A digital microfluidic MALBAC device based on temperature measurement and control
By combining a closed digital microfluidic chip with a temperature measurement and control system, the problems of reagent contamination and temperature control in single-cell genome amplification using microfluidic chips have been solved, achieving efficient and accurate single-cell genome amplification, reducing costs and time, and adapting to diverse research scenarios.
Patent Information
- Application Number
- CN202411951298.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-27
- Publication Date
- 2025-10-28
- Estimated Expiration
- 2044-12-27
AI Technical Summary
Existing microfluidic chips suffer from problems such as reagent contamination, limited temperature control, complex structural design, and low capture efficiency during single-cell genome amplification, which affect the accuracy and efficiency of single-cell sequencing.
It adopts a closed digital microfluidic chip combined with a temperature measurement and control system, including a temperature conduction module and a sensing module. It achieves precise temperature control through thermoelectric coolers and heat conduction blocks, designs multiple independent temperature zones, optimizes thermal cycling efficiency, and simplifies the operation process.
It provides a pollution-free operating environment, improves the accuracy and efficiency of single-cell genome amplification, reduces experimental costs and time, adapts to diverse research needs, and enables high-throughput integrated experiments.
Smart Images
Figure CN119746967B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of digital microfluidics technology, and more specifically, relates to a digital microfluidic MALBAC device based on temperature measurement and control. Background Technology
[0002] The rise of single-cell analysis technology has overcome the limitations of traditional population cell analysis in distinguishing subtle differences between cells, providing a new perspective for the study of cellular heterogeneity. With the continuous development of this technology, it has propelled the in-depth development of single-cell omics research, covering multiple dimensions such as genomics, transcriptomics, epigenetics, proteomics, and metabolomics. In these studies, the genome, as the core repository of genetic information within an organism, plays a decisive role in revealing the biological characteristics and individual traits of organisms; its decoding is of paramount importance 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, solving the challenge of extracting and amplifying DNA from tiny samples. Currently, this field employs various techniques, such as degenerate oligonucleotide primer PCR amplification, multiple substitution amplification, multiple annealing circular amplification (MALBAC), and linear amplification achieved through transposon insertion.
[0003] MALBAC technology uses specially designed primers to induce the amplified fragments to form a closed loop structure, avoiding the exponential amplification effect during DNA amplification and reducing genome amplification bias, thus significantly reducing the template requirements 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 variant detection and the integrity of genome data.
[0004] While microchannel technology has improved the automation of single-cell whole-genome library preparation in microfluidic chips, it still faces challenges such as reagent contamination due to chamber dead volume, low single-cell capture efficiency, complex pump and valve design, and limitations of traditional temperature control. Digital microfluidics, as a type of microfluidic technology, eliminates complex components such as micropumps, microvalve, and micromixers, achieving droplet movement, generation, splitting, and mixing through continuous electrical signal control, providing an ideal platform for single-cell sequencing library preparation. Therefore, there is an urgent need to develop a low-cost, easy-to-operate, and highly integrated temperature-controlled digital microfluidic MALBAC device. Summary of the Invention
[0005] In view of the above-mentioned defects or improvement needs of the existing technology, 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 limitations.
[0006] To achieve the above objectives, according to one aspect of the present invention, a temperature-controlled digital microfluidic MALBAC device is provided, comprising a digital microfluidic chip 10 and a temperature control system 20; the digital microfluidic chip 10 adopts a closed structure, including an upper cover plate 111 and a lower chip 112 arranged in parallel; the temperature control system 20 includes a temperature conduction module 21 and a temperature sensing module 22; the temperature conduction module 21 includes an upper heat conduction block 211, a thermoelectric cooler 212, a lower heat conduction block 213, and a heat pipe 214 arranged sequentially from top to bottom; the digital microfluidic chip 10 is disposed on top of the temperature conduction module 21 and in contact 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 connected to the processing system 222 for data exchange and execution of temperature control commands.
[0007] Preferably, the upper cover plate 111 includes an upper substrate 107, an upper electrode layer 106, and an upper hydrophobic layer 105 arranged sequentially 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 sequentially from bottom to top; the upper hydrophobic layer 105 and the lower hydrophobic layer 104 face each other, and a droplet channel is formed between them.
[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-low temperature area 18.
[0009] Preferably, the left reservoir 11 is located to the left of the temperature gradient zone and is used to store single-cell lysis buffer and washing buffer; the right reservoir 13 is located to the lower right of the temperature gradient zone and is used to store MALBAC amplification solution and replenishment solution.
[0010] Preferably, the waste liquid zone 12 is located to the upper right of the temperature gradient zone and is used to collect waste liquid generated during the reaction; the product outlet zone 14 is located below the temperature gradient zone and is used as the outlet for the product of the subsequent measurement unit.
[0011] Preferably, the upper cover plate 111 and the lower chip 112 are encapsulated by a gasket 108 or 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, model PT1000, and the temperature sensor 221 is fixed to the center of the upper heat-conducting block 211 by thermal grease.
[0014] Preferably, the temperature of the low-temperature zone 15 is controlled between -1℃ and 3℃, and more preferably 0℃; the temperature of the medium-low temperature zone 18 is controlled between 55℃ and 60℃, and more preferably 58℃.
[0015] Preferably, the temperature of the high-temperature zone 17 is controlled at 90℃~98℃, more preferably 94℃; the temperature of the medium-temperature zone 16 is controlled at 62℃~68℃, more preferably 65℃.
[0016] In summary, 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 Contamination-Free Operating Environment: The digital microfluidic chip of this 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 adapt to the library construction needs of various genetic material samples, and through the interconnection of electrode design, it realizes high-throughput, integrated, and customized experiments, meeting the needs of diverse research scenarios.
[0018] 2. Innovative Temperature Control Capabilities: The temperature measurement and control system of this invention breaks through the limitations of traditional temperature control technology. It adopts advanced components capable of bidirectional temperature adjustment, enabling not only heating above room temperature but also cooling close to freezing point, providing precise temperature conditions for the MALBAC reaction. This system can create multiple independent temperature zones on a single chip, greatly improving the integration and operational flexibility of the device.
[0019] 3. Optimized thermal cycling efficiency: This invention optimizes the thermal cycling efficiency in MALBAC technology by precisely controlling each temperature region, ensuring that the temperature requirements at each stage of the amplification process are accurately met, thereby improving the uniformity and reliability of amplification. This is crucial for improving the quality and accuracy of single-cell genome amplification.
[0020] 4. Dual savings in cost and time: The integrated design of this invention reduces reliance on external equipment, simplifies experimental procedures, and reduces reagent and sample consumption, thereby reducing experimental costs. Simultaneously, due to the automation and simplification of operations, researchers can dedicate more time to data analysis and result interpretation rather than tedious experimental procedures, effectively saving experimental time and improving research efficiency. Attached Figure Description
[0021] Figure 1 This is a schematic cross-sectional view of the digital microfluidic MALBAC chip in this invention.
[0022] Figure 2 This is a schematic diagram of the driving electrode arrangement on the digital microfluidic MALBAC chip in this invention;
[0023] Figure 3 This is a schematic diagram of the structure of the digital microfluidic MALBAC chip and the temperature measurement and control system in this invention;
[0024] Figure 4 This is a schematic diagram of the working process of the temperature measurement and control system in this invention;
[0025] Figure 5 This is a trend graph showing the temperature change over time during operation of the temperature measurement and control system in this invention.
[0026] In all the accompanying drawings, the same reference numerals are used to denote the same elements or structures, wherein:
[0027] 10—Digital microfluidic chip; 111—Upper cover plate; 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—Pad; 109—Droplet; 11—Left reservoir area; 12—Waste liquid area; 13—Right reservoir area; 14—Product outlet area; 15—Low temperature area; 16—Medium temperature area; 17—High temperature area; 18—Medium-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 Implementation
[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.
[0029] The accompanying drawings provided in this invention document are for illustrative purposes only and do not represent actual dimensions or proportions. In practical applications, details such as the thickness of the film layers and the spacing between them need to be adjusted according to the specific implementation. Therefore, the structures shown in the drawings are merely schematic diagrams intended to help understand the core concepts of the invention and do not imply that the embodiments of the invention are limited to the specific forms or values shown in the drawings. The implementation of the invention is not limited to the specific details shown in the drawings.
[0030] Please see Figure 1 and Figure 3 A temperature-controlled digital microfluidic MALBAC device includes a digital microfluidic chip 10 and a temperature control system 20. The digital microfluidic chip 10 adopts a closed structure, including an upper cover plate 111 and a lower chip 112 arranged in parallel. The temperature control system 20 includes a temperature conduction module 21 and a temperature sensing module 22. The temperature conduction module 21 achieves thermal management through a multi-layer structure, including an upper heat-conducting block 211, a thermoelectric cooler 212, a lower heat-conducting block 213, and a heat pipe 214 arranged sequentially from top to bottom. The digital microfluidic chip 10 is located on top of the temperature conduction module 21 and is in contact with the upper heat-conducting 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-conducting block 211 and connected to the processing system 222 for data exchange and execution of temperature control commands. This structural design ensures that the temperature control system 20 can operate efficiently and provide precise 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 sequentially 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 sequentially from bottom to top; the upper hydrophobic layer 105 and the lower hydrophobic layer 104 face each other, forming a droplet channel between them. The upper cover plate 111 and the lower chip 112 are encapsulated by a gasket 108 or photocurable adhesive to form a closed structure.
[0032] The upper heat-conducting block 211 and the lower heat-conducting block 213 are made of metals with excellent thermal conductivity, such as copper or aluminum, and the thickness is set to 0.1 to 80 mm.
[0033] Temperature sensor 221 is a platinum resistance temperature sensor with excellent long-term stability and a wide temperature measurement range, preferably a PT1000 model. Temperature sensor 221 is fixed to the center of the upper heat-conducting block 211 with thermally conductive silicone grease to ensure good thermal contact. Temperature sensor 221 is led out from the upper heat-conducting block 211 via a wire and connected to the processing system 222, which involves connection to the signal input terminal of the microcontroller. To reduce heat loss near the sensor and improve the accuracy of temperature measurement, the length of the wire connecting temperature sensor 221 and processing system 222 should be appropriate to optimize thermal management.
[0034] Please see Figure 2 The driving electrode 102 is patterned with multiple functional regions: a left reservoir 11, a waste liquid region 12, a right reservoir 13, a product outlet region 14, and a temperature gradient region. The temperature gradient region is a rectangular loop composed of a low-temperature region 15, a medium-temperature region 16, a high-temperature region 17, and a medium-low-temperature region 18. Within the closed structure formed by the upper cover plate 111 and the lower chip 112, the droplet 109 undergoes dielectric wetting along the pattern of the driving electrode 102, thereby enabling manipulation actions such as droplet movement, splitting, and merging. The low-temperature region 15 is used for the annealing step of primer binding to the DNA template; the medium-low-temperature region 18 and the medium-temperature region 17 are used to ensure the occurrence of linear amplification cycles and strand displacement polymerization reactions, respectively; while the high-temperature region 17 is used for DNA denaturation, causing double strands to split into single strands, preparing for the next round of amplification reactions.
[0035] The left reservoir 11, located to the left of the temperature gradient zone, stores single-cell lysis buffer and washing buffer, providing the necessary starting materials for the MALBAC reaction. The right reservoir 13, located to the lower right of the temperature gradient zone, stores MALBAC amplification solution and replenishment solution to maintain the continuity and stability of the reaction. The waste liquid zone 12, located to the upper right of the temperature gradient zone, collects waste liquid generated during the reaction, ensuring the cleanliness of the entire system and the efficiency of the reaction. The product outlet zone 14, located below the temperature gradient zone, is a key channel connecting the inside of the chip to the external environment, serving as the outlet for the products of subsequent measurement units, facilitating subsequent analysis and research.
[0036] The temperature conduction module 21 and the temperature sensing module 22 work together to maintain multiple regions with different temperature settings for the chip's temperature gradient region. The temperature of these regions can be controlled independently, including not only areas above room temperature but also areas requiring temperatures below room temperature, to accommodate the complex 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 temperature changes in real time through the temperature sensor 221 and feeds the data back to the processing system 222. Therefore, the temperature control system 20 can accurately adjust the temperature of each region, ensuring that the temperature of each region reaches and remains at the preset optimal conditions, whether high temperatures are needed to promote DNA denaturation or low temperatures are needed for primer annealing.
[0037] Please see Figure 4 The diagram illustrates the workflow of the temperature measurement and control system 20 in this invention. Here, TS represents the target set temperature, TR represents the measured actual temperature, E represents the temperature error (E = TS - TA), and dE / dt represents the error change rate. The temperature sensing module employs a PID fuzzy control algorithm. The calculated set value is input into the temperature rise / fall module, and after passing through the temperature measurement module, the actual temperature value is obtained. This actual temperature value is then compared with the target temperature value. Specifically, the temperature control method is as follows: when the deviation between the two is large, fuzzy control is used, resulting in fast response and good dynamic performance; when the deviation is small, PID control is used to ensure good static performance and meet the system accuracy requirements.
[0038] Please see Figure 5 This is a graph showing the temperature change over time in the temperature measurement and control system 20 during operation, as described in this invention. Figure 5 The temperature measurement data shows that the temperature in all temperature zones remains very uniform, with no temperature interference between zones. Specifically, the temperature in low-temperature zone 15 is controlled at 0±0.2℃; the temperature in medium-low-temperature zone 18 is controlled at 58±0.7℃; the temperature in high-temperature zone 17 is controlled at 94±1℃; and the temperature in medium-temperature zone 16 is controlled at 65±0.8℃. Because a large temperature difference intensifies 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 control system under different temperature settings, proving that the system can provide accurate and stable temperature control according to the needs of different temperature zones.
[0039] Those skilled in the art will readily understand that the above description is merely 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 within the scope of protection of the present invention.
Claims
1. A digital microfluidic MALBAC device based on temperature measurement and control, characterized in that: The system includes a digital microfluidic chip (10) and a temperature control system (20). The digital microfluidic chip (10) has a closed structure, including an upper cover plate (111) and a lower chip (112) arranged in parallel. The temperature control system (20) includes a temperature conduction module (21) and a temperature sensing module (22). The temperature conduction module (21) includes an upper heat conduction block (211), a thermoelectric cooler (212), a lower heat conduction block (213), and a heat pipe (214) arranged from top to bottom. The digital microfluidic chip (10) is located on top of the temperature conduction module (21) and is in contact 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 connected to the processing system (222) to exchange data and execute temperature control commands. The upper cover plate (111) includes an upper substrate (107), an upper electrode layer (106), and an upper hydrophobic layer (105) arranged sequentially 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 sequentially from bottom to top; the upper hydrophobic layer (105) and the lower hydrophobic layer (104) face each other, and a droplet channel is formed between them; 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), and 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-low temperature area (18). The temperature of the low-temperature zone (15) is controlled at -1℃ to 3℃; the temperature of the medium-low temperature zone (18) is controlled at 55℃ to 60℃; The temperature of the high-temperature zone (17) is controlled at 90℃~98℃; the temperature of the medium-temperature zone (16) is controlled at 62℃~68℃.
2. The digital microfluidic MALBAC device based on temperature measurement and control as described in claim 1, characterized in that: The left reservoir (11) is located to the left of the temperature gradient zone and is used to store single-cell lysis buffer and washing buffer; the right reservoir (13) is located to the lower right of the temperature gradient zone and is used to store MALBAC amplification solution and replenishment solution.
3. The digital microfluidic MALBAC device based on temperature measurement and control as described in claim 1, characterized in that: The waste liquid zone (12) is located to the upper right of the temperature gradient zone and is used to collect the waste liquid generated during the reaction process; the product outlet zone (14) is located below the temperature gradient zone and is used as the outlet of the product of the subsequent measurement unit.
4. The digital microfluidic MALBAC device based on temperature measurement and control as described in claim 1, characterized in that: The upper cover plate (111) and the lower chip (112) are encapsulated by a gasket (108) or photocurable adhesive to form a closed structure.
5. The digital microfluidic MALBAC device based on temperature measurement and control as described 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.
6. The digital microfluidic MALBAC device based on temperature measurement and control as described in claim 1, characterized in that: The temperature sensor (221) is a platinum resistance temperature sensor, model PT1000. The temperature sensor (221) is fixed to the center of the upper heat-conducting block (211) with thermal grease.