TWI831393B - Pcr detection device and system - Google Patents

Abstract

The present disclosure provide a detection device of microfluidic polymerase chain reaction (PCR) and a detection system including the same. This all-in-one device and system may be used to detect at least one biological detection chip, so that can amplify gene fragments at the front-end and detect them at the back-end immediately, decreasing the time required for the analysis, enabling real-time, low-cost, and rapid detection of various viruses, such as EBV and COVID-19, without compromising accuracy or sensitivity.

Description

PCR detection device and system

This case relates to a polymerase chain reaction (PCR) detection device and a system including the detection device. Specifically, this case relates to a microfluidic PCR detection device, a detection system including the detection device, and a detection method using the detection system of the detection device.

In recent years, disease diagnosis often uses DNA and mRNA gene amplification technology, such as polymerase chain reaction (Polymerase Chain Reaction, PCR), and further developed Fluorescence Based Real-Time PCR, real-time polymerase chain reaction lock reaction (real-time PCR, RT-PCR) or quantitative polymerase chain reaction (quantitative PCR, qPCR). The principle of traditional PCR technology is to amplify DNA molecules through cyclic reactions at different temperatures, and then put the products into a gel electrophoresis device to detect the results. This detection method itself is time-consuming and has the disadvantage of accuracy. RT-PCR or qPCR embeds fluorescent molecules on the amplified target gene fragment. When the amplification process amplifies the gene fragment, it also amplifies the fluorescent signal. At the same time, real-time detection can be obtained by detecting fluorescent signals. However, the disadvantage is that qPCR detection reagents are expensive because they contain fluorescent labels.

In order to improve the shortcomings of conventional PCR device detection, the purpose of this disclosure is to provide a microfluidic PCR and biological detection chip (for example, surface plasmon resonance (SPR) chip) integrated device to shorten PCR detection time. PCR detection device and detection system.

This case provides a PCR device, which includes a constant temperature unit, which includes a first heating plate and a second heating plate, wherein the first heating plate and the second heating plate are configured to be parallel and juxtaposed with a distance d; A microfluidic channel substrate, which includes a microfluidic channel, wherein the microfluidic channel is configured in the microfluidic channel substrate in a plurality of equal-length parallel back-and-forth cycles; wherein the microfluidic channel substrate is placed in the constant temperature unit and is in contact with the first heating plate to form a first heating zone, and is in contact with the second heating plate to form a second heating zone, wherein the first heating plate can apply a first temperature T1 to heat the first heating zone , and the second heating plate can apply a second temperature T2 to heat the second heating zone, wherein when T1 is different from T2, the area of the microfluidic substrate above the distance d forms a third heating zone, and A third temperature T3 formed in the third heating zone is between T1 and T2, and each equal-length parallel back-and-forth cycle in the microfluidic substrate passes through the first heating zone and the second heating zone. and the third heating zone.

This case also provides a PCR detection device, which includes a constant temperature unit, which includes a first heating plate and a second heating plate, wherein the first heating plate and the second heating plate are configured to be parallel with a distance d. Parallel; a microfluidic channel substrate, which includes a microfluidic channel, wherein the microfluidic channel is configured in the microfluidic channel substrate in a plurality of equal-length parallel back-and-forth cycles; and a biological detection chip, which can be connected with a fluid The method is loaded on the end of the microfluidic channel of the microfluidic channel substrate; wherein the microfluidic channel substrate is placed on the constant temperature unit and is in contact with the first heating plate to form a first heating area, and with the second The heating plates contact to form a second heating zone, wherein the first heating plate can apply a first temperature T1 to heat the first heating zone, and the second heating plate can apply a second temperature T2 to heat the second heating zone, When T1 is different from T2, the area of the microfluidic substrate above the distance d forms a third heating zone, and a third temperature T3 formed in the third heating zone is between T1 and T2 , wherein each equal-length parallel back-and-forth cycle in the microfluidic substrate passes through the first heating zone, the second heating zone, and the third heating zone.

This case also provides a PCR detection system, which includes a constant temperature unit, which includes a first heating plate and a second heating plate, wherein the first heating plate and the second heating plate are arranged in parallel with a distance d; A microfluidic channel substrate, which includes a microfluidic channel, wherein the microfluidic channel is configured in the microfluidic channel substrate in a plurality of equal-length parallel back-and-forth cycles, wherein the microfluidic channel The microfluidic substrate is placed on the constant temperature unit, and is in contact with the first heating plate to form a first heating area, and is in contact with the second heating plate to form a second heating area, wherein the first heating plate can A first temperature T1 is applied to heat the first heating zone, and the second heating plate can apply a second temperature T2 to heat the second heating zone; a biological detection chip is loaded in the microfluidic channel in a fluid-connected manner. At the end of the microfluidic channel of the substrate; a flow control unit that can control the flow rate of the liquid in the microfluidic substrate; a temperature control unit that is electrically connected to the constant temperature unit and can respectively control the first heating plate and the heating temperature of the second heating plate; and a detection unit for detecting the biological detection chip; wherein when T1 is greater than T2, the area above the distance d in the microfluidic substrate forms a third heating area, And a third temperature T3 formed in the third heating zone is between T1 and T2.

This case also provides a PCR detection method, which method includes: providing the above-mentioned PCR detection system; using a temperature control unit to adjust the first heating plate and the second heating plate in the constant temperature unit for a period of time, so that the first heating zone and the second heating zone and the third heating zone are stabilized at the temperatures of T1, T2 and T3 respectively; the flow control unit is used to continuously pump the sample to be tested at a fixed flow rate into the inlet end of the microfluidic channel of the microfluidic substrate for a period of time, and the detection unit is used to detect and analyze unit analysis.

10: Microfluidic substrate

11: Microfluidic channel

12: Entrance port

12a: Chip microchannel connection port

12b: Chip microchannel connection port

13:Export end

13a: Chip microchannel connection port

14:Export end

14a: Chip microchannel connection port

15: First heating zone

16: Second heating zone

17: The third heating zone

20: Constant temperature unit

21:First heating plate

21a: Temperature control port

22:Second heating plate

22a: Temperature control port

31:The first biological detection chip

32: Second biological detection chip

40:Flow control unit

50:Temperature control unit

60:Detection unit

61:Objective lens

62:Polarizer

63:Lens

64: Optical fiber

70:Analysis unit

d: spacing

L:UV light

Lc:laser cutting machine

Le:Laser engraving machine

P: pressure

P1: First acrylic substrate

P2: Second acrylic substrate

T: Heat-resistant double-sided tape

Further features and advantages of the present invention will become apparent from the following and more particular description of the preferred embodiments, as illustrated in the accompanying drawings, in which like reference characters generally refer to the same portions throughout the text. Or a component, wherein: FIG. 1 is a schematic diagram of a microfluidic substrate illustrated in this disclosure.

Figure 2 is a schematic diagram of a PCR detection device illustrated in the present disclosure.

Figure 3 is a schematic diagram of a PCR detection system exemplified in this disclosure.

FIG. 4 is a flow chart of microfluidic substrate preparation illustrated in this disclosure.

Figure 5 is another preparation flow chart of the microfluidic channel substrate illustrated in this disclosure.

6A to 6C are temperature distribution diagrams of the heating system of the present disclosure. Figure 6A shows that when the distance d is 8, 9 and 10mm respectively, the temperature in the third heating zone detected by the thermocouple The measured temperatures were 72, 66 and 60°C respectively; Figure 6B shows the temperature stability of the first heating zone, the second heating zone and the third heating zone when the interval d is 9mm. Figure 6C shows three temperature zones for operating the microfluidic device.

Figure 7 shows the red shift changes in the resonance wavelength of the detection chip exemplified in the present disclosure before modification, after modification with a probe, and after the probe binds to the target nucleic acid.

Figure 8 shows a comparison of the amplification capabilities of the disclosed microfluidic channel PCR and traditional PCR machines.

It is to be understood that this disclosure is not limited to the specifically illustrated materials, structures, procedures, methods, or constructions listed herein. Therefore, although some options are listed here, any that are similar or have the same effect can be applied to the implementation or embodiments of the present disclosure, and this article only describes the preferred materials and methods.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the present disclosure only and is not intended to be limiting.

The detailed description set forth below in connection with the accompanying drawings is considered to be a description of exemplary embodiments of the disclosure and is not intended to represent the only exemplary embodiments in which the disclosure may be practiced. As used throughout this specification, the term "illustrative" means "serving as an instance, example, or illustration" and should not necessarily be construed as being preferred or advantageous over other exemplary embodiments. The detailed description includes specific details for the purpose of providing a thorough understanding of the exemplary embodiments of the present description. It will be apparent to one of ordinary skill in the art to which this disclosure pertains that the exemplary embodiments described herein may be practiced without these specific details.

Definition:

As used herein, each of the following terms has the meaning set forth in this paragraph. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In general, the nomenclature and laboratory procedures used in this article are those of molecular biology, optics, organic chemistry, etc. Known and commonly used. It should be understood that the order of steps or the order in which certain actions are performed is immaterial so long as the teachings remain operable. The use of any section headings is to aid in reading the document and should not be construed as limiting; information associated with a section heading may appear within or outside that particular section. All publications, patents, and patent documents mentioned in this document are incorporated by reference in their entirety to the same extent as if individually incorporated by reference.

For purposes of convenience and clarity only, directional terms such as up, down, left, right, below, above, above, below, below, behind, behind, and forward may be used with respect to the accompanying drawings. These similar directional terms should not be construed in any way as limiting the scope of this disclosure.

In the methods described herein, the actions may be performed in any order, except where the timing or order of operations is explicitly recited. Furthermore, the actions referred to may be performed simultaneously unless the explicit claim language states that they are performed separately. For example, performing the requested action of X and performing the requested action of Y can be performed simultaneously in a single operation, and the resulting method will fall within the context of the requested method.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one familiar with the art to which this disclosure belongs. Furthermore, as used in this specification and the accompanying patent claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the term "about" will be understood by those of ordinary skill in the art and will vary to some extent depending on the context in which it is used. As used herein, when referring to a measurable value such as an amount, a length of time, or the like, "about" is meant to include a variation of ±20%, ±10%, ±5%, ±1%, or ±0.1% of that particular value. . Because these changes are suitable for carrying out the disclosed method.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific procedures, embodiments, patent claims, and examples described herein. Such equivalents are deemed to be within the scope of this disclosure and are covered by the claims appended hereto. For example, it should be understood that, including but not limited to reaction time, reaction size/volume and experimental reagents (such as solvents, catalysts), pressure, gas pressure conditions (such as nitrogen pressure) Modifications of reaction conditions such as reducing/oxidizing agents, substitutions approved by the technical field, and the use of no more than routine experiments are within the scope of this application.

It should be understood that wherever numerical values and ranges are provided herein, the range format is described for convenience and brevity only and should not be construed as limiting the scope of the present disclosure. Accordingly, all values and ranges encompassed by such values and ranges are included within the scope of this disclosure. In addition, all numerical values falling within these ranges, as well as the upper or lower limits of the numerical ranges, are also contemplated by this application. Descriptions of ranges should be considered to specifically disclose all possible subranges and individual values within such ranges, and where appropriate, partial integers of values are also included within the range. For example, description of a range from 1 to 6 should be deemed to specifically disclose subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, from 3 to 6, etc., and within that range Individual numbers within, such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6. For example, a range of “about 0.1% to about 5%” or “about 0.1% to 5%” should be construed to include not only about 0.1% to about 5%, but also individual values (e.g., 1%, 2%, 3% and 4%) and sub-ranges within the indicated range (for example, 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%). Unless otherwise specified, the expression "about X to Y" has the same meaning as "about X to about Y". Likewise, the expression "about X, Y or about Z" has the same meaning as "about X, about Y or about Z" unless otherwise specified. This applies regardless of the width of the range.

Microfluidic substrate

The PCR detection device of the present disclosure includes a microfluidic channel substrate (as shown in Figure 1), which contains at least one microfluidic channel, wherein the at least one microfluidic channel is configured in a plurality of equal-length parallel back-and-forth cycles on the microfluidic channel. in the flow channel base plate. In the operating procedure of the PCR detection device, the PCR sample flows through an equal-length parallel back-and-forth cycle, which is equivalent to one PCR thermal cycle. There is no limit to the number of equal-length parallel back-and-forth cycles included in the microfluidic channel substrate. It can be designed based on the number of thermal cycles required to amplify the target in the PCR sample. For example, 10 to 50 equal-length parallel back-and-forth cycles can be designed. , preferably 20 to 40 cycles, more preferably 30 cycles, such as 10, 15, 20, 25, 30, 35, 40, 45, 50 cycles.

"Equal-length parallel back-and-forth" or "equal-length parallel back-and-forth cycle" as used herein refers to the fact that each circulation path on the microfluidic substrate in the PCR detection device of the present disclosure is of equal length and Capable of spanning each heating zone to maintain the same reaction time in each heating zone during each cycle during PCR amplification, and the paths within the microfluidic channels for PCR amplification zones are substantially parallel to each other to reduce microfluidics The area of the substrate. In other words, for each circulation path, it can be a simple parallel back and forth, or for a specific sample, the microfluidic channel can be designed to add several parallel back and forth in a specific heating zone, for example, one can be added in the third heating zone or A second parallel round trip is performed to increase the actual time required for nucleic acid extension and is suitable for samples containing longer nucleic acids.

The microfluidic channel in the microfluidic channel substrate of the present disclosure may include an inlet port and at least one outlet port. In one embodiment, the end of the microfluidic channel in the microfluidic substrate of the present disclosure can include a plurality of outlet ports (as shown in Figure 2) to provide simultaneous detection of multiple biological detection chips, wherein the biological detection chips can be the same Or different.

In one embodiment, a laser engraving machine can be used to engrave an appropriate carrier with appropriate power, speed and focal length to create the microfluidic channel of the present disclosure, and then the carrier is encapsulated between two plastic substrates through encapsulation technology. There are no specific restrictions on the plastic substrate. Any plastic substrate that can be used for hot-pressing packaging to prepare microfluidics can be used in this process. For example, acrylic (PMMA), polycarbonate (PC), etc. can be used. In another embodiment, a laser engraving machine can be used to directly engrave the designed microfluidic channels on two plastic substrates of the same size, and then the two plastic substrates are bonded and encapsulated. The encapsulation technology can, for example, use organic solvents. Adhesion method, but not limited to this. In another embodiment, a laser engraving machine can be used to directly engrave the designed microfluidic channel on a piece of plastic substrate, and then bond and package it with another piece of plastic substrate of the same size. In one embodiment, the microfluidic channels of the present disclosure can also be produced using injection molding.

There are no special restrictions on the width, depth, and spacing of the microfluidic channels. They can be optimally designed based on many factors such as the biological target to be detected, the type of probe, the fluid flow rate, and the detection chip. In one embodiment, the width of the microfluidic channel of the present disclosure is above 50 μm, for example, 100 to 400 μm, preferably 150 to 300 μm, and may be, for example, 100, 150, 200, 250, 300, 350, 400 μm or in between. of any size. In one embodiment, the depth of the microfluidic channel of the present disclosure may be more than 100 μm, such as 100 to 200 μm, and may be, for example, 100, 150, 200 μm, or any size therebetween.

There is no special restriction on the cross-sectional area shape of the microfluidic channel, and it can be processed according to general processing. It is convenient to design and use, and can be designed in other cross-sectional shapes such as square, semicircle, hexagon, trapezoid, etc.

In one embodiment, the prepared microfluidic channel is encapsulated between two acrylic plates and sealed with cured glue on all sides. One acrylic plate corresponds to the inlet end and at least one outlet of the microfluidic channel. Each end is provided with a through hole, and each through hole corresponds to the inlet end and at least one outlet end of the microfluidic channel in a fluid-communicable manner.

As used herein, the term "fluid-communicable" or "fluid-communicable means" refers to channels and channels, channels and through-holes, or through-holes and through-holes that can still allow liquid to flow therethrough after bonding and encapsulation.

Constant temperature unit

The PCR detection device of the present disclosure includes a constant temperature unit, which is composed of at least two heating plates. When composed of two heating plates, the first heating plate and the second heating plate are arranged in parallel and juxtaposed with a distance d. The microfluidic substrate can be placed on the constant temperature unit, that is, straddling between the first heating plate and the second heating plate, and in contact with the first heating plate to form a first heating area, and with the first heating plate. The second heating plate contacts to form a second heating zone. The area of the microfluidic substrate located above the distance d forms a third heating area. The first heating plate can apply a first temperature T1 to heat the first heating zone, and the second heating plate can apply a second temperature T2 to heat the second heating zone. When T1 and T2 are different, the third heating zone formed in the third heating zone The three temperatures T3 will be between T1 and T2.

Since the distance d can be adjusted by configuring the distance between the first heating plate and the second heating plate, and the contact area between the microfluidic channel substrate and the first and second heating plates can be adjusted, the microfluidic channel can be used to The length of the path in the first heating zone, the second heating zone and the third heating zone is adjusted to adjust the heating time, thereby corresponding to the time required for denaturation, primer adhesion and extension of various PCR samples.

The constant temperature unit in the PCR detection device of the present disclosure can be controlled by a temperature control unit, which is electrically connected to the constant temperature unit and can respectively control the heating temperatures of the first heating plate and the second heating plate. In addition, the distance d can vary within the range of 1 to 10 mm. Therefore, by controlling the heating temperature of the first heating plate and the second heating plate and the range of the distance d, and T1, T2 and T3 can be easily fixed at the desired temperature. In one embodiment, the PCR sample is a salt-free sample, the preferred temperature of T1 is 80°C to 90°C, the preferred temperature of T2 is 55°C to 65°C, and the distance d is in the range of 1 to 6mm. In another embodiment, the PCR sample is a salt sample, the preferred temperature of T1 is 90°C to 100°C, the preferred temperature of T2 is 55°C to 65°C, and the distance d is in the range of 6 to 10mm.

PCR detection device and detection system

In one embodiment, the present disclosure provides a PCR detection device, as shown in FIG. 2 , which at least includes the aforementioned microfluidic substrate 10, a constant temperature unit 20, and first and second biological detection chips 31 and 32. The microfluidic channel substrate 10 includes a microfluidic channel 11 , and the microfluidic channel 11 includes an inlet end 12 and two outlet ends 13 and 14 . The constant temperature unit 20 includes a first heating plate 21 and a second heating plate 22, wherein the first heating plate 21 and the second heating plate 22 are arranged in parallel with a distance d. The first heating plate 21 and the second heating plate 22 respectively include temperature control connection ports 21a and 22a that can be electrically connected to the temperature control unit. The biological detection chips 31 and 32 are arranged near the ends of the microfluidic channel 11 on the microfluidic channel substrate 10 . Both ends of the first biological detection chip 31 are respectively loaded in the chip microchannel connection ports 11a and 13a at the end of the microfluidic channel 11 in a fluid-connected manner. Both ends of the second biological detection chip 32 are loaded in a fluid-connectable manner in the microfluidic channel. Chip microchannel connection ports 11b and 14a at the end of lane 11.

The microfluidic channel substrate 10 is placed on the constant temperature unit 20 and is in contact with the first heating plate 21 to form a first heating area 15 and in contact with the second heating plate 22 to form a second heating area 16. The microfluidic channel substrate 10 The upper region between the first heating zone 15 and the second heating zone 16 is the third heating zone 17 . The heating area of the third heating zone 17 can be adjusted by adjusting the distance d between the first heating plate 21 and the second heating plate 22 . In addition, the heating areas of the first heating zone 15 and the second heating zone 16 can be adjusted by adjusting the position of the microfluidic channel substrate 10 placed on the constant temperature unit 20 . For example, taking FIG. 2 as an example, if the microfluidic channel substrate 10 is placed to the left without changing the distance d, a larger first heating area 15 and a smaller second heating area 16 can be obtained.

Since the microfluidic channels in the microfluidic channel substrate of the present disclosure have consistent diameters, the flow rate of the sample in the microfluidic channel can be used to calculate the time required for the sample to flow through the unit distance. because Therefore, the PCR detection device of the present disclosure can simply and accurately determine the areas of the three heating zones through the above adjustments, thereby adjusting the heating time of the sample. Specifically, in addition to controlling the time for the sample to pass through the three heating zones based on the amount of sample pumped into the microfluidic channel per unit time, the position and spacing of the microfluidic channel substrate 10 placed on the constant temperature unit 20 can be further adjusted. d to more optimally adjust the time the sample spends in the three heating zones to meet the temperatures and times required for denaturation, annealing/primer binding, extension and other steps required for PCR amplification of different biological samples. In other words, the PCR detection device and detection system of the present disclosure have a wider range of applications.

In one embodiment, the present disclosure provides a PCR detection system, as shown in FIG. 3 , which includes, in addition to the above-mentioned PCR detection device, a flow control unit 40 , a temperature control unit 50 , a detection unit 60 and an analysis unit 70 .

The flow control unit 30 of the present disclosure can be a micropump or other similar device, which can fluidly connect the inlet end of the micropump to the sample bottle through the microtube, and connect the outlet end of the micropump to the test bottle. The inlet end 12 of the microfluidic channel (as shown in Figure 2). The flow control unit 40 may have an independent control unit to control the feed rate, or may be integrated into other units, such as the analysis unit 70 .

The temperature control unit 50 of the present disclosure is electrically connected to the constant temperature unit 20, and can independently control at least two heating units, and preferably can independently control three to four heating units. The temperature control unit 50 is electrically connected to the temperature control connection port 21a on the first heating plate 21 and the temperature control connection port 22a on the second heating plate 22 in the constant temperature unit 20 respectively (see Figure 2), and can be controlled independently. The temperature of the first heating plate 21 and the second heating plate 22.

The detection unit 60 of the present disclosure is used to detect the detection results of the biological chip (such as the chips 31 and 32 shown in Figure 2) on the microfluidic substrate 10, so it can be changed according to the best detection method of the biological chip, for example The objective lens 61, the polarizer 62, the lens 63 and/or the optical fiber 64 are further combined for specific detection of the sample.

The analysis unit 70 of the present disclosure can be electrically connected to the detection unit 60, can analyze the detection results of the detection unit 60, and can cooperate with various analysis software and databases on itself, the Internet or the cloud to perform analysis and comparison and provide various comparative analysis results. , such as various analysis charts. In one embodiment, the analysis unit 70 of the present disclosure can be integrated with other units in the system, For example, the flow control unit 40, the temperature control unit 50 and the detection unit 60 can be integrated into a single control, detection and analysis unit.

Detection method

The present disclosure also provides a PCR detection method, which method includes: providing the above-mentioned PCR detection system; using the temperature control unit 50 to adjust the first heating plate 21 and the second heating plate 22 in the constant temperature unit 20 for a period of time, so that the first heating area 15. The second heating zone 16 and the third heating zone 17 are stabilized at the temperatures of T1, T2 and T3 respectively; use the flow control unit 40 to continuously pump the sample to be measured into the entrance of the microfluidic channel 11 of the microfluidic substrate 10 at a fixed flow rate. The terminal 12 is detected for a period of time by the detection unit 60 and analyzed by the analysis unit 70 .

The PCR detection method disclosed in the present disclosure can select biological detection chips based on the samples to be tested and assemble them into the system for detection. For example, a probe for detecting the nucleic acid can be selected or designed based on the type of nucleic acid to be detected, and attached to the nanochannel of the biochip using known techniques. When a trace amount of the nucleic acid to be tested passes through the microfluidic channel, it can reach the lowest detectable amount that can be combined with the probe on the biological detection chip through multiple equal-length parallel back-and-forth cycle increases. Then it is detected by the detection unit 60 and analyzed by the analysis unit 70 .

Example

Example 1: Materials and methods

1. Preparation of test samples

This disclosure uses Epstein-Barr virus (EBV) related nucleic acids and proteins as test samples. EBV is a human herpes virus associated with several cancers, including nasopharyngeal carcinoma, Burkitt lymphoma, and Hodgkin lymphoma. Six nuclear antigens and three membrane proteins synergistically induce the proliferation and transformation of EBV-infected cells. Among them, latent membrane protein 1 (LMP1) is expressed in most EBV-related malignancies and is considered a prognostic biomarker for these diseases.

Test samples were taken from nasopharyngeal cancer patients, and the samples were preprocessed to obtain LMP1 DNA. In addition, LMP1 DNA was amplified by PCR and analyzed by electrophoresis. In this study, the primer and template were modified, and the total length of LMP1 DNA was 311 base pairs (bp). Details of primers and LMP1 DNA are as follows in Table 1:

2.DNA extraction

Cells from 4 EBV-positive cell lines and 1 EBV-negative cell line were used. HKNPC-C43 or NPC43 is an EBV-positive nasopharyngeal carcinoma cell line (gifted by Professor George Sai Wah Tsao); Akata, P3HR1 and NAMALWA are EBV-positive Burkitt lymphoma cell lines. DB is an EBV-negative diffuse large cell lymphoma cell line. DNA was extracted from these cells using a commercial kit (AxyPrep Multisource Genomic DNA Miniprep Kit, 50prep), following the manufacturer's instructions. After extraction, samples were collected and measured with NanoDrop (Thermo Scientific™) to determine DNA concentration and purity.

3.PCR parameters

PCR solution (Taq PCR Master Mix) was purchased from Qiagen (Germantown, MD, USA), including forward and reverse primers, DNA template, and nuclease-free water. In the device, use 50 μL of a mixed solution for each reaction, which includes 25 μL of master mix, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 1 μL of 10 μg/ml DNA template, and 22 μL of nuclease-free water.

The reaction parameters of the traditional PCR machine as the control group are as follows in Table 2:

4. Nano-slit surface plasmon resonance (SPR) chip

By the method proposed in the literature (Chuang, C.-S., Wu, C.-Y., Juan, P.-H., Hou, N.-C., Fan, Y.-J., Wei, P.-K., Sheen, H.-J., 2020. Analyst 145, 52-60, incorporated herein by reference in its entirety) prepared gold-coated nanoslit wafers. The defined nanoslit structure (spacing 500nm, width 80nm) is accurately etched on the silicon wafer, and then transferred to a nickel-cobalt (Ni-Co) alloy mold through the electrode positioning method. Then, according to the method proposed in the literature (Lee, K.-L., Chen, P.-W., Wu, S.-H., Huang, J.-B., Yang, S.-Y., Wei ,P.-K.,2012.ACS Nano 6,2931-2939; and Lee,K.-L.,You,M.-L.,Tsai,C.-H.,Lin,E.-H., Hsieh, S.-Y., Ho, M.-H., Hsu, J.-C., Wei, P.-K., 2016., incorporated herein by reference in their entirety), by hot pressing Nanoimprint lithography (at a temperature of 165°C and a working pressure of 690kPa) imprints the nanostructures on the Ni-Co mold onto the polycarbonate (PC) film. Finally, gold was deposited onto the nanoslit PC film using a DC sputtering system (Ulvac, Methuen, MA, USA) (deposition time was 70 s, working pressure was 4 × 10 ^-6 kPa, and air flow was 50 standard cm ³ /min (sccm), power is 0.06kW). To integrate the microfluidic PCR with the nanoslit SPR chip, a heat-resistant double-sided tape with a chamber opening was used to fluidically bond the SPR chip to the end of the PCR microchannel.

5.Surface modification of SPR wafer

To detect the LMP1 gene for testing, the SPR chip in the microfluidic device was further modified with an LMP1 DNA probe (5′-GTCATAGTAGCTTAGCTGAACTGGGCCGT-3′).

A cysteamine solution (100 μg/ml) was flowed into the device prepared above and incubated for 2 hours, allowing the cysteamine to bind to the gold-covered surface of the SPR wafer through the thiol groups. After washing with deionized (DI) water, the LMP1 probe was pumped into the device for various time intervals to allow the LMP1 probe to electrostatically adsorb to the SPR wafer. Finally, the device was cleaned with deionized water to complete the surface modification.

6. Optical measurement device

An optical device for measuring the transparent resonance spectrum of the above SPR wafer was constructed. When broadband white light passes through the nanoslit SPR chip, a polarizer located on the other side of the SPR chip filters out the resonant wavelength in the transverse magnetic (TM) direction. The transmitted light was collected with an optical fiber through a fiber optic lens and then measured with a spectrometer (B&W Tek, Newark, DE, USA).

7.SPR data analysis

The sensing mechanism is shown in Figure 7. The resonant wavelength (λ) in the TM direction can be expressed as λ = a.dp; (1) where a is the refractive index of the environment near the gold-plated nanoslit, and dp is the period of the nanoslit.

First, the nano-slit SPR wafer is immersed in water, when a is equal to 1.33. When the LMP1 probe is modified onto the gold-covered nanoslit surface, the refractive index increases and the resonance wavelength red-shifts. Subsequently, when the LMP1 target is sensed and bound to the probe, the resonant wavelength is red-shifted again due to the increase in refractive index. To quantify the red shift, spectral processing was performed using the spectral centroid method to locate the resonance wavelength. The calculated resonance wavelengths at different time intervals were recorded every 2 seconds and plotted in a time correlation graph to observe the changes in resonance wavelengths at different DNA binding stages. The red shift value was evaluated by subtracting the average wavelength before and after DNA was bonded to the nanoslit SPR chip.

Example 2: Preparation of microfluidic channel substrate

Use AutoCAD (Autodesk, USA) software to design the microfluidic channel pattern (for example, see Figure 1). The microfluidic channel is designed into a single parallel loop of multiple equal lengths, and the microfluidic channel is generated by laser scribing technology. flow channel.

Referring to the preparation flow chart in Figure 4, a carrier is used to prepare a microfluidic substrate. First, heat-resistant double-sided tape T (thickness 400 μm) is used as a carrier to adhere to the first acrylic substrate (acrylic plate) P1, and then the laser beam of the laser engraving machine Le is used to engrave the microfluidic channel in the designed microfluidic pattern. The microfluidic design portion of the heat-resistant double-sided tape is removed to create a channel (width 300 μm) of microfluidic channel 11 (Fig. 4(a)). Multiple parallel back-and-forth cycles of equal length in a single strip provide different zones for corresponding PCR temperatures, which can heat or cool the sample 30 times between 95 and 60°C. Then, another second acrylic substrate P2 with reserved inlet and outlet hole openings is bonded to the other side of the pattern tape in such a way that the inlet and outlet are aligned with the starting point and end point of the microfluidic channel respectively, using uniform pressure. P (100kPa) is pressed (Figure 4(b)) and irradiated with ultraviolet L (10 minutes) to solidify and seal the glue (Figure 4(c)), and the microfluidic substrate can be made (Figure 4(d)).

Referring to the preparation flow chart in Figure 5, the microfluidic substrate is prepared without using a carrier. First, a laser cutting machine Lc is used to cut two pieces of the first acrylic substrate (acrylic plate) P1 and the second acrylic substrate P2 of the same size (length 87 mm, width 50 mm) (Fig. 5(a)). A laser engraving machine Le (CNC engraving machine, EGX-400, Roland) was used to directly engrave the first acrylic substrate P1 with the designed microfluidic pattern (Figure 5(b)). The microfluidic design has a width of 300 μm and a depth of 100 μm. Multiple parallel loops of equal length in a single strip provide different areas for corresponding PCR temperatures. The sample can be heated or cooled 30 times between 95 and 60°C. Then, the first acrylic substrate P1 and the second acrylic substrate P2 are packaged as microfluidic substrates using an organic solvent bonding method. Prepare a mixture of 20% acetone and 80% alcohol as an organic solvent, and coat it on an acrylic substrate without microfluidic channels. Then closely fit the two acrylic substrates, and finally put them into the hot stamping machine and set the target temperature to 60 ℃, pressure P is 5kg/ cm ² , continue to solidify under these conditions for about 3 minutes (Figure 5(c)), take it out after cooling, and complete the microfluidic substrate packaging.

The size of the detection chamber where the detection chip is set is designed to be a 10x10mm square that fits the chip. The interior is designed into a polygon to reduce the generation of bubbles and ensure that the fluid fills the detection chamber stably. Using a process similar to the above, the heat-resistant double-sided tape with a thickness of 400 μm is cut by a laser engraving machine.

Example 3: Setting of heating device

When performing PCR procedures on the microfluidic device, the three temperature zones used for denaturation, primer bonding and extension of PCR samples are respectively set as follows: T1 is 95°C, used for denaturation; T2 is 60°C, used for annealing and primer bonding; and T3 is 65°C for extension and detection.

First, the first heating plate and the second heating plate are aluminum blocks with electric heating films. The first heating zone and the second heating zone are set at 95 and 60°C respectively, and the third heating zone of 65°C in the middle of the device is adjusted by The distance d and the heating plates on both sides are maintained. A proportional integral derivative controller (PID controller) is used to adjust the current to achieve the desired temperature. A resistance temperature detector connected to the heating plate sends a signal back to the PID controller to stabilize the heating process. Use a thermal imaging camera (Flir, Wilsonville, OR, United States, USA) to check the actual temperature distribution of the device.

Example 4: Temperature distribution of microfluidic substrate

Thermal imaging cameras and thermocouples are used to determine the stability of temperature distribution in microfluidic devices.

First, when the distance d between the two thermal heating plates changes, the thermocouple monitors the temperature of each heating zone. Temperatures were recorded every minute and spacing distances of 8, 9 and 10 cm were applied. The results showed that the temperature stabilized within 10 minutes (shown in Figure 6A). When the distance d is smaller, there is a higher temperature in the third heating zone.

For microfluidic PCR, the reagents need to be thermally cycled between 95°C and 60°C for DNA amplification, while for SPR wafers, the temperature should be maintained at around 63.7°C, That is, the melting temperature (Tm) of the probe for DNA detection. Therefore, the distance d is set to 9cm. Thermocouples monitor the temperatures of denaturation (first heating zone), primer adhesion (second heating zone), and elongation and detection (third heating zone) (shown in Figure 6B ).

The results showed that after 5 minutes of heating, the device reached the desired temperature (as shown in Figure 6C). Uniform temperature distribution was observed in the microfluidic PCR area lasting at least 1 hour. In addition, when the heating plates are 10 mm apart, the third heating zone is maintained at 65°C.

Example 5: Amplification capability of microfluidic PCR device

Gel electrophoresis was used to confirm the products of the disclosed microfluidic PCR device at different flow rates. First, a PCR solution containing LMP1 DNA with an initial concentration of 10 μg/mL was pumped into the microchannel PCR, and the syringe pump was set to different flow rates, namely 3, 4, 5, 6, and 7 μL/min. The products were then analyzed by gel electrophoresis.

The results showed that at flow rates of 3, 4 and 5 μL/min, the fluorescent signal of gel electrophoresis appeared at the correct position (300 bp), indicating that the microfluidic device of the present disclosure can successfully amplify DNA under these flow conditions. In contrast, flow rates of 6 and 7 μL/min showed no fluorescent signal, indicating that the flow rate was too fast to produce product, resulting in low concentrations that could not be detected by gel electrophoresis. These results indicate that the microfluidic PCR device of the present disclosure can successfully amplify LMP1 DNA.

Example 6: Detection capability of microfluidic PCR device integrated with biological detection chip

In order to confirm the detection capability of the integrated device, the red shift produced by the PCR detection device of the present disclosure was recorded at different flow rates with an incubation time of 10 minutes, and then juxtaposed with the red shift of the product amplified by a traditional PCR machine to evaluate the detection integrated device capabilities (Figure 8). The red shift of the device with flow rates set to 3 and 4 μL/min is similar to that of the traditional machine after 30 cycles, and the red shift of the device is still in a stable state, which indicates that the SPR sensor has reached its maximum detection capability. In comparison, the red shift of devices with flow rates set to 6 and 7 μL/min was lower than that of 30 cycles of a conventional machine. In particular, the red shift of the device with a flow rate of 7 μL/min is even lower than that of the traditional Redshift of 20 cycles of the machine.

On the other hand, at a flow rate of 5 μL/min, the PCR detection device of the present disclosure exhibits similar amplification capabilities to traditional machines, but the reaction time is significantly shortened. The reaction time of the disclosed PCR detection equipment only takes 36 minutes, while the traditional PCR machine takes 105 minutes to perform 30 cycles of amplification. Therefore, under the conditions of this embodiment, 5 μL/min can be considered as the optimal reaction flow rate of the PCR detection device of the present disclosure.

In addition, the SPR chip can show corresponding red shifts at different flow rates before reaching its maximum capacity, which indicates that the SPR chip can detect LMP1 DNA and can perform quantitative analysis based on concentration to a certain extent.

effect

Compared with traditional PCR machines, the PCR detection device of the present disclosure can significantly reduce the required sample volume. In addition, since the contact area between the PCR solution and the heat source is increased and the temperature control is more effective, the use of the PCR detection device of the present disclosure reduces the overall PCR amplification time. After amplification, the target DNA can be captured by the DNA probe on the gold-coated nanoslit used and sensitively detected by the SPR method. In short, the device disclosed in the present disclosure can amplify the LMP1 gene fragment at the front end and perform detection at the back end, thereby speeding up PCR and having high specificity and sensitivity for detecting target DNA.

Listed implementations

The following illustrative embodiments are provided, and their numbering should not be construed as designating a degree of significance.

Embodiment 1 provides a PCR device, which includes a constant temperature unit, which includes a first heating plate and a second heating plate, wherein the first heating plate and the second heating plate are configured to be parallel with a distance d. Parallel; a microfluidic channel substrate, which includes a microfluidic channel, wherein the microfluidic channel is configured in the microfluidic channel substrate in a plurality of parallel back-and-forth cycles of equal length; and wherein the microfluidic channel substrate is placed on the on the constant temperature unit and in contact with the first heating plate forming a first heating area, and contacting the second heating plate to form a second heating area, wherein the first heating plate can apply a first temperature T1 to heat the first heating area, and the second heating plate can apply a The second temperature T2 heats the second heating zone, wherein when T1 is different from T2, the area of the microfluidic substrate located above the distance d forms a third heating zone, and one of the third heating zones formed The third temperature T3 is between T1 and T2, wherein each equal-length parallel back-and-forth cycle in the microfluidic substrate passes through the first heating zone, the second heating zone, and the third heating zone.

Embodiment 2 provides the PCR device as described in Embodiment 1, wherein the distance d can vary within the range of 1 to 10 mm.

Embodiment 3 provides the PCR device as described in Embodiment 1, wherein the microfluidic channel in the microfluidic channel substrate includes an inlet end and at least one outlet end.

Embodiment 4 provides a PCR detection device, which includes a constant temperature unit, which includes a first heating plate and a second heating plate, wherein the first heating plate and the second heating plate are configured to be separated by a distance d. Parallel and juxtaposed; a microfluidic channel substrate, which includes a microfluidic channel, wherein the microfluidic channel is configured in the microfluidic channel substrate in a plurality of equal-length parallel back-and-forth cycles; and a biological detection chip, which can be measured with a fluid The communication method is loaded on the end of the microfluidic channel of the microfluidic channel substrate; wherein the microfluidic channel substrate is placed on the constant temperature unit and contacts the first heating plate to form a first heating area, and is connected with the third heating plate. The two heating plates contact to form a second heating zone, wherein the first heating plate can apply a first temperature T1 to heat the first heating zone, and the second heating plate can apply a second temperature T2 to heat the second heating zone. , when T1 is different from T2, the area of the microfluidic substrate above the distance d forms a third heating zone, and a third temperature T3 formed in the third heating zone is between T1 and T2 time, wherein each equal-length parallel back-and-forth cycle in the microfluidic substrate passes through the first heating zone, the second heating zone, and the third heating zone.

Embodiment 5 provides the PCR detection system as described in Embodiment 4, wherein the biological detection chip is a surface plasmon resonance chip.

Embodiment 6 provides the PCR detection device as described in Embodiment 4, which further includes a flow control unit that can control the flow rate of the liquid in the microfluidic channel substrate.

Embodiment 7 provides the PCR detection device as described in Embodiment 4, which further includes a temperature control unit that is electrically connected to the constant temperature unit and can respectively control the heating temperatures of the first heating plate and the second heating plate. .

Embodiment 8 provides the PCR detection device as described in Embodiment 4, wherein the distance d can vary within the range of 1 to 10 mm.

Embodiment 9 provides the PCR detection device as described in Embodiment 4, wherein when the test sample is a salt-free sample, the distance d is in the range of 1 to 6 mm.

Embodiment 10 provides the PCR detection device as described in Embodiment 4, wherein when the test sample is a salt sample, the distance d is in the range of 6 to 10 mm.

Embodiment 11 provides the PCR detection device as described in Embodiment 4, wherein when T1 is greater than T2, the temperature control range of the first heating plate is 80°C to 100°C.

Embodiment 12 provides the PCR detection device as described in Embodiment 4, wherein when T1 is greater than T2, the temperature control range of the second heating plate is 55°C to 65°C.

Embodiment 13 provides the PCR detection device as described in Embodiment 4, wherein when the test sample is a salt-free sample, the temperature control range of the first heating plate is 80°C to 90°C, and the temperature of the second heating plate The control range is 55℃ to 65℃.

Embodiment 14 provides the PCR detection device as described in Embodiment 4, wherein when the test sample is a salt sample, the temperature control range of the first heating plate is 90°C to 100°C, and the temperature of the second heating plate The control range is 55℃ to 65℃.

Embodiment 15 provides the PCR detection device as described in Embodiment 4, wherein the microfluidic channel in the microfluidic substrate includes an inlet end and at least one outlet end.

Embodiment 16 provides a PCR detection system, which includes a constant temperature unit, which includes a first heating plate and a second heating plate, wherein the first heating plate and the second heating plate are arranged in parallel with a distance d; A microfluidic channel substrate, which includes a microfluidic channel, wherein the microfluidic channel is configured in the microfluidic channel substrate in a plurality of equal-length parallel back-and-forth cycles, wherein the microfluidic channel substrate is placed on the constant temperature unit on, and in contact with the first heating plate to form a first heating area, and in contact with the second heating plate to form a second heating area, wherein the first heating plate can apply a first temperature T1 to heat the first heating area area, and the second heating plate can apply a second temperature T2 to heat the second heating area; a biological detection chip, which is loaded in a fluid-connectable manner at the end of the microfluidic channel of the microfluidic channel substrate; a flow rate A control unit that can control the flow rate of the liquid in the microfluidic channel substrate; a temperature control unit that is electrically connected to the constant temperature unit and can respectively control the heating temperatures of the first heating plate and the second heating plate; and A detection unit for detecting the biological detection chip; wherein when T1 is greater than T2, the area of the microfluidic substrate above the distance d forms a third heating area, and one of the areas formed in the third heating area The third temperature T3 is between T1 and T2.

Embodiment 17 provides the PCR detection system as claimed in claim 16, wherein the biological detection chip is a surface plasmon resonance chip.

Embodiment 18 provides the PCR detection system as described in Embodiment 16, wherein the distance d can vary in the range of 1 to 10 mm.

Embodiment 19 provides the PCR detection system as described in Embodiment 16, wherein when the test sample is a salt-free sample, the distance d is in the range of 1 to 6 mm.

Embodiment 20 provides the PCR detection system as described in Embodiment 16, wherein when the test sample is a salt sample, the distance d is in the range of 6 to 10 mm.

Embodiment 21 provides the PCR detection system as described in Embodiment 16, wherein when T1 is greater than T2, the temperature control range of the first heating plate is 80°C to 100°C.

Embodiment 22 provides the PCR detection system as described in Embodiment 16, wherein when T1 is greater than T2, the temperature control range of the second heating plate is 55°C to 65°C.

Embodiment 23 provides the PCR detection system as described in Embodiment 16, When the test sample is a salt-free sample, the temperature control range of the first heating plate is 80°C to 90°C, and the temperature control range of the second heating plate is 55°C to 65°C.

Embodiment 24 provides the PCR detection system as described in Embodiment 16, wherein when the test sample is a salt sample, the temperature control range of the first heating plate is 90°C to 100°C, and the temperature of the second heating plate The control range is 55℃ to 65℃.

Embodiment 25 provides the PCR detection system as described in Embodiment 16, wherein the microfluidic channel in the microfluidic substrate includes an inlet end and at least one outlet end.

Embodiment 26 provides a PCR detection method, which method includes: providing a PCR detection system in any one of embodiments 16 to 25; using the temperature control unit to adjust the first heating plate and the second heating in the constant temperature unit plate for a period of time to stabilize the first heating zone, the second heating zone and the third heating zone at the temperatures of T1, T2 and T3 respectively; use the flow control unit to continuously pump a sample to be measured into the plate at a fixed flow rate. The inlet end of the microfluidic channel of the microfluidic channel substrate is detected by the detection unit and analyzed by an analysis unit for a period of time.

Embodiment 27 provides the PCR detection method as described in Embodiment 26, wherein the distance d can vary within the range of 1 to 10 mm.

Embodiment 28 provides the PCR detection method as described in Embodiment 26, wherein when the test sample is a salt-free sample, the distance d is in the range of 1 to 6 mm.

Embodiment 29 provides the PCR detection method as described in Embodiment 26, wherein when the test sample is a salt sample, the distance d is in the range of 6 to 10 mm.

Embodiment 30 provides the PCR detection method as described in Embodiment 26, wherein when T1 is greater than T2, the temperature control range of the first heating plate is 80°C to 100°C.

Embodiment 31 provides the PCR detection method as described in Embodiment 26, wherein when T1 is greater than T2, the temperature control range of the second heating plate is 55°C to 65°C.

Embodiment 32 provides the PCR detection method as described in Embodiment 26, wherein when the test sample is a salt-free sample, the temperature control range of the first heating plate is 80°C to 90°C, and the temperature of the second heating plate The control range is 55℃ to 65℃.

Embodiment 33 provides the PCR detection method as described in Embodiment 26, wherein when the test sample is a salt sample, the temperature control range of the first heating plate is 90°C to 100°C, and the temperature of the second heating plate The control range is 55℃ to 65℃.

The exemplary embodiments disclosed above are merely intended to illustrate the various uses of the present invention. It should be understood that according to the above teachings, various modifications, changes and combinations can be made to the functional elements and forms of the present application. Therefore, within the scope of the appended claims, the contents of this application can be implemented in a manner different from that specifically disclosed, and can be implemented for other applications. The principles of this instruction can be easily extended by applying them with appropriate modifications.

All patents and published applications are herein incorporated by reference to the same extent as if each individual published application was specifically and individually indicated to be incorporated by reference. It should be understood that although the content of this case has been specifically disclosed through preferred embodiments and optional features, those skilled in the art can make modifications and changes to the concepts disclosed herein, and it should be considered that these modifications and changes are within the scope of this case.

11: Microfluidic channel

12: Entrance port

12a: Chip microchannel connection port

12b: Chip microchannel connection port

13:Export end

13a: Chip microchannel connection port

14:Export end

14a: Chip microchannel connection port

15: First heating zone

16: Second heating zone

17: The third heating zone

20: Constant temperature unit

21:First heating plate

21a: Temperature control port

22:Second heating plate

22a: Temperature control port

31:The first biological detection chip

32: Second biological detection chip

d: spacing

Claims (9)

A PCR detection device, which includes a constant temperature unit, which includes a first heating plate and a second heating plate, wherein the first heating plate and the second heating plate are configured to be parallel and juxtaposed with a distance d; a A microfluidic channel substrate, which includes a microfluidic channel, wherein the microfluidic channel includes an inlet end and at least one outlet end, and the microfluidic channel is configured in the microfluidic channel substrate in a plurality of equal-length parallel back-and-forth cycles ; and a biological detection chip, which is loaded in a fluid-connectable manner at the end of the microfluidic channel of the microfluidic channel substrate; wherein the microfluidic channel substrate is placed on the constant temperature unit and in contact with the first heating plate A first heating area is formed, and a second heating area is formed in contact with the second heating plate, wherein the first heating plate can apply a first temperature T1 to heat the first heating area, and the second heating plate can apply A second temperature T2 heats the second heating zone, wherein when T1 is different from T2, the area of the microfluidic substrate above the distance d forms a third heating zone, and the area formed in the third heating zone A third temperature T3 is between T1 and T2, wherein each equal-length parallel back-and-forth cycle in the microfluidic substrate passes through the first heating zone, the second heating zone and the third heating zone. The PCR detection device as claimed in claim 1, wherein the distance d can vary within the range of 1 to 10 mm. The PCR detection device as claimed in claim 1, wherein when the test sample is a salt-free sample, the distance d is in the range of 1 to 6 mm. The PCR detection device as claimed in claim 1, wherein when the test sample is a salt sample, the distance d is in the range of 6 to 10 mm. The PCR detection device of claim 1, wherein when T1 is greater than T2, the temperature control range of the first heating plate is 80°C to 100°C, and the temperature control range of the second heating plate is 55°C to 65°C. . The PCR detection device as described in claim 1, wherein when the detection sample is When there is no salt sample, the temperature control range of the first heating plate is 80°C to 90°C, and the temperature control range of the second heating plate is 55°C to 65°C. The PCR detection device of claim 1, wherein when the test sample is a salt sample, the temperature control range of the first heating plate is 90°C to 100°C, and the temperature control range of the second heating plate is 55 ℃ to 65℃. A PCR detection system, which includes a constant temperature unit, which includes a first heating plate and a second heating plate, wherein the first heating plate and the second heating plate are parallel and juxtaposed with a distance d; a microfluidic channel The substrate includes a microfluidic channel, wherein the microfluidic channel includes an inlet end and at least one outlet end, and the microfluidic channel is configured in the microfluidic channel substrate in a plurality of equal-length parallel back-and-forth cycles, wherein the microfluidic channel The microfluidic substrate is placed on the constant temperature unit, and is in contact with the first heating plate to form a first heating area, and is in contact with the second heating plate to form a second heating area, wherein the first heating plate can A first temperature T1 is applied to heat the first heating zone, and the second heating plate can apply a second temperature T2 to heat the second heating zone; a biological detection chip is loaded in the microfluidic channel in a fluid-communicable manner At the end of the microfluidic channel of the substrate; a flow control unit that can control the flow rate of the liquid in the microfluidic substrate; a temperature control unit that is electrically connected to the constant temperature unit and can respectively control the first heating plate and the heating temperature of the second heating plate; and a detection unit for detecting the biological detection chip; wherein when T1 is greater than T2, the area in the microfluidic substrate above the distance d forms a third heating area, And a third temperature T3 formed in the third heating zone is between T1 and T2. The PCR detection system of claim 8, wherein the biological detection chip is a surface plasmon resonance chip.