Digital microfluidic MALBAC device based on temperature measurement and control

Abstract

The invention belongs to the related technical field of digital microfluidics, and discloses a digital microfluidics MALBAC device based on temperature measurement and control, which comprises a digital microfluidics chip 10 and a temperature measurement and control system 20, the digital micro-fluidic chip 10 adopts a closed structure and comprises an upper cover plate 111 and a lower chip 112 which are arranged in parallel relatively, and the temperature measurement and control system 20 comprises a temperature conduction module 21 and a temperature sensing module 22; according to the device, a totally-closed operation environment is provided for the MALBAC process, the risk of cross contamination is effectively avoided, the device can create a plurality of independent temperature areas on a single chip, the thermal cycle efficiency in the MALBAC technology is optimized, it is ensured that the temperature requirement of each stage in the amplification process is accurately met, and the amplification efficiency is improved. The integration level, the operation flexibility and the expandability of the device are greatly improved, so that the device can adapt to library construction requirements of various genetic material samples.

Description

Technical Field

[0001] The present invention belongs to the technical field related to digital microfluidics, and more specifically, relates to a digital microfluidics MALBAC device based on temperature measurement and control. Background Art

[0002] The rise of single-cell analysis technology has broken through the limitations of traditional population cell analysis in distinguishing subtle differences between cells, and provided a new perspective for the study of cell heterogeneity. With the continuous development of this technology, it has promoted the in-depth study of single-cell omics, covering multiple dimensions such as genome, transcriptome, epigenome, proteome and metabolome. In these studies, the genome, as the core library of genetic information in organisms, plays a decisive role in revealing the biological characteristics and individual characteristics of organisms. Its decoding is of great significance for the study of genetics and biological evolution. In the field of single-cell sequencing, single-cell whole genome amplification technology is one of the key steps, which solves the problem of extracting and amplifying DNA from tiny samples. At present, a variety of technologies are used in this field, such as degenerate oligonucleotide primer PCR amplification, multiple displacement amplification, multiple annealing circular amplification (MALBAC), and linear amplification achieved by transposon insertion.

[0003] MALBAC technology uses special primers to force the amplified fragments to form a closed-loop structure, avoiding the exponential amplification effect during DNA amplification, reducing genome amplification deviation, and significantly reducing the template demand for genome sequencing. However, MALBAC technology has limitations in terms of fidelity and preference for repetitive sequences, which may affect the accuracy of single nucleotide variation detection and the integrity of genomic data.

[0004] Although microchannel technology has improved the automation level of single-cell whole genome library preparation in microfluidic chips, it still faces problems such as reagent contamination caused by cavity dead volume, low single-cell capture efficiency, complex pump and valve structure design, and limitations of traditional temperature control. As a type of microfluidic technology, digital microfluidics abandons complex components such as micropumps, microvalves, and micromixers, and realizes the movement, generation, splitting, and mixing of droplets through continuous electrical signal control, providing an ideal platform for the library preparation process in single-cell sequencing. Therefore, it is urgent to develop a low-cost, easy-to-operate, and highly integrated digital microfluidic MALBAC device based on temperature measurement and control. Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the present invention provides a digital microfluidic MALBAC device based on temperature measurement and control, thereby solving the technical problems of reagent contamination and temperature control limitation.

[0006] To achieve the above-mentioned purpose, according to one aspect of the present invention, a digital microfluidic MALBAC device based on temperature measurement and control is provided, comprising a digital microfluidic chip 10 and a temperature measurement and control system 20; the digital microfluidic chip 10 adopts a closed structure, comprising an upper cover plate 111 and a lower chip 112 arranged relatively in parallel, the temperature measurement and control system 20 comprises a temperature conduction module 21 and a temperature sensing module 22, the temperature conduction module 21 comprises an upper heat conduction block 211, a thermoelectric cooler 212, a lower heat conduction block 213, and a heat pipe 214 arranged in sequence from top to bottom; the digital microfluidic chip 10 is arranged on the top of the temperature conduction module 21 and contacts the upper heat conduction block 211; the temperature sensing module 22 comprises a temperature sensor 221 and a processing system 222, the temperature sensor 221 is fixed on the upper heat conduction block 211, and is connected to the processing system 222 so as to exchange data and execute temperature control instructions.

[0007] Preferably, the upper cover plate 111 includes an upper substrate 107, an upper electrode layer 106, and an upper hydrophobic layer 105 arranged in sequence from top to bottom; the lower chip 112 includes a lower substrate 101, a driving electrode 102, a lower dielectric layer 103, and a lower hydrophobic layer 104 arranged in sequence from bottom to top; the upper hydrophobic layer 105 and the lower hydrophobic layer 104 face each other, and a droplet channel is formed therebetween.

[0008] Preferably, the pattern arrangement of the driving electrode 102 includes multiple functional areas: a left liquid storage area 11, a waste liquid area 12, a right liquid storage area 13, a product outlet area 14, and a temperature gradient area; the temperature gradient area is a rectangular loop composed of a low temperature area 15, a medium temperature area 16, a high temperature area 17, and a medium and low temperature area 18.

[0009] Preferably, the left liquid storage area 11 is located on the left side of the temperature gradient zone, and is used to store single cell lysis solution and washing solution; the right liquid storage area 13 is located at the lower right side of the temperature gradient zone, and is used to store MALBAC amplification solution and supplementary solution.

[0010] Preferably, the waste liquid zone 12 is located at the upper right of the temperature gradient zone, and is used to collect waste liquid generated during the reaction process; the product outlet zone 14 is located below the temperature gradient zone, and is used as an outlet for subsequent measurement unit products.

[0011] Preferably, the upper cover plate 111 and the lower chip 112 are packaged by a gasket 108 or a photocurable adhesive to form a closed structure.

[0012] Preferably, the upper heat conducting block 211 and the lower heat conducting block 213 are made of copper or aluminum, and the thickness is set to 0.1 to 80 mm.

[0013] Preferably, the temperature sensor 221 is a platinum resistance temperature sensor, the model of which is set to PT1000. The temperature sensor 221 is fixed to the center of the upper heat-conducting block 211 by heat-conducting silicone grease.

[0014] Preferably, the temperature of the low temperature zone 15 is controlled at -1°C to 3°C, preferably 0°C; the temperature of the medium and low temperature zone 18 is controlled at 55°C to 60°C, preferably 58°C.

[0015] Preferably, the temperature of the high temperature zone 17 is controlled at 90°C to 98°C, preferably 94°C; the temperature of the medium temperature zone 16 is controlled at 62°C to 68°C, preferably 65°C.

[0016] In general, compared with the prior art, the digital microfluidic MALBAC device based on temperature measurement and control provided by the present invention has the following beneficial effects:

[0017] 1. Closed and pollution-free operating environment: The digital microfluidic chip of the present invention provides a fully closed operating environment for the MALBAC process, effectively avoiding the risk of cross contamination. Its high flexibility and scalability enable the chip to meet the library construction requirements of various genetic material samples, and through the interconnection of electrode design, it realizes high-throughput, integrated customized experiments, meeting the needs of diversified research scenarios.

[0018] 2. Innovative temperature control capability: The temperature measurement and control system of the present invention breaks the limitations of traditional temperature control technology and adopts advanced components that can adjust the temperature in both directions. It can not only achieve heating above room temperature, but also achieve cooling close to the freezing point, providing precise temperature conditions for the MALBAC reaction. The system can create multiple independent temperature zones on a single chip, greatly improving the integration of the equipment and the flexibility of operation.

[0019] 3. Optimized thermal cycle efficiency: The present invention optimizes the thermal cycle efficiency in the MALBAC technology by precisely controlling each temperature zone, ensuring that the temperature requirements at each stage of the amplification process are accurately met, thereby improving the uniformity and reliability of amplification, which is crucial to improving the quality and accuracy of single-cell genome amplification.

[0020] 4. Double savings in cost and time: The integrated design of the present invention reduces the reliance on external equipment, simplifies the experimental operation process, reduces the consumption of reagents and samples, and thus reduces the experimental cost. At the same time, due to the automation and simplification of the operation, the experimenter can devote more time to data analysis and result interpretation instead of tedious experimental operations, which effectively saves experimental time and improves research efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the cross-sectional structure of the digital microfluidic MALBAC chip of the present invention;

[0022] Figure 2 It is a schematic diagram of the arrangement of driving electrodes on the digital microfluidic MALBAC chip of the present invention;

[0023] Figure 3 It is a schematic diagram of the structure of the digital microfluidic MALBAC chip and the temperature measurement and control system of the present invention;

[0024] Figure 4 It is a schematic diagram of the working process of the temperature measurement and control system in the present invention;

[0025] Figure 5 It is a trend diagram of the temperature change over time when the temperature measurement and control system of the present invention is working;

[0026] Throughout the drawings, the same reference numerals are used to denote the same elements or structures, wherein:

[0027] 10—digital microfluidic chip; 111—upper cover; 112—lower chip; 101—lower substrate; 102—driving electrode; 103—lower dielectric layer; 104—lower hydrophobic layer; 105—upper hydrophobic layer; 106—upper electrode layer; 107—upper substrate; 108—gasket; 109—droplet; 11—left liquid storage area; 12—waste liquid area; 13—right liquid storage area; 14—product outlet area; 15—low temperature area; 16—medium temperature area; 17—high temperature area; 18—medium and low temperature area; 20—temperature measurement and control system; 21—temperature conduction module; 22—temperature sensing module; 211—upper heat conduction block; 212—thermoelectric cooler; 213—lower heat conduction block; 214—heat pipe; 221—temperature sensor; 222—processing system. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0029] In the description document of the present invention, the drawings provided are only for schematic display and do not represent the actual size ratio. In practical applications, details such as the thickness of the film layer and the spacing between them need to be adjusted accordingly according to the specific implementation. Therefore, the structure shown in the drawings is only a schematic diagram, which is intended to help understand the core concept of the present invention and does not mean that the implementation method of the present invention is limited to the specific form or value shown in the drawings. The implementation of the present invention is not limited to the specific details shown in the drawings.

[0030] See also Figure 1 and Figure 3 A digital microfluidic MALBAC device based on temperature measurement and control includes a digital microfluidic chip 10 and a temperature measurement and control system 20; the digital microfluidic chip 10 adopts a closed structure, including an upper cover plate 111 and a lower chip 112 arranged relatively parallel, the temperature measurement and control system 20 includes a temperature conduction module 21 and a temperature sensing module 22, the temperature conduction module 21 realizes thermal management through a multi-layer structure, including an upper heat conduction block 211, a thermoelectric cooler 212, a lower heat conduction block 213, and a heat pipe 214 arranged in sequence from top to bottom; the digital microfluidic chip 10 is arranged on the top of the temperature conduction module 21, and contacts with the upper heat conduction block 211; the temperature sensing module 22 includes a temperature sensor 221 and a processing system 222, the temperature sensor 221 is fixed on the upper heat conduction block 211, and is connected to the processing system 222, so as to exchange data and execute temperature control instructions. This structural design ensures that the temperature measurement and control system 20 can operate efficiently and provide accurate temperature control for the digital microfluidic chip 10.

[0031] The upper cover plate 111 includes an upper substrate 107, an upper electrode layer 106, and an upper hydrophobic layer 105 arranged in sequence from top to bottom; the lower chip 112 includes a lower substrate 101, a driving electrode 102, a lower dielectric layer 103, and a lower hydrophobic layer 104 arranged in sequence from bottom to top; the upper hydrophobic layer 105 faces the lower hydrophobic layer 104, and a droplet channel is formed therebetween. The upper cover plate 111 and the lower chip 112 are packaged by a gasket 108 or a photocurable adhesive to form a closed structure.

[0032] The upper heat-conducting block 211 and the lower heat-conducting block 213 are made of metal materials with excellent thermal conductivity such as copper or aluminum, and the thickness is set to 0.1 to 80 mm.

[0033] The temperature sensor 221 is a platinum resistance temperature sensor with excellent long-term stability and a wide temperature measurement range. Preferably, the model is PT1000. The temperature sensor 221 is fixed to the center of the upper heat-conducting block 211 by thermal grease to ensure good thermal contact. The temperature sensor 221 is led out from the upper heat-conducting block 211 by a wire and connected to the processing system 222, which involves the connection with the signal input terminal of the single-chip controller. In order to reduce the heat loss near the sensor part and improve the accuracy of temperature measurement, the length of the wire connecting the temperature sensor 221 and the processing system 222 should be appropriate to optimize thermal energy management.

[0034] See also Figure 2 , the pattern arrangement of the driving electrode 102 includes multiple functional areas: left liquid storage area 11, waste liquid area 12, right liquid storage area 13, product outlet area 14, temperature gradient area; the temperature gradient area is a rectangular loop composed of low temperature area 15, medium temperature area 16, high temperature area 17, and medium and low temperature area 18. The droplet 109 undergoes dielectric wetting along the pattern of the driving electrode 102 in the closed structure composed of the upper cover plate 111 and the lower chip 112, thereby realizing the movement, splitting and merging of the droplet and other manipulation actions. The low temperature area 15 is used for the annealing step of the primer and the DNA template, the medium and low temperature area 18 and the medium temperature area 17 are used to ensure the occurrence of linear amplification cycle and chain displacement polymerization reaction, respectively, and the high temperature area 17 is used for DNA denaturation, so that the double strand is split into a single strand, ready for the next round of amplification reaction.

[0035] The left liquid storage area 11 is located on the left side of the temperature gradient area, and is used to store single cell lysis solution and washing solution, providing the necessary starting materials for the MALBAC reaction; the right liquid storage area 13 is located at the lower right of the temperature gradient area, and is used to store MALBAC amplification solution and supplementary solution to maintain the continuity and stability of the reaction. The waste liquid area 12 is located at the upper right of the temperature gradient area, and is used to collect waste liquid generated during the reaction process, ensuring the environmental cleanliness and reaction efficiency of the entire system; the product outlet area 14 is located below the temperature gradient area, and is a key channel connecting the inside of the chip with the external environment. It is used as the outlet for subsequent measurement unit products, providing convenience for subsequent analysis and research.

[0036] The temperature conduction module 21 and the temperature sensing module 22 work together to maintain multiple areas with different temperature settings for the temperature gradient area of ​​the chip. The temperature of these areas can be controlled independently, including not only areas above room temperature, but also areas that need to be below room temperature, to meet the complex requirements for temperature changes during MALBAC amplification. The temperature conduction module 21 is responsible for the actual heating or cooling process, while the temperature sensing module 22 monitors the temperature changes in real time through the temperature sensor 221 and feeds the data back to the processing system 222. Therefore, the temperature measurement and control system 20 can accurately adjust the temperature of each temperature zone to ensure that the temperature of each area can reach and remain at the preset optimal conditions, whether it is high temperature to promote DNA denaturation or low temperature to achieve primer annealing.

[0037] See also Figure 4 , is the working process of the temperature measurement and control system 20 in the present invention, where TS is the target set temperature, TR is the actual measured temperature, E represents the temperature error (E=TS-TA), and dE / dt represents the error change rate. The temperature sensing module adopts the PID fuzzy control algorithm, and the set calculated value is transmitted to the temperature lifting module. After passing through the temperature measurement module, the actual temperature value is obtained and compared with the target temperature value. The specific temperature control method is: when the deviation between the two is large, fuzzy control is used, which has fast response speed and good dynamic performance; when the deviation between the two is small, PID control is used to ensure good static performance and meet the system accuracy requirements.

[0038] See also Figure 5 , which is a trend diagram of the temperature change over time of the temperature measurement and control system 20 in the present invention when it is working, through Figure 5 From the temperature measurement data, we can see that the temperatures in all temperature zones are maintained very uniformly, and there is no temperature interference. Specifically, the temperature of low temperature zone 15 is controlled at 0±0.2℃; the temperature of medium and low temperature zone 18 is controlled at 58±0.7℃; the temperature of high temperature zone 17 is controlled at 94±1℃; the temperature of medium temperature zone 16 is controlled at 65±0.8℃. Since a large temperature difference will intensify the convective heat transfer effect of the air, the measured temperature difference increases with the increase of the set temperature. This trend reflects the performance of the temperature measurement and control system under different temperature settings, proving that the system can provide accurate and stable temperature control effects according to the needs of different temperature zones.

[0039] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A digital microfluidic MALBAC device based on temperature measurement and control, characterized in that: The invention comprises a digital microfluidic chip (10) and a temperature measurement and control system (20); the digital microfluidic chip (10) adopts a closed structure, comprising an upper cover plate (111) and a lower chip (112) arranged relatively in parallel; the temperature measurement and control system (20) comprises a temperature conduction module (21) and a temperature sensing module (22); the temperature conduction module (21) comprises an upper heat conduction block (211), a thermoelectric cooler (212), a lower heat conduction block (213), and a heat pipe (214) arranged in sequence from top to bottom; the digital microfluidic chip (10) is arranged on the top of the temperature conduction module (21) and is in contact with the upper heat conduction block (211); the temperature sensing module (22) comprises a temperature sensor (221) and a processing system (222); the temperature sensor (221) is fixed on the upper heat conduction block (211) and is connected to the processing system (222) so as to exchange data and execute temperature control instructions.

2. A digital microfluidic MALBAC device based on temperature measurement and control as claimed in claim 1, characterized in that: The upper cover plate (111) comprises an upper substrate (107), an upper electrode layer (106), and an upper hydrophobic layer (105) which are arranged in sequence from top to bottom; the lower chip (112) comprises a lower substrate (101), a drive electrode (102), a lower dielectric layer (103), and a lower hydrophobic layer (104) which are arranged in sequence from bottom to top; the upper hydrophobic layer (105) and the lower hydrophobic layer (104) face each other, and a liquid droplet channel is formed therebetween.

3. A digital microfluidic MALBAC device based on temperature measurement and control as claimed in claim 2, characterized in that: The pattern arrangement of the driving electrode (102) includes multiple functional areas: a left liquid storage area (11), a waste liquid area (12), a right liquid storage area (13), a product outlet area (14), and a temperature gradient area; the temperature gradient area is a rectangular loop consisting of a low temperature area (15), a medium temperature area (16), a high temperature area (17), and a medium and low temperature area (18).

4. A digital microfluidic MALBAC device based on temperature measurement and control as claimed in claim 3, characterized in that: The left liquid storage area (11) is located on the left side of the temperature gradient area and is used to store single cell lysis solution and washing solution; the right liquid storage area (13) is located at the lower right side of the temperature gradient area and is used to store MALBAC amplification solution and supplementary solution.

5. A digital microfluidic MALBAC device based on temperature measurement and control as claimed in claim 3, characterized in that: The waste liquid area (12) is located at the upper right of the temperature gradient area and is used to collect waste liquid generated during the reaction process; the product outlet area (14) is located below the temperature gradient area and is used as an outlet for subsequent measurement unit products.

6. A digital microfluidic MALBAC device based on temperature measurement and control as claimed in claim 1, characterized in that: The upper cover plate (111) and the lower chip (112) are packaged via a gasket (108) or a photocurable adhesive to form a closed structure.

7. A digital microfluidic MALBAC device based on temperature measurement and control as claimed in claim 1, characterized in that: The upper heat conducting block (211) and the lower heat conducting block (213) are made of copper or aluminum, and the thickness is set to 0.1 to 80 mm.

8. A digital microfluidic MALBAC device based on temperature measurement and control as claimed in claim 1, characterized in that: The temperature sensor (221) is a platinum resistance temperature sensor, the model of which is set to PT1000. The temperature sensor (221) is fixed at the center of the upper heat-conducting block (211) by heat-conducting silicone grease.

9. A digital microfluidic MALBAC device based on temperature measurement and control as claimed in claim 3, characterized in that: The temperature of the low temperature zone (15) is controlled at -1°C to 3°C; the temperature of the medium and low temperature zone (18) is controlled at 55°C to 60°C.

10. A digital microfluidic MALBAC device based on temperature measurement and control as claimed in claim 3, characterized in that: The temperature of the high temperature zone (17) is controlled at 90°C to 98°C; the temperature of the medium temperature zone (16) is controlled at 62°C to 68°C.

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