WO2024108537A1 - Biochemical reaction method and system for digital quantitative volume measurement - Google Patents

Abstract

A biochemical reaction method and system for digital quantitative volume measurement. The method comprises: according to a digital microflow measurement principle, using a volumetric bar-chart chip with pressure-based distance readout to contain a reactant; using a digital on-chip flowmeter based on microfluidic chip technology to discretize a continuous liquid stream of the reactant into digital droplets, and directly reading out the digitization frequency of the reactant by using a visualization method. The system comprises: a volumetric bar-chart chip and a digital on-chip flowmeter, wherein the volumetric bar-chart chip is a volumetric bar-chart chip with pressure-based distance readout used for containing a reactant according to the digital microflow measurement principle, and the digital on-chip flowmeter is a digital on-chip flowmeter based on microfluidic chip technology used for discretizing a continuous liquid stream of the reactant into digital droplets, and directly reading out the digitization frequency of the reactant by using the visualization method.

Description

A biochemical reaction method and system for digital quantitative volume measurement Technical Field

The invention relates to the field of biochemical reactions, and in particular to a biochemical reaction method and system for digital quantitative volume measurement.

Background technique

In modern biomedical research and industrial monitoring, accurate monitoring and control of liquid flow at low flow rates are becoming increasingly important. Among the existing micro-flow meters, thermal flow meters are one of the most commonly used low-flow flow sensors due to their simple structure and circuit design. However, thermal flow meters have many disadvantages. For example, their nonlinear part in the temperature range requires additional calibration steps and compensation design. They are also very susceptible to fluid contamination and complex fluids (such as non-Newtonian fluids). More seriously, the above disadvantages may cause damage to the measured fluid or change in characteristics, and are easily affected by changes in ambient temperature. In addition, distance reading flow meters using microfluidic technology change the channel pressure through the gas produced by biochemical reactions, thereby moving the liquid, and then read the liquid movement distance by the scale marked on the chip to calculate the volume change. However, this method is limited by the resolution of the scale and can only read up to 0.1mm at most, which cannot achieve more accurate quantitative readings.

Summary of the invention

The embodiments of the present invention provide a biochemical reaction method and system for digital quantitative volume measurement, so as to at least solve the technical problem that more accurate quantitative readings cannot be achieved in the prior art.

According to one embodiment of the present invention, a biochemical reaction method for digital quantitative volume measurement is provided, comprising the following steps:

Using the principle of digital micro-flow measurement, a volume bar graph chip with pressure-based distance readout is used to hold the reactants;

A digital on-chip flowmeter based on microfluidic chip technology is used to discretize the continuous liquid flow of the reactants into digital droplets, and a visualization method is used to directly read out the digital frequency.

The technical solution adopted by the embodiment of the present application also includes: using a visualization method to directly read out the digitized frequency, including;

The digitized frequency is directly read out by electrical or optical visualization methods, achieving accurate measurement of liquid volume resolution of a few nanoliters and liquid flow rate of hundreds of nanoliters to tens of microliters.

According to another embodiment of the present invention, a biochemical reaction system for digital quantitative volume measurement is provided, comprising:

A volume bar graph chip for holding reactants using a pressure-based distance readout volume bar graph chip utilizing the principle of digital microflow measurement;

A digital on-chip flowmeter is used for discretizing the continuous liquid flow of a reactant into digital droplets using a digital on-chip flowmeter based on microfluidic chip technology, and directly reading out the digital frequency thereof by a visualization method.

The technical solution adopted by the embodiment of the present application also includes: the preparation of the digital on-chip flow meter includes:

Spin coating the photoresist on the silicon wafer, followed by soft baking, UV exposure using a mask aligner, and post-exposure baking to transfer the pre-designed digital on-chip flow meter pattern from the high-resolution glass photomask to the deposited photoresist;

Spin-coat a second layer of photoresist with controllable thickness on the first coating, and perform the same baking and UV exposure process as the previous step to transfer the designed second layer pattern of the digital on-chip flow meter to the second layer of the photoresist;

The double-layer coated silicon wafer is immersed in a developer to dissolve the uncrosslinked photoresist to form a master with a three-dimensional structure; then the matrix and the curing agent are mixed according to a mass ratio and poured on the master placed in a culture dish to prepare a polydimethylsiloxane elastomer PDMS;

After degassing in a desiccator, the Petri dish containing the master and PDMS is placed in an oven to thermally cure the PDMS; once completed, the PDMS mold is peeled off the master;

Another PDMS mold was prepared using the same method, and the inlet and outlet of the droplet were made using a PDMS punch; two identical PDMS layers were bonded together using a plasma cleaner, and ethanol was added to form the final digital on-chip flowmeter device.

The technical solution adopted in the embodiment of the present application also includes: the preparation of the digital on-chip flowmeter specifically includes:

Transfer the pre-designed digital on-chip flowmeter pattern from the high-resolution glass photomask to the deposited photoresist using SU-8 3050 photoresist spin-coated on a 4-inch silicon wafer followed by a standard soft bake, UV exposure using a mask aligner, and post-exposure baking;

Spin-coat a second layer of SU-8 photoresist with controllable thickness on the first coating, and perform the same baking and UV exposure process as in the previous step to transfer the designed second layer pattern of the digital on-chip flow meter to the second layer of the photoresist;

The double-layer SU-8 coated silicon wafer was immersed in SU-8 developer to dissolve the uncrosslinked photoresist to form a SU-8 master with a three-dimensional structure; then the matrix and the curing agent were mixed in a mass ratio of 10:1 and poured on the SU-8 master placed in a culture dish to prepare polydimethylsiloxane elastomer PDMS;

After degassing in a desiccator, the Petri dish containing the SU-8 master and PDMS was placed in an 80°C oven to thermally cure the PDMS; upon completion, the PDMS mold was peeled off from the SU-8 master.

Another PDMS mold was prepared using the same method, and a PDMS punch was used to make the inlet and outlet of the droplet. Two identical PDMS layers were bonded together using a plasma cleaner, and a drop of ethanol was added under a microscope to enhance the adhesion of the PDMS and restore the hydrophobicity of the PDMS to form the final digital on-chip flowmeter device.

The technical solution adopted by the embodiment of the present application also includes: the size design of the digital on-chip flow meter includes:

Add reactants to the reaction area of the volume bar graph chip, and use nano-platinum as a catalyst; the reaction produces oxygen to increase the pressure in the microchannel, and the reaction liquid is pushed toward the nozzle upstream of the digital on-chip flow meter under pressure driving;

When the liquid flow moves to the nozzle, under the action of surface tension, the liquid flow forms a spherical droplet in the air buffer chamber. When the droplet expands to the maximum, the volume is V _C ; it contacts the interface of the downstream collection pool to form a liquid meniscus neck, and the liquid in the droplet is discharged into the downstream microchannel;

After a brief capillary action and rebalancing of forces, the liquid bridge is automatically pinched off, and the remaining suspended droplet volume V _r is proportional to V _c ; thus, the droplet transfer volume V _t = V _c -V _r , which is the volume resolution; this volume is a constant under low flow rate conditions and is only related to the nozzle's dimensions of height H _n and width W _n and the distance D _a from the nozzle to the downstream collection pool.

The technical solution adopted by the embodiment of the present application also includes: the size design of the digital on-chip flow meter specifically includes:

Add reactants to the reaction area of the volume bar graph chip, and use nano-platinum as a catalyst; the reaction produces oxygen to increase the pressure in the microchannel, and the reaction liquid is pushed toward the nozzle upstream of the digital on-chip flow meter under pressure; a cone-angle nozzle is used to prevent side wall wetting; the nozzle depth range is 100 to 200 μm;

When the liquid flow moves to the nozzle, under the action of surface tension, the liquid flow forms a spherical droplet in the air buffer chamber. When the droplet expands to the maximum, the volume is V _c ; it contacts the interface of the downstream collection pool to form a liquid meniscus neck, that is, a liquid bridge, which discharges the liquid in the droplet into the downstream microchannel;

After a brief capillary action and rebalancing of forces, the liquid bridge is automatically pinched off, and the remaining suspended droplet volume _Vr is also spherical and proportional to _Vc ; thus the droplet transfer volume _Vt = _Vc - _Vr , which is also the volume resolution; this volume is a constant under low flow conditions and is only related to the size of the nozzle height _Hn and width _Wn and the distance _Da from the nozzle to the downstream collection pool.

The technical solution adopted in the embodiment of the present application also includes: the nozzle height _Hn is selected to be 150μm, and the width _Wn is selected to be in the range of 125μm to 175μm; when _Da is selected to be 150μm and _Wn is at least 125μm, the highest volume resolution is 5.3nL, and when _Wn is at most 175μm, the lowest volume resolution is 6.3nL;

The distance _Da from the nozzle to the downstream collection pool ranges from 125μm to 175μm; when _Wn is selected as 150μm and _Da is the minimum value of 125μm, the highest volume resolution is 2.5nL, and when _Da takes the maximum value of 175μm, the lowest volume resolution is 5.8nL.

The technical solution adopted by the embodiment of the present application also includes: using a visualization method to directly read out the digitized frequency includes:

The droplet generation frequency is read out by an electrical method: two electrodes are inserted upstream of the nozzle and downstream of the collector, respectively, and the electrical impedance between the two electrodes is measured. The droplet digitization frequency f can be obtained by measuring the time interval between two adjacent fluctuations of the impedance; therefore, the flow rate Q is calculated by Q = f × V _t .

The technical solution adopted by the embodiment of the present application also includes: using a visualization method to directly read out the digitized frequency includes:

An optical visualization method is used to read out the frequency of droplet generation: a smartphone is connected to an optical microscope to take pictures, and the MATLAB Canny function is used to detect the transient edges of the droplets in each frame to determine the frequency of droplet discretization.

Compared with the prior art, the beneficial effects of the embodiments of the present application are: the biochemical reaction method and system for digital quantitative volume measurement in the embodiments of the present invention, the microfluidic chip of the present invention can use trace reagents for chemical and biological analysis while greatly shortening the reaction time, thereby changing the cumbersome laboratory operation, and the scope of application is becoming wider and wider. The present invention uses the principle of digital micro-flow measurement, uses a pressure-based distance readout microfluidic device-volume bar chart chip to hold reactants, uses a digital on-chip flowmeter based on microfluidic chip technology to discretize continuous liquid flow into "digital" droplets, and uses electrical methods for direct readout, achieving a few nanoliters of liquid volume resolution and accurate measurement of liquid flow rates from hundreds of nanoliters to tens of microliters.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

FIG1 is a schematic diagram of a biochemical reaction system for digital quantitative volume measurement according to the present invention.

Detailed ways

In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

Microfluidic chips can be used to perform chemical and biological analysis using trace reagents while greatly shortening the reaction time, thereby changing the cumbersome laboratory operation and having an increasingly wide range of applications. The present invention utilizes the principle of digital micro-flow measurement, uses a pressure-based distance readout microfluidic device, a volume bar graph chip, to hold reactants, uses a digital on-chip flowmeter based on microfluidic chip technology to discretize continuous liquid flow into "digital" droplets, and uses electrical methods for direct readout, achieving a few nanoliters of liquid volume resolution and accurate measurement of liquid flow rates from hundreds of nanoliters to tens of microliters.

In view of this, the present invention proposes a biochemical reaction method and system for digital quantitative volume measurement, which is composed of a volume bar graph chip and a digital on-chip flowmeter. The system can discretize a continuous liquid flow into "digital" droplets, and can directly read out its digital frequency using electrical methods or optical visualization methods, thereby achieving accurate measurement of liquids ranging from a few nanoliters to hundreds of microliters. The system can also achieve multi-channel quantitative volume measurement.

The technical solution of the present invention specifically includes:

1. Preparation of digital on-chip flowmeter device

1. Transfer the pre-designed digital on-chip flowmeter pattern from the high-resolution glass photomask to the deposited photoresist using SU-8 3050 photoresist spin-coated on a 4-inch silicon wafer, followed by a standard soft bake, UV exposure using a mask aligner, and post-exposure baking.

2. Spin-coat a second layer of SU-8 photoresist with controllable thickness on the first coating, and perform the same baking and UV exposure process as step 1 to transfer the designed digital on-chip flowmeter second layer pattern to the second layer of the photoresist.

3. Soak the double-layer SU-8 coated silicon wafer in SU-8 developer to dissolve the uncrosslinked photoresist and form a SU-8 master with a three-dimensional structure. Then, mix the matrix and curing agent in a mass ratio of 10:1 and pour it on the SU-8 master placed in a petri dish to prepare a polydimethylsiloxane (PDMS) elastomer.

4. After degassing in the desiccator, place the Petri dish containing the SU-8 master and PDMS in an 80°C oven to thermally cure the PDMS. When finished, peel the PDMS mold off the SU-8 master.

5. Use the same method to prepare another PDMS mold and use a PDMS punch to make the inlet and outlet of the droplet. Use a plasma cleaner to bond the two identical PDMS layers together and add a drop of ethanol under a microscope to enhance the PDMS adhesion and restore the hydrophobicity of PDMS to form the final digital on-chip flowmeter device.

2. Digital on-chip flow meter device size design and data readout

6. The schematic diagram of the structure of the system is shown in FIG1 , which includes a reaction zone 1, an optical visualization detection device 2, a signal generator 3, etc. Reactants such as hydrogen peroxide (H ₂ O ₂ ) are added to the reaction zone 1 of the volume bar graph chip, and nano-platinum (PtNPs) is used as a catalyst. The reaction produces oxygen (O ₂ ) to increase the air pressure in the microchannel, and the reaction liquid is driven by pressure to the nozzle upstream of the digital on-chip flowmeter. A conical nozzle is used to prevent side wall wetting. The nozzle depth ranges from 100 to 200 μm. A small depth of less than 100 μm will result in inconsistent droplet digitization process, and a depth greater than 200 μm will increase manufacturing difficulty.

7. When the liquid flow moves to the nozzle, under the action of surface tension, the liquid flow forms a spherical droplet in the air buffer chamber. When the droplet expands to the maximum, the volume is V _c . It contacts the interface of the downstream collection pool to form a liquid meniscus neck, that is, a liquid bridge, which discharges the liquid in the droplet into the downstream microchannel.

8. After a brief capillary action and rebalancing of forces, the liquid bridge automatically pinches off, and the remaining suspended droplet volume V _r is also spherical and proportional to V _c . Therefore, the droplet transfer volume V _t = V _c -V _r , which is also the volume resolution. This volume is a constant under low flow conditions and is only related to the size of the nozzle (height H _n and width W _n ) and the distance _Da from the nozzle to the downstream collection pool.

9. The nozzle height _Hn is selected as 150μm, and the width _Wn is selected in the range of 125μm to 175μm. When _Wn is greater than 175μm, the droplets will completely coalesce and cannot be pinched off. When _Wn is less than 125μm, the nozzle will be wetted during the pinching process, affecting the accuracy. When _Da is selected as 150μm and _Wn is minimum 125μm, the highest volume resolution is 5.3nL. When _Wn is maximum 175μm, the lowest volume resolution is 6.3nL.

10. The distance _Da from the nozzle to the downstream collection pool ranges from 125μm to 175μm. _Da less than 125μm will cause the droplets to completely coalesce and cannot be pinched off. Da greater than 175μm will cause the droplets to contact the chamber wall, affecting the accuracy. When _Wn is selected as 150μm and _Da is the minimum value of 125μm, the highest volume resolution is 2.5nL. When _Da takes the maximum value of 175μm, the lowest volume resolution is 5.8nL.

11. Use electrical methods to read the droplet generation frequency: insert two electrodes into the upstream of the nozzle and the downstream of the collector, measure the electrical impedance between the two electrodes, and obtain the droplet digitization frequency f by measuring the time interval between two adjacent fluctuations of the impedance. Therefore, the flow rate Q can be calculated by Q = f × V _t . To accurately measure the volume change of the reaction liquid, it is only necessary to measure the flow rate within a certain period of time. For example, when _Da = 125um, f _max is about 524Hz, so Q _max = f _max × V _t = 524Hz × 2.5nL = 524s ^-1 × 2.5 × 10 ^-3 uL = 78.6uL min ^-1 , which is about 80uL min ^-1 .

12. In order to achieve accurate measurement of liquid by multi-channel digital on-chip flowmeter, it is necessary to integrate the circuits of each digital on-chip flowmeter.

13. Use optical visualization method to read the frequency of droplet generation: Use optical visualization detection device 2 such as a smartphone connected to an optical microscope to take pictures at a frame rate of 960fps, use MATLAB Canny function to detect the transient edge of the droplet in each frame, and determine the frequency of droplet discretization.

The key points and intended protection points of the present invention are:

1. A method and system for realizing digital quantitative volume measurement of biochemical reactions using a digital on-chip flow meter.

2. The reactants used include but are not limited to H ₂ O ₂ , and also include various gas generation reactions, such as catalytic generation of O ₂ , N ₂ , NH ₃ , CO ₂ and H ₂ , etc.

3. It can be applied to various biochemical reaction scenarios, including but not limited to nucleic acids, proteins, ions (such as heavy metal ions), antibiotic reactions, etc.

4. In Article 6 of the technical solution, the nozzle depth range is 50 to 500 μm.

5. In item 9 of the technical solution, the nozzle height is 10 to 200 μm, and the nozzle width ranges from 100 μm to 500 μm.

6. In item 10 of the technical solution, the distance from the nozzle to the downstream collection tank ranges from 100 μm to 500 μm.

7. In Article 11 of the technical solution, an electrical method is used to measure the digitized frequency and save the data.

8. In Article 12 of the technical solution, the direct readout circuit design for multi-channel digital quantitative volume measurement.

9. In Article 13 of the technical solution, an optical visualization method is used to measure the digitized frequency and save the data.

10. Use computer methods to automatically calculate the flow rate and flow rate using the saved data.

The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

In the above embodiments of the present invention, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

In the several embodiments provided in this application, it should be understood that the disclosed technical content can be implemented in other ways. Among them, the system embodiments described above are only schematic. For example, the division of units can be a logical function division. There may be other division methods in actual implementation. For example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be through some interfaces, indirect coupling or communication connection of units or modules, which can be electrical or other forms.

The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed over multiple units. Some or all of the units may be selected according to actual needs to achieve the purpose of the present embodiment.

In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, each unit may exist physically separately, or two or more units may be integrated into one unit. The above integrated unit may be implemented in the form of hardware or in the form of software functional unit.

If the integrated unit is implemented in the form of a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, or all or part of the technical solution can be embodied in the form of a software product. The computer software product is stored in a storage medium, including several instructions for a computer device (which can be a personal computer, server or network device, etc.) to perform all or part of the steps of the methods of each embodiment of the present invention. The aforementioned storage medium includes: U disk, read-only memory (ROM, Read-Only Memory), random access memory (RAM, Random Access Memory), mobile hard disk, magnetic disk or optical disk, etc. Various media that can store program codes.

The above description of the disclosed embodiments enables those skilled in the art to implement or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims (10)

1. A biochemical reaction method for digital quantitative volume measurement, characterized in that it comprises the following steps:

   Using the principle of digital micro-flow measurement, a volume bar graph chip with pressure-based distance readout is used to hold the reactants;

   A digital on-chip flowmeter based on microfluidic chip technology is used to discretize the continuous liquid flow of the reactants into digital droplets, and a visualization method is used to directly read out the digital frequency.
2. The biochemical reaction method for digital quantitative volume measurement according to claim 1 is characterized in that the direct reading of the digitized frequency by using a visualization method comprises:

   The digitized frequency is directly read out by electrical or optical visualization methods, achieving accurate measurement of liquid volume resolution of a few nanoliters and liquid flow rate of hundreds of nanoliters to tens of microliters.
3. A biochemical reaction system for digital quantitative volume measurement, characterized in that it comprises: a volume bar graph chip and a digital on-chip flow meter, wherein:

   A volume bar graph chip for holding reactants using a pressure-based distance readout volume bar graph chip utilizing the principle of digital microflow measurement;

   A digital on-chip flowmeter is used for discretizing the continuous liquid flow of a reactant into digital droplets using a digital on-chip flowmeter based on microfluidic chip technology, and directly reading out the digital frequency thereof by a visualization method.
4. The biochemical reaction system for digital quantitative volume measurement according to claim 3 is characterized in that the preparation of the digital on-chip flowmeter comprises:

   Spin coating the photoresist on the silicon wafer, followed by soft baking, UV exposure using a mask aligner, and post-exposure baking to transfer the pre-designed digital on-chip flow meter pattern from the high-resolution glass photomask to the deposited photoresist;

   Spin-coat a second layer of photoresist with controllable thickness on the first coating, and perform the same baking and UV exposure process as the previous step to transfer the designed second layer pattern of the digital on-chip flow meter to the second layer of the photoresist;

   The double-layer coated silicon wafer is immersed in a developer to dissolve the uncrosslinked photoresist to form a master with a three-dimensional structure; then the matrix and the curing agent are mixed according to a mass ratio and poured on the master placed in a culture dish to prepare polydimethylsiloxane elastomer PDMS;

   After degassing in a desiccator, the Petri dish containing the master and PDMS is placed in an oven to thermally cure the PDMS; once completed, the PDMS mold is peeled off the master;

   Another PDMS mold was prepared using the same method, and the inlet and outlet of the droplet were made using a PDMS punch; two identical PDMS layers were bonded together using a plasma cleaner, and ethanol was added to form the final digital on-chip flowmeter device.
5. The biochemical reaction system for digital quantitative volume measurement according to claim 3 is characterized in that the preparation of the digital on-chip flowmeter specifically comprises:

   Transfer the pre-designed digital on-chip flowmeter pattern from the high-resolution glass photomask to the deposited photoresist using SU-8 3050 photoresist spin-coated on a 4-inch silicon wafer followed by a standard soft bake, UV exposure using a mask aligner, and post-exposure baking;

   Spin-coat a second layer of SU-8 photoresist with controllable thickness on the first coating, and perform the same baking and UV exposure process as in the previous step to transfer the designed second layer pattern of the digital on-chip flow meter to the second layer of the photoresist;

   The double-layer SU-8 coated silicon wafer was immersed in SU-8 developer to dissolve the uncrosslinked photoresist to form a SU-8 master with a three-dimensional structure; then the matrix and the curing agent were mixed in a mass ratio of 10:1 and poured on the SU-8 master placed in a culture dish to prepare polydimethylsiloxane elastomer PDMS;

   After degassing in a desiccator, the culture dish containing the SU-8 master and PDMS was placed in an 80°C oven to thermally cure the PDMS. After completion, the PDMS mold was peeled off from the SU-8 master.

   Another PDMS mold was prepared using the same method, and a PDMS punch was used to make the inlet and outlet of the droplet. Two identical PDMS layers were bonded together using a plasma cleaner, and a drop of ethanol was added under a microscope to enhance the adhesion of the PDMS and restore the hydrophobicity of the PDMS to form the final digital on-chip flowmeter device.
6. The biochemical reaction system for digital quantitative volume measurement according to claim 3 is characterized in that the size design of the digital on-chip flowmeter includes:

   Add reactants to the reaction area of the volume bar graph chip, and use nano-platinum as a catalyst; the reaction produces oxygen to increase the pressure in the microchannel, and the reaction liquid is pushed toward the nozzle upstream of the digital on-chip flow meter under pressure driving;

   When the liquid flow moves to the nozzle, under the action of surface tension, the liquid flow forms a spherical droplet in the air buffer chamber. When the droplet expands to the maximum, the volume is V _c ; it contacts the interface of the downstream collection pool to form a liquid meniscus neck, and the liquid in the droplet is discharged into the downstream microchannel;

   After a brief capillary action and rebalancing of forces, the liquid bridge is automatically pinched off, and the remaining suspended droplet volume V _r is proportional to V _c ; thus, the droplet transfer volume V _t = V _c -V _r , which is the volume resolution; this volume is a constant under low flow rate conditions and is only related to the nozzle's dimensions of height H _n and width W _n and the distance D _a from the nozzle to the downstream collection pool.
7. The biochemical reaction system for digital quantitative volume measurement according to claim 3 is characterized in that the size design of the digital on-chip flowmeter specifically includes:

   Add reactants to the reaction area of the volume bar graph chip, and use nano-platinum as a catalyst; the reaction produces oxygen to increase the pressure in the microchannel, and the reaction liquid is pushed toward the nozzle upstream of the digital on-chip flow meter under pressure; a cone-angle nozzle is used to prevent side wall wetting; the nozzle depth range is 100 to 200 μm;

   When the liquid flow moves to the nozzle, under the action of surface tension, the liquid flow forms a spherical droplet in the air buffer chamber. When the droplet expands to the maximum, the volume is V _c ; it contacts the interface of the downstream collection pool to form a liquid meniscus neck, that is, a liquid bridge, which discharges the liquid in the droplet into the downstream microchannel;

   After a brief capillary action and rebalancing of forces, the liquid bridge is automatically pinched off, and the remaining suspended droplet volume _Vr is also spherical and proportional to _Vc ; thus the droplet transfer volume _Vt = _Vc - _Vr , which is also the volume resolution; this volume is a constant under low flow conditions and is only related to the size of the nozzle height _Hn and width _Wn and the distance _Da from the nozzle to the downstream collection pool.
8. The biochemical reaction system for digital quantitative volume measurement according to claim 6 is characterized in that the nozzle height _Hn is selected as 150 μm, and the width _Wn ranges from 125 μm to 175 μm; when _Da is selected as 150 μm and _Wn is a minimum of 125 μm, the highest volume resolution is 5.3 nL, and when _Wn is a maximum of 175 μm, the lowest volume resolution is 6.3 nL;

   The distance _Da from the nozzle to the downstream collection pool ranges from 125μm to 175μm; when _Wn is selected as 150μm and _Da is the minimum value of 125μm, the highest volume resolution is 2.5nL, and when _Da takes the maximum value of 175μm, the lowest volume resolution is 5.8nL.
9. The biochemical reaction system for digital quantitative volume measurement according to claim 3 is characterized in that the direct reading of the digitized frequency by using a visualization method comprises:

   The droplet generation frequency is read out by an electrical method: two electrodes are inserted upstream of the nozzle and downstream of the collector, respectively, and the electrical impedance between the two electrodes is measured. The droplet digitization frequency f can be obtained by measuring the time interval between two adjacent fluctuations of the impedance; therefore, the flow rate Q is calculated by Q = f × V _t .
10. The biochemical reaction system for digital quantitative volume measurement according to claim 3 is characterized in that the direct reading of the digitized frequency by using a visualization method comprises:

    An optical visualization method is used to read out the frequency of droplet generation: a smartphone is connected to an optical microscope to take pictures, and the MATLAB Canny function is used to detect the transient edges of the droplets in each frame to determine the frequency of droplet discretization.

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