Ceramic microfluidic chip for automated PCR detection

Abstract

This invention relates to the field of biomedical detection technology, specifically to a ceramic microfluidic chip for automated PCR detection. The ceramic microfluidic chip consists of a chip body and a chip holder. The chip body comprises a ceramic substrate and a transparent cover plate. The ceramic substrate includes a substrate base plate and several parallel-arranged reaction grooves, each reaction groove being a recessed space etched into the substrate base plate. The substrate base plate is made of high-purity aluminum nitride ceramic or alumina ceramic. The transparent cover plate is bonded and sealed to the ceramic substrate, forming several parallel-arranged reaction chambers with the reaction grooves. This invention is the first to incorporate aluminum nitride ceramic material into a PCR chip. Compared to silicon-based chips, aluminum nitride ceramic chips offer 50-100% higher temperature control and production yield, making them an ideal material for PCR carriers and preventing substrate cracking or edge chipping.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, specifically to a ceramic microfluidic chip for automated PCR detection. Background Technology

[0002] PCR technology is a core detection method in the field of molecular biology. Automated PCR detection utilizes large-scale automated PCR equipment for automatic sample introduction, sealing, and PCR (polymerase chain reaction) reactions, significantly reducing manpower and improving detection efficiency. However, the reaction efficiency of automated PCR detection is highly dependent on the thermal cycling speed of the PCR vector, the chamber's airtightness, the ease of solution dispensing, and its anti-contamination capabilities.

[0003] Materials suitable for PCR vectors include silicon-based, glass-based, plastic-based, and ceramic-based materials. For example, Chinese Patent 202410558702.5 discloses a PCR sealed detection box. In this patent, the PCR reaction vector uses silicon as a substrate, and microchannels are fabricated on the silicon substrate to form a chamber bonded to glass, utilizing the thermal conductivity of the silicon substrate to improve the heating and cooling rates. However, the inventors of this application discovered in practice that the intrinsic thermal conductivity of silicon-based materials is approximately... However, large-size processing (≥30mm×28mm) is prone to edge chipping, and tolerance control is difficult (usually ±10μm or more). Furthermore, the surface oxide layer increases thermal resistance, resulting in poor actual thermal uniformity. The brittleness of silicon-based materials also makes the cavity prone to cracking, increasing the risk of leakage. Glass-based materials have good transparency but low thermal conductivity, only… The thermal cycling speed is slow; plastic-based materials (such as conventional EP tubes) are low in cost, but have low thermal conductivity and poor heat resistance; the technical difficulties of ordinary ceramic-based materials lie in achieving precision machining, efficient bonding, convenient liquid injection structure integration and anti-pollution design.

[0004] Therefore, in order to promote the rapid development of automated PCR detection, it is urgent to design a high thermal conductivity PCR chip that not only simplifies the processing and bonding process, ensures processing accuracy, meets the processing requirements of different channel sizes, and is suitable for large-scale production, but also solves the problems of poor thermal conductivity uniformity, easy cracking, and easy edge breakage of silicon-based materials. Summary of the Invention

[0005] To address the above problems, this invention proposes a ceramic microfluidic chip for automated PCR detection.

[0006] This invention provides a ceramic microfluidic chip for automated PCR detection. The ceramic microfluidic chip consists of a chip body and a chip holder. The chip body comprises a ceramic substrate and a transparent cover plate. The ceramic substrate includes a substrate base plate and several parallel-arranged reaction grooves, each reaction groove being a recessed space etched into the substrate base plate. One side of each of the parallel-arranged reaction grooves opens onto one side of the substrate base plate. The substrate base plate is made of high-purity aluminum nitride ceramic or alumina ceramic. The transparent cover plate is bonded and sealed to the ceramic substrate, forming several parallel-arranged reaction chambers with the reaction grooves. The chip holder is a chip fixing structure adapted to an automated PCR detection instrument. One side of the chip holder is detachably fixed to the automated PCR detection instrument, and the other side is fixed to the side of the reaction groove opening of the chip body. The chip holder is a solid structure, with an injection port and an vent only at positions corresponding to each reaction chamber.

[0007] Furthermore, the dimensions of the reaction tank are ≥30mm×28mm.

[0008] Furthermore, the aluminum nitride ceramic plate has a purity of ≥98% and a thermal conductivity of [missing information]. .

[0009] Furthermore, the alumina ceramic plate has a purity of ≥98% and a thermal conductivity of [missing information]. .

[0010] Furthermore, the process of etching reaction grooves on the substrate includes one or more of the following: precision sandblasting, CNC engraving, femtosecond laser processing, and laser sandblasting.

[0011] Furthermore, the transparent cover is made of glass or plastic with a transparency of ≥90%.

[0012] Furthermore, the bonding and sealing process between the transparent cover and the ceramic substrate can be as follows: sealing with UV adhesive followed by UV lamp irradiation for 30-60 seconds for bonding and curing; sealing with thermosetting adhesive followed by heat preservation at 80-100℃ for 30-60 minutes for bonding and curing; or sealing with UV adhesive followed by UV lamp irradiation for 30-60 seconds and then heat preservation at 80-100℃ for 30-60 minutes for bonding and curing.

[0013] Furthermore, one side of the chip holder is bonded and fixed to one side of the reaction groove opening of the chip body with adhesive.

[0014] Furthermore, the injection well is compatible with the pipette tip of the automated PCR detection instrument.

[0015] The beneficial effects of this invention are as follows:

[0016] 1. This invention is the first to incorporate aluminum nitride or alumina ceramic materials into a PCR chip. It specifies the use of high-purity aluminum nitride or alumina ceramic substrates to fabricate the reaction chambers. Compared to silicon-based materials, ceramic chip materials exhibit higher rigidity and better chemical stability, solving the problems of easy cracking and edge chipping associated with silicon-based materials. Furthermore, it significantly improves thermal conductivity. For the same size, aluminum nitride ceramic chips can achieve 50-100% higher heating and cooling rates compared to silicon-based chips, resulting in higher production yields. Traditional PCR instruments on the market typically use PCR tubes or PP materials as reaction carriers, with a maximum heating rate generally of 4-6°C / s. Patent 202410558702.5 discloses a silicon-based microfluidic chip for a sealed PCR detection box that can reach 30°C / s, while the aluminum nitride chip of this invention has been measured to have a maximum heating rate of 50°C / s. The thermal conductivity of aluminum nitride ceramic materials can reach [missing information]. The above-mentioned materials exhibit high rigidity, good chemical stability, and superior large-size processing tolerance compared to silicon-based materials, making them ideal for PCR vectors. In practice, it has been found that even when the reaction tank size is ≥30mm × 28mm using the technical solution of this invention, no substrate cracking or edge chipping occurs.

[0017] 2. The PCR chip processing of the present invention does not require complex processes such as photolithography and deep silicon etching. Precision sandblasting, CNC engraving, femtosecond laser, and laser sandblasting are all mature mass production technologies. Bonding adopts a low-temperature and simple solution, and the entire processing technology is simple and easy to implement.

[0018] 3. The PCR chip of the present invention is easy to mass-produce and is compatible with manual sample addition detection or automated PCR detection. It can be used alone or in combination of multiple chips. It supports manual sample addition and PCR instrument detection, and can also be used with fully automated equipment to achieve automatic sample addition, sealing and detection. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the ceramic microfluidic chip structure according to an embodiment of the present invention;

[0020] Figure 2 This is a schematic diagram of the chip body structure according to an embodiment of the present invention;

[0021] Figure 3 This is a schematic diagram of the ceramic substrate structure according to an embodiment of the present invention. Detailed Implementation

[0022] The present invention will be further described below with reference to the embodiments.

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1: Preparation of a ceramic microfluidic chip for automated PCR detection:

[0025] like Figures 1 to 3 As shown, a ceramic microfluidic chip for automated PCR detection is fabricated. The ceramic microfluidic chip consists of a chip body 1 and a chip holder 2. The chip body 1 consists of a ceramic substrate 11 and a transparent cover plate 12. The ceramic substrate 11 includes a substrate base plate 111 and four parallel reaction grooves 112. Each reaction groove 112 is a recessed space etched into the substrate base plate 111. One side of each of the four parallel reaction grooves 112 opens onto one side of the substrate base plate 111. The substrate base plate 111 is made of high-purity... The material is made of aluminum nitride ceramic plate; the transparent cover plate 12 is bonded and sealed to the ceramic substrate 11, and the transparent cover plate 12 and the reaction tank 112 form several parallel reaction chambers 3; the chip holder 2 is a chip fixing structure adapted to an automated PCR detector; one side of the chip holder 2 is detachably fixed to the automated PCR detector, and the other side is fixed to the side of the opening of the reaction tank 112 of the chip body 1; the chip holder 2 is a solid structure, with an injection hole 4 and an exhaust hole 5 only at the position corresponding to each of the reaction chambers 3.

[0026] Specifically, the aluminum nitride ceramic plate in this embodiment has a purity of ≥98% and a thermal conductivity of [missing information]. It can bring about a rapid heating effect.

[0027] In some other embodiments, high-purity (≥98%) alumina ceramics (thermally conductive) can be used. It can replace aluminum nitride ceramics, reduce costs, and is suitable for routine PCR scenarios with lower requirements for heating and cooling rates.

[0028] The process of etching reaction grooves on the substrate 111 includes one or more of the following: precision sandblasting, CNC engraving, femtosecond laser processing, and laser sandblasting. Grooving of the ceramic substrate is not limited to a single process; a combination of multiple processes can be used, such as "precision sandblasting roughing + femtosecond laser finishing," which can balance mass production efficiency and precision. Specifically, this embodiment uses femtosecond laser processing.

[0029] The transparent cover plate 12 is made of glass or plastic with a transparency of ≥90%, and is used to seal the reaction tank and ensure the light transmittance of fluorescence detection. Specifically, this embodiment uses glass with a transparency of ≥90%.

[0030] The bonding and sealing process between the transparent cover plate 12 and the ceramic substrate 11 can be achieved by sealing with UV adhesive followed by UV lamp irradiation for 30-60 seconds for bonding and curing, or by sealing with thermosetting adhesive followed by heat treatment at 80-100℃ for 30-60 minutes for bonding and curing, or by first sealing with UV adhesive, then irradiating with UV lamp for 30-60 seconds, and finally heat treatment at 80-100℃ for 30-60 minutes for bonding and curing. Low-temperature methods are simple and do not require high-temperature sintering. Specifically, in this embodiment, the UV adhesive used is bonded and cured by UV lamp irradiation for 60 seconds.

[0031] One side of the chip holder 2 is bonded and fixed to one side of the opening of the reaction groove 112 of the chip body 1 with glue (UV glue or AB glue), which serves to fix and seal the chip.

[0032] The injection port 4 is compatible with the pipette tip of the automated PCR detector. The injection port 4 facilitates sample addition. The injection port 4 and the vent port 5 serve as guides. During sample addition, the gas in the chamber is compressed and exits from the vent port 5.

[0033] Example 2: Comparison of the thermal conductivity of PCR chips prepared from different ceramic materials and silicon-based materials:

[0034] This embodiment aims to verify the difference in thermal conductivity between aluminum nitride ceramics and alumina ceramics, zirconia ceramics (approximate ceramic materials), and silicon materials under the same large size (30mm×28mm×2.5mm), and to clarify the material advantages of aluminum nitride ceramics in PCR chip applications.

[0035] Experimental steps:

[0036] 2.1 Experimental Samples: Four test substrates were prepared, made of aluminum nitride ceramic (purity ≥98%), alumina ceramic (purity ≥98%), zirconia ceramic (purity ≥95%), and single crystal silicon (intrinsic type). All substrates were uniformly sized at 30mm × 28mm × 2.5mm and had the same surface treatment process (no additional coating, only precision polishing to a surface roughness Ra ≤ 0.1μm) to ensure that the only test variable was the material itself.

[0037] 2.2 Test equipment: high-precision temperature acquisition instrument (error ≤ ±2%), programmable temperature control heating platform (temperature control accuracy ±0.1℃), infrared thermometer (response time ≤ 10ms), heat insulation device (to reduce ambient temperature interference).

[0038] 2.3 Test Principle: The steady-state heat flow method is adopted. First, the temperature of the heating stage is set to a constant value (100℃). After the temperature of the heating stage stabilizes, the substrate is tightly attached to the surface of the heating stage (the bonding surface is coated with thermal grease with a thickness of ≤0.1mm to eliminate the thermal resistance caused by the contact gap).

[0039] 2.4 Temperature sensor arrangement: On the lower surface of the substrate (the side that is in contact with the heating stage), in the non-contact area at the edge (with a reserved sensor mounting groove, 0.2mm deep, not penetrating the substrate), one K-type thermocouple sensor is attached to measure the initial temperature of the substrate receiving heat; on the upper surface of the substrate (the free surface away from the heating stage), one K-type thermocouple sensor is attached to the center position to measure the final temperature after heat conduction (the sensor is fixed to the substrate surface with thermally conductive adhesive to ensure lossless heat conduction).

[0040] 2.5 Detection: After the entire system reaches thermal steady state (the temperature readings of the upper and lower surfaces fluctuate by ≤0.05℃ for 30 seconds), record the temperature values ​​of the two sensors and calculate the temperature difference ΔT.

[0041] The thermal conductivity is calculated using the formula λ=Q×d / (A×ΔT) by combining the input thermal power (Q, which is monitored in real time by a thermal constant analyzer), the substrate thickness (d=2.5mm), and the heated area (A=30mm×28mm=840mm²).

[0042] 2.6 Experimental Results:

[0043] Table 1. Comparison of thermal conductivity of PCR chips prepared from different ceramic and silicon-based materials

[0044]

[0045] 2.7 Experimental Conclusion:

[0046] In this embodiment, the measured thermal conductivity of aluminum nitride ceramic is... Significantly higher than alumina ceramics Zirconia ceramics It is 8-9 times that of the latter two; and also higher than that of silicon materials. It is about 1.6 times that of silicon, demonstrating extremely strong thermal conductivity.

[0047] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0048] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A ceramic microfluidic chip for automated PCR detection, characterized in that, The ceramic microfluidic chip consists of a chip body (1) and a chip holder (2). The chip body (1) consists of a ceramic substrate (11) and a transparent cover plate (12). The ceramic substrate (11) includes a substrate base plate (111) and several parallel reaction grooves (112). The reaction grooves (112) are recessed spaces etched on the substrate base plate (111). One side of each of the several parallel reaction grooves (112) opens onto one side of the substrate base plate (111). The substrate base plate (111) is made of high-purity aluminum nitride ceramic plate or alumina ceramic plate. The transparent cover plate (12) is bonded and sealed to the ceramic substrate (11), and the transparent cover plate (12) and the reaction tank (112) form several parallel reaction chambers (3); the chip holder (2) is a chip fixing structure adapted to the automated PCR detector; one side of the chip holder (2) is detachably fixed to the automated PCR detector, and the other side is fixed to the side of the opening of the reaction tank (112) of the chip body (1); the chip holder (2) is a solid structure, and only has a liquid injection hole (4) and an air outlet hole (5) at the position corresponding to each of the reaction chambers (3).

2. The ceramic microfluidic chip for automated PCR detection according to claim 1, characterized in that, The dimensions of the reaction tank (112) are ≥30mm×28mm.

3. The ceramic microfluidic chip for automated PCR detection according to claim 1, characterized in that, The aluminum nitride ceramic plate has a purity of ≥98% and a thermal conductivity of [missing information]. .

4. The ceramic microfluidic chip for automated PCR detection according to claim 1, characterized in that, The alumina ceramic plate has a purity of ≥98% and a thermal conductivity of [missing information]. .

5. The ceramic microfluidic chip for automated PCR detection according to claim 1, characterized in that, The process of etching reaction grooves on the substrate base plate (111) includes one or more of the following: precision sandblasting, CNC engraving, femtosecond laser processing, and laser sandblasting.

6. The ceramic microfluidic chip for automated PCR detection according to claim 1, characterized in that, The transparent cover (12) is made of glass or plastic with a transparency of ≥90%.

7. The ceramic microfluidic chip for automated PCR detection according to claim 1, characterized in that, The bonding and sealing process between the transparent cover plate (12) and the ceramic substrate (11) can be as follows: sealing with UV glue and then irradiating with a UV lamp for 30-60 seconds for bonding and curing; sealing with thermosetting adhesive and then holding at 80-100℃ for 30-60 minutes for bonding and curing; or sealing with UV glue, then irradiating with a UV lamp for 30-60 seconds and then holding at 80-100℃ for 30-60 minutes for bonding and curing.

8. The ceramic microfluidic chip for automated PCR detection according to claim 1, characterized in that, The chip holder (2) is glued to one side of the opening of the reaction groove (112) of the chip body (1).

9. The ceramic microfluidic chip for automated PCR detection according to claim 1, characterized in that, The injection port (4) is compatible with the pipette tip of the automated PCR detector.

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