescent particles include one or any combination of quantum dots, fluorescent particles, nano-silicon spheres, or nano-diamonds.

In one 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 the fluorescent particles falls in a range of about 1ppm 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 one embodiment of the present disclosure, the transparent substrate has a viscosity of less than 5500centipoise (cP) when in a liquid state.

Another technical embodiment of the present disclosure is a method for preparing 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 of a qPCR system comprises: preparing a liquid transparent substrate; mixing a liquid transparent substrate with fluorescent particles to form a mixture; vacuumizing the mixture; and curing the mixture to form a solid transparent substrate, wherein the fluorescent particles are uniformly dispersed in the solid transparent substrate.

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

In an embodiment of the present disclosure, the method of making an optical correction tool for a qPCR system further comprises loading the mixture into a container prior to curing the mixture.

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

In one embodiment of the present disclosure, the shore D hardness of the solid transparent substrate falls within a range of about 90D to 100D.

In an embodiment of the present disclosure, curing the mixture is performed by moisture curing, thermal curing, or photo curing.

Another technical embodiment of the present disclosure is a method of calibrating a qPCR system.

In one embodiment of the present disclosure, a method for calibrating a qPCR system comprises placing an optical calibration tool on the qPCR system; turning on a light source of the qPCR system to excite fluorescent particles in the light correction tool; comparing the emitted light of the fluorescent particles with the built-in data to generate a comparison result; and adjusting the setting of the qPCR system according to the comparison result.

In an embodiment of the present disclosure, the setting of the qPCR system comprises 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 excitation light wavelength of the fluorescent particles.

In the above embodiments, the fluorescent particles have high stability in light emission intensity, and thus the optical correction tool of the present disclosure can be reused, is 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 correction performance of the qPCR system.

Drawings

Fig. 1 is a schematic diagram of an optical correction tool of a qPCR system according to an embodiment of the present disclosure.

Fig. 2A-2D are schematic diagrams of excitation and emission spectra of fluorescent particles of an optical calibration tool of a qPCR system according to an embodiment of the disclosure.

Fig. 3A-3C are graphs of concentration of fluorescent particles versus emitted light intensity for an optical correction tool of a qPCR system according to another embodiment of the present disclosure.

Fig. 4 is an emission light intensity and signal-to-noise ratio of fluorescent particles of an optical correction tool of a qPCR system according to another embodiment of the present disclosure.

Fig. 5 is a schematic diagram of an optical correction tool of a qPCR system according to another embodiment of the present disclosure.

Fig. 6 is thermal test data of an optical correction tool of a qPCR system according to an embodiment of the present disclosure.

Fig. 7 is light stability data for an optical correction tool of a qPCR system according to an embodiment of the present disclosure.

Fig. 8 is a flow chart of a method of making an optical correction tool of a qPCR system according to an embodiment of the present disclosure.

Fig. 9-10 are schematic diagrams of intermediate steps of a method of making an optical correction tool of the qPCR system of fig. 8.

Fig. 11 is a flow chart of a method of optical correction of a qPCR system according to an embodiment of the present disclosure.

Wherein the reference numerals are as follows:

100,200: optical correction tool

110,210: transparent substrate

120: fluorescent particles

130: container with a lid

140: centrifugal machine

300,400: method of producing a composite material

EX1, EX2, EX3, EX4: excitation light

EM1, EM2, EM3, EM4: emitting light

R1, R2, R3: coefficient of correlation

G: air bubble

S11 to S14, S21 to S29: step (ii) of

Detailed Description

In the following description, numerous implementation details are set forth in order to provide a thorough understanding of the present invention. It should be understood, however, that these implementation details are not to be interpreted as limiting the invention. That is, in some embodiments of the invention, such implementation details are not necessary. In addition, some conventional structures and elements are shown in simplified schematic form in the drawings. And the thickness 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 correction 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 contain the transparent substrate 110 and the fluorescent particles 120. The optical correction tool 100 is applied to correct the optical quality of a qPCR system. The fluorescent particles 120 are configured to be excited by a light source emitted by the qPCR system. The container 130 is an optical grade container, and may be, for example, a centrifuge tube. Thus, the light source of the qPCR system may penetrate the container 130 to excite the fluorescent particles 120 in the transparent substrate 110 such that the fluorescent particles 120 emit light of a particular wavelength for performing a calibration of the qPCR system.

The material of the transparent substrate 110 includes Polydimethylsiloxane (PDMS), polymethylmethacrylate (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 when in a liquid state is less than 5500centipoise (centripoise). By this feature, bubbles can be prevented from being generated when the fluorescent particles 120 are mixed into the liquid transparent substrate 110. In this way, bubbles or impurities can be prevented from remaining in the transparent substrate 110 and the optical calibration performance of the qPCR system can be reduced.

The difference between the refractive index of the container 130 and the refractive index of the transparent substrate 120 falls within a range of about 0.2 to 0.3. Thus, the aberration problem caused by the difference in medium refractive index can be reduced, and the optical correction accuracy of the qPCR system can be reduced.

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

For example, a portion of the phosphor particles 120 may 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 wavelength peak of about 470 nanometers, the fluorescent particles 120 can be excited to emit emitted light EM1 (blue-green light) with a wavelength peak of about 520 nanometers. As shown in fig. 2B, another portion of the fluorescent particles 120 can be excited by a light source EX2 (blue green light) with a peak wavelength of about 520 nm to emit an emission EM2 (green light) with a peak wavelength of about 570 nm. As shown in fig. 2C, another portion of the fluorescent particles 120 can be excited by a light source EX3 (yellow light) with a wavelength peak of about 590 nm to emit an emission EM3 (yellow light) with a wavelength peak of about 590 nm. As shown in fig. 2D, another portion of the fluorescent particles 120 can be excited by a light source EX4 (red light) with a peak wavelength of about 630 nm to emit an emission EM4 (red light) with a peak wavelength of about 640 nm.

As can be seen from 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 to perform the optical calibration of the qPCR system, and the detailed calibration steps will be described in detail later. It should be understood that the above-mentioned light source wavelength is only an example, and those skilled in the art can select the appropriate fluorescent particles 120 accordingly according to the light source wavelength of the qPCR system.

The Fluorescent Particles 120 may comprise one or any combination of Quantum dots (Quantum dots), fluorescent Particles (Fluorescent Particles), silicon Nanoparticles (silicon Nanoparticles), or Nanodiamonds (Nanodiamonds). The average diameter of the fluorescent particles 120 falls within a range of about 1 micron to 10 microns. The concentration of the fluorescent particles 120 falls within a range of about 1 million parts per million (ppm) to 10000 ppm.

Fig. 3A-3C are graphs of concentration of fluorescent particles versus emitted light intensity for an optical correction tool of a qPCR system according to another embodiment of the present disclosure. FIG. 3A is a graph showing the relationship between the concentration of the fluorescent particles 120 excited by blue light and the intensity of the emitted light, corresponding to the graph shown in FIG. 2A. For example, as shown in FIG. 3A, the concentration of the fluorescent particles 120 is proportional to the intensity of the fluorescent signal in the range of 0 to 1000ppm, and the correlation coefficient R1 of the linear regression analysis is about 1. FIG. 3B is a graph showing the relationship between the concentration of the fluorescent particles 120 excited by yellow light and the intensity of emitted light (millivolts, mV) corresponding to the graph shown in FIG. 2C. The concentration of the fluorescent particles 120 is proportional to the intensity of the fluorescent signal in the range of 0 to 1000ppm, and the correlation coefficient R2 of the linear regression analysis is about 0.9853. Fig. 3C is a graph of the concentration of the fluorescent particles 120 excited in red light versus the intensity of the emitted light, corresponding to that shown in fig. 2D. The concentration of the fluorescent particles 120 is proportional to the intensity of the fluorescent signal in the range of 0 to 1000ppm, 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 1000ppm, the light intensity of the excitation light of the fluorescent particles 120 can be accurately controlled.

Fig. 4 is a graph of the emitted light intensity and signal-to-noise ratio of the fluorescent particles 120 of an optical correction tool of a qPCR system according to another embodiment of the present disclosure. In fig. 4 different fluorescent particles 120 are exemplified with a concentration of 1000 ppm. In the example of FIG. 4, the excitation light intensities of the fluorescent particles 120 emitting blue, green, yellow and red light can all have high SNR to improve the optical calibration performance of the qPCR system.

Fig. 5 is a schematic diagram of an optical correction 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 holding the transparent substrate 210 and the fluorescent particles 120. The transparent substrate 110 of the optical calibration tool 200 can be shaped by high precision machining such as diamond cutting machine, so that the outer surface of the transparent substrate 110 can meet the quality required by optical calibration. The transparent substrate 110 of the optical calibration tool 200 can have different shapes, such as a centrifuge tube shape, a cone shape, a cylinder shape, a square shape, a flat shape, and the like similar to the optical calibration tool 100 shown in FIG. 1. In other words, as long as the shape of the optical correction tool 200 is compatible with the space used by the qPCR system to place the optical correction tool 200.

Fig. 6 is thermal test data of an optical correction tool of a qPCR system according to an embodiment of the present disclosure. Data in fig. 6 experimental data taken two days after the optical calibration tool 100 of fig. 1 was placed in a 65 degree environment. As can be seen from the experimental data in FIG. 6, in the present embodiment, the variation of the emission intensities of the fluorescent particles 120 emitting yellow and blue light is less than 10%, and the variation of the emission intensity of the fluorescent particles 120 with a concentration of 1000ppm is substantially less than 2%. Accordingly, the phosphor particles 120 of the optical calibration tool of the present disclosure can withstand high temperature without a light emission intensity degradation phenomenon.

Fig. 7 is light stability data for an optical correction tool of a qPCR system according to an embodiment of the present disclosure. Data in fig. 7 experimental data for the optical correction tool 100 shown in fig. 1 placed under ambient light source illumination for about five months. As can be seen from the experimental data of fig. 7, the emission intensity of the fluorescent particles 120 emitting red light and yellow light remains substantially constant. Therefore, the fluorescent particles 120 of the optical calibration tool of the present disclosure can be reused for a long time, so as to reduce resource waste.

In summary, since the fluorescent particles 120 have high stability of the light intensity, the optical calibration tool of the present disclosure can be reused, is not prone to photobleaching, and has high accuracy. In addition, the design of the container 130 or the shaped transparent substrate 110 can be conveniently used by a user, and the complexity of the optical calibration step is reduced.

Fig. 8 is a flow diagram of a method 300 of making an optical correction tool for 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, in step S12, the liquid transparent substrate and the plurality of fluorescent particles are mixed to form a mixture. Next, step S13 is to vacuumize the mixture. Finally, step S14 is to cure the mixture to form a solid transparent substrate, wherein the fluorescent particles are uniformly dispersed in the solid transparent substrate.

Fig. 9-10 are schematic diagrams illustrating intermediate steps of a method 300 for making an optical correction tool of the qPCR system of fig. 8. As shown in fig. 9, in steps S11 to S12, a liquid transparent base material 110L is prepared, and the fluorescent particles 120 are added to the liquid transparent base material 110L. Then, the transparent base 110L and the fluorescent particles 120 are mixed. In the present 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 rotation speed of centrifugal stirring is less than 100rpm. In other embodiments, the fluorescent particles 120 in the transparent base 110L may be stirred by an electric stirrer.

As shown in fig. 10, in step S13, the mixture is evacuated to remove the bubbles G in the transparent substrate 110L. The mixture is then loaded into a container 130 before curing the mixture. Finally, in step S14, the mixture is cured by light L to form the optical correction tool 100. In other embodiments, curing the mixture may also be performed by moisture curing or thermal curing, among other means.

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

Fig. 11 is a flow diagram of a method 400 for optical correction of a qPCR system according to an embodiment of the present disclosure. Step S21 of method 400 begins by placing an optical correction tool on the qPCR system. Next, in step S22, the light source of the qPCR system is turned on to excite the fluorescent particles in the light correction tool. Then, in step S23, the qPCR system receives the emission light signal of the fluorescent particle, and compares the emission light intensity of the fluorescent particle with the built-in data to generate a comparison result. For example, the built-in data includes the normal range of the emitted light intensity of the 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 correction tool, which can be excited by the excitation light of different wavelengths, is within the normal range.

If the comparison result obtained in step S24 indicates that the emission intensity is within the normal range, it is proceeded to step S25 to return the qPCR system to the preset mode. Then, the process proceeds to step S26, and the correction routine of the qPCR system is ended.

If the comparison result obtained in step S24 shows that the emission light intensity is not within the normal range, the setting of the qPCR system is adjusted according to the comparison result. The settings of the qPCR system include the light intensity of the light source at each wavelength. For example, step S24 may continue to step S27, adjusting the light source intensity of the qPCR system. Next, step S28 is performed to determine whether the number of times of light source intensity adjustment of the qPCR system is less than 10. If the result of step S28 is positive, go back to step S24 to compare the adjusted emission light intensity with the built-in data. If the result of step S28 is no, proceed to step S29, execute the maintenance program of the qPCR system.

In summary, the optical calibration tool of the present disclosure can be reused and has high accuracy due to the high stability of the light intensity of the fluorescent particles. The design of the container or the shaped transparent substrate can be conveniently used by a user, and the complexity of an optical calibration step is reduced. By controlling the concentration of the fluorescent particles in the range of 0 to 1000ppm, the light intensity of the fluorescent particle excitation light can be accurately controlled. In addition, the excitation light intensity of the fluorescent particles can have high signal-to-noise ratio, and the optical correction performance of the qPCR system can be improved. The viscosity of the transparent base material is less than 5500 centiphase, so that bubbles can be prevented from being generated when the fluorescent particles are mixed into the liquid transparent base material. The difference between the refractive index of the container and the refractive index of the transparent substrate falling in the range of about 0.2 to 0.3 can reduce the optical correction accuracy of the qPCR system due to aberration problems caused by the difference in the refractive index of the medium.

Although the present invention has been described with reference to the above embodiments, it should be understood that various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the invention.

Claims

1. An optical calibration tool for a 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.

2. The optical calibration kit for quantitative real-time polymerase chain reaction system according to claim 1, further comprising:

a container configured to contain the transparent substrate and the plurality of fluorescent particles.

3. The optical calibration kit of claim 2, wherein a difference between a refractive index of the container and a refractive index of the transparent substrate is in a range of about 0.2 to 0.3.

4. The optical calibration kit according to claim 1, wherein the plurality of fluorescent particles are configured to be excited by a light source having a wavelength in a range of about 400 nm to about 800 nm.

5. The optical calibration kit of claim 1, wherein the plurality of fluorescent particles comprise one or any combination of quantum dots, fluorescent particles, nano-silica spheres, or nano-diamonds.

6. The optical correction tool of quantitative polymerase chain reaction system according to claim 1, wherein the average diameter of the plurality of fluorescent particles falls within a range of about 1 to 10 microns.

7. The optical correction tool of quantitative polymerase chain reaction system according to claim 1, wherein the concentration of the plurality of fluorescent particles falls within a range of about 1ppm to 10000 ppm.

8. The optical calibration kit for real-time quantitative polymerase chain reaction system according to claim 1, wherein the material of the transparent substrate comprises Polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polycarbonate or resin.

9. The optical calibration kit for real-time quantitative polymerase chain reaction system according to claim 1, wherein the viscosity of the transparent substrate is less than 5500centipoise (centripose) when the transparent substrate is in a liquid state.

10. A method for preparing an optical calibration tool for a quantitative polymerase chain reaction (qPCR) system on-the-fly, comprising:

preparing a liquid transparent substrate;

mixing the liquid transparent substrate and a plurality of fluorescent particles to form a mixture;

evacuating the mixture; and

curing the mixture to form a solid transparent substrate, wherein the plurality of fluorescent particles are uniformly dispersed in the solid transparent substrate.

11. The method of claim 10, wherein the step of mixing the transparent liquid substrate and the fluorescent particles is performed by centrifugal stirring at a speed of less than 100rpm.

12. The method for preparing an optical calibration kit for real-time quantitative polymerase chain reaction system according to claim 10, further comprising:

the mixture is filled into a container before curing the mixture.

13. The method for preparing an optical calibration tool of an in-line quantitative polymerase chain reaction system according to claim 10, further comprising:

the solid transparent substrate is shaped by diamond carving.

14. The method of claim 10, wherein the shore D hardness of the transparent solid substrate is in the range of about 90D to 100D.

15. The method for preparing an optical calibration tool of an in-line quantitative polymerase chain reaction system according to claim 10, wherein curing the mixture is performed by moisture curing, thermal curing or photo curing.

16. A method of calibrating a quantitative polymerase chain reaction (qPCR) system in real time, comprising:

placing an optical calibration tool in the qPCR system;

turning on a light source of the qPCR system to excite a plurality of fluorescent particles in the optical calibration tool;

comparing the emitted light of the plurality of fluorescent particles with a built-in data and generating a comparison result; and

and adjusting the setting of the qPCR system according to the comparison result.

17. The method of claim 16, wherein the setting of the qPCR system comprises a light intensity of the light source.

18. The method of claim 16, wherein the wavelength of the light source of the qPCR system corresponds to the wavelength of the excitation light of the fluorescent particles.