Microfluidic channel integrated microwell array chip and periodic bidirectional oscillation sampling mode

Abstract

The application relates to a microfluidic channel integrated micropore array chip and a periodic bidirectional oscillation sampling method, the chip comprising a micropore array and a microfluidic channel; sampling inlets and sampling outlets are arranged at two ends of the microfluidic channel; a single micropore array comprises 220000-225000 micropore units, the diameter of the micropore unit is 5.0-5.4 mu m, the spacing between adjacent micropore units is 4.5 mu m, and the micropore units between two adjacent rows in the micropore array are staggered. In the application, the chip can be combined with a microparticle sensitive element to realize single-molecule detection of a biomolecule target; and the application further provides a periodic bidirectional oscillation sampling method based on the chip. Without high-cost methods such as process improvement or large external equipment, only by improving the sampling method and sampling parameters, a significantly higher filling rate and filling uniformity compared with traditional sampling methods can be obtained.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip technology, and specifically relates to a microfluidic channel integrated micropore array chip and a periodic bidirectional oscillation sample introduction method. Background Technology

[0002] Single-molecule array (SIMOA) technology is a detection technique for achieving ultrasensitive detection of low-abundance biomarkers at the fg level. This technology utilizes microwell arrays and the principle of single-molecule detection. Its core principle is to capture the analyte molecule through microparticle-sensitive elements. In SIMOA, each microwell in the microwell array captures a maximum of one microparticle-sensitive element. An optical detection system detects and counts the signal from each microwell, enabling quantitative analysis of the target molecule. SIMOA technology offers significantly higher detection sensitivity than traditional detection techniques such as ELISA, with a detection limit reaching fg / ml. Effective microwell filling is a crucial prerequisite for achieving single-molecule counting of the target molecule and ensuring both detection sensitivity and data accuracy in SIMOA technology.

[0003] Currently, most single-molecule array technologies for microporous array chips still rely on manual, simple sample introduction, which easily leads to problems such as bubble generation and pressure imbalance within the microchannels during the sample introduction process. These problems severely affect the fill rate of the microporous array chip. Low fill rate in SIMOA technology causes several issues: 1. Decreased detection sensitivity: Low fill rate significantly reduces the number of micropores effectively participating in detection within the chip, resulting in insufficient capture of sensitive elements; 2. Decreased accuracy of monitoring data: Uneven fill rate causes significant differences in the fill rate of micropores in different regions of the microporous array, easily leading to the loss of information from effective sensitive elements; 3. Reduced chip utilization efficiency and higher detection costs: SIMOA microporous array chips are precision micro / nano fabricated devices with certain manufacturing costs. Low fill rate means that a large number of micropores are not effectively utilized, resulting in a serious waste of chip resources. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a microfluidic channel integrated micropore array chip and a periodic bidirectional oscillation injection method to improve the fill rate of the micropore array chip, thereby solving the problems of decreased detection sensitivity, decreased accuracy of monitoring data, low chip utilization efficiency and high cost in SIMOA technology caused by low fill rate.

[0005] This invention provides a microfluidic channel integrated micropore array chip, the chip comprising a micropore array and a microfluidic channel; the microfluidic channel has a strictly distinct inlet and outlet at both ends; the micropore array is positioned near the outlet of the microfluidic channel; a single micropore array contains 220,000-225,000 micropore units, the diameter of the micropore units is 5.0-5.4 μm, and the spacing between adjacent micropore units is 4.5 μm, with adjacent rows of micropore units interleaved within the micropore array.

[0006] Preferably, the micropore array is used to achieve physical isolation of individual sensitive elements; the microfluidic channel is used for transporting sensitive elements and assisting in filling sensitive elements.

[0007] Preferably, the chip has an array of eight micropores and corresponding integrated microfluidic channels. The design of the eight-micropore array and microfluidic channels offers the following advantages: 1. It facilitates simultaneous parallel detection of different samples in experimental research and clinical applications. In specific experimental studies, the same chip can be used to perform detection experiments on the same target at up to eight concentration gradients, and multiple samples can also be detected in parallel; 2. The design of integrating eight micropores and microfluidic channels on the same chip also reduces manufacturing costs.

[0008] Preferably, the fabrication method of the microfluidic channel integrated micropore array chip includes two methods: Method 1: oxygen plasma treatment is performed on the surface of the microfluidic channel and the micropore array; alignment marks are set on the microfluidic channel, the alignment marks are aligned with the micropore array, and bonding is performed; after bonding, the microfluidic channel and the micropore array are heated in an oven at 80 °C for 2 hours to make the microfluidic channel and the micropore array firmly bonded.

[0009] Method 2: The microfluidic channel and the microporous array chip are combined using a fixture; the fixture consists of two square plastic clamps, each with a thread at each of its four corners for connection; one of the clamps has a slot to expose the inlet and outlet; screws are used to tightly connect the microfluidic chip and the microporous array chip, thereby producing a microfluidic channel integrated microporous array chip.

[0010] Furthermore, the micropore array is fabricated using micro-nano fabrication technology, specifically including the following steps: selecting a quartz substrate as the substrate for the micropore array, organically cleaning the quartz substrate, and performing oxygen plasma treatment on the surface; subsequently, spin-coating photoresist as an etching protective layer, and exposing and developing the micropore pattern; then etching the quartz substrate using an ICP etching process, selecting Freon CHF3 and Argon Ar as working gases; after etching, immersing the quartz substrate in acetone and alcohol for ultrasonic cleaning, and drying it with nitrogen.

[0011] Furthermore, the height of the microfluidic channel ranges from 65 to 145 μm, the distance between the inlet and outlet is 29.0 to 29.2 mm, and the channel substrate is any one of COP, PC thermoplastic plastic, or PDMS, Flexdym polymer material; the microfluidic channel is prepared by soft lithography, precision machining or precision injection molding process.

[0012] The present invention also provides a periodic bidirectional oscillation injection method based on the above-mentioned microfluidic channel integrated micropore array chip, including: bidirectional oscillation, that is, the sensitive element solution is alternately driven in the direction of the inlet and outlet of the microfluidic channel, and this step is repeated periodically. Before each drive, the chip is left to stand still so that the sensitive element solution is completely stable and the sensitive element is allowed to settle naturally to the bottom of the micropore array.

[0013] Furthermore, the specific process of completing a single bidirectional oscillation is as follows:

[0014] S1. Shake the sensitive element solution for 30 s to distribute the sensitive element evenly, and then aspirate it into the pipette tube; connect the inlet of the microfluidic channel to the pipette tube and the rigid gas tube to connect to the gas pump, and introduce gas pressure into the chip at 8 mbar. After all the sensitive element solution has entered the microfluidic channel and covered the micropore array area, stop the gas pressure and let the chip stand for 90 s to allow the sensitive element to settle to the bottom of the micropore array;

[0015] S2. After sedimentation, insert another pipette into the outlet to collect the sensing element solution flowing out of the microfluidic channel; apply a pressure of 5 mbar at the inlet. Initially, the liquid flow rate is relatively fast, but it decreases significantly as the liquid reaches the micropore array at the edge. This is because a large number of sensing elements are clustered together, and the slower flow rate allows the sensing elements already at the bottom to be carried into the micropore array by the liquid. The sensing elements just falling into the micropores will exert some resistance on the liquid; at a pressure of 5 mbar, the liquid passes through the entire micropore array in about 4 minutes; after the sensing element solution has completely passed through the micropore array area, stop applying pressure and allow it to settle for 90 seconds.

[0016] S3. After sedimentation, apply 5 mbar air pressure to the other end of the sample outlet to allow the liquid to pass through the micropore array relatively slowly. Stop applying air pressure when the liquid edge at the sample outlet just reaches the edge of the micropore array near the sample outlet. Let it stand for 90 seconds to allow the sensitive elements to settle. At this time, a considerable number of sensitive elements have fallen into the micropores, and the single bidirectional oscillation takes 10 minutes to complete.

[0017] Further, the bidirectional oscillation operation is repeated to improve the final filling rate, and then the excess liquid is aspirated to complete the entire injection process.

[0018] Preferably, the settling time is controlled to shorten the settling time while ensuring complete settling of the sensing element, thereby optimizing the sample injection time efficiency; considering the number of micropore units in the micropore array and the volume of the microfluidic channels, the concentration of the sensing element solution is set to 2.5 × 10⁻⁶. 5 Samples were injected at 8 μL per chip via an air pump. The air pump was connected to the chip via a 20 μL pipette tube and a 0.6 mm diameter rigid tubing.

[0019] The sample introduction method of this invention effectively improves the problems of low detection efficiency and signal deviation caused by uneven filling in specific experimental studies and clinical tests by increasing the filling rate.

[0020] Beneficial effects

[0021] (1) The microfluidic channel integrated micropore array chip in this invention can be combined with microparticle sensitive elements to realize single-molecule detection of biomolecular targets.

[0022] (2) This invention proposes a periodic bidirectional oscillating injection method based on a microfluidic channel integrated microporous array chip. This injection method does not require expensive methods such as improved preparation process or large external equipment. By simply improving the injection method and injection parameters, a significantly higher filling rate and filling uniformity can be obtained compared with the traditional injection method. This provides a solution to the problems encountered by existing microfluidic channel integrated microporous array chips in single molecule detection applications. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the structure of the microfluidic channel integrated micropore array chip of the present invention.

[0024] Figure 2 This is a flowchart illustrating the fabrication process of the microfluidic channel integrated micropore array chip of this invention.

[0025] Figure 3 This is a schematic diagram of the microfluidic channel integrated micro-hole array chip fabricated by combining microfluidic channels and micro-hole arrays using a fixture according to the present invention.

[0026] Figure 4 This is a schematic diagram of the periodic bidirectional oscillation sample introduction method based on a microfluidic channel integrated micropore array chip according to the present invention.

[0027] Figure 5 These are fluorescence field and bright field images of the microporous array under a microscope after the sample introduction experiment in Example 2.

[0028] Figure 6 The results of the filling rate in different parts in Example 2 are statistically analyzed. Detailed Implementation

[0029] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0030] Example 1

[0031] This embodiment provides a microfluidic channel integrated micropore array chip, the structure of which is as follows: Figure 1 As shown, it includes 8 microwell arrays and microfluidic channels integrated with each microwell array. Each microfluidic channel has an inlet and an outlet at each end. A single microwell array contains 220,000-225,000 microwell units.

[0032] The fabrication method of the above-mentioned microfluidic channel integrated micropore array chip is as follows: Figure 2 As shown, the specific steps include:

[0033] (1) The micro-hole array was fabricated using micro-nano fabrication technology. A 500-micrometer-thick quartz substrate was selected as the substrate for the micro-hole array. The quartz substrate was organically cleaned and then subjected to oxygen plasma treatment on its surface. After oxygen plasma treatment, SPR200 photoresist was spin-coated as an etching protective layer, and the micro-hole pattern was exposed and developed. Subsequently, ICP etching was used to etch the substrate, with Freon CHF3 and Argon Ar selected as the working gases. After etching, the quartz substrate was immersed in acetone and alcohol for ultrasonic cleaning and then dried with nitrogen to obtain the micro-hole array.

[0034] (2) The substrate of the microfluidic channel is a thermoplastic (such as COP, PC, etc.) or a polymer material (such as PDMS and Flexdym, etc.), the channel height is 65-145 μm, and the distance between the inlet and outlet is 29.0-29.2 mm. The microfluidic channel is prepared by soft lithography, precision machining or precision injection molding.

[0035] (3) The microfluidic channel and the micropore array are bonded after oxygen plasma treatment or combined using a clamp. 1. The surfaces of the microfluidic channel and the micropore array are treated with oxygen plasma. Alignment marks are present on the microfluidic channel. The alignment marks are aligned with the micropore array and bonded. After bonding, the microfluidic channel and the micropore array are heated in an 80 ℃ oven for 2 hours to ensure a stable bond. 2. The micropore array and the microfluidic channel are clamped together using a clamp, such as... Figure 3 As shown, the clamp consists of two square plastic clamps, each with a thread at each of its four corners for connection; one of the clamps has two slots, each 2.5 mm wide and 30.0 mm long, with a center-to-center distance of 28.7 mm, to expose the inlet and outlet.

[0036] Example 2

[0037] This embodiment provides a periodic bidirectional oscillation sample introduction method based on the microfluidic channel integrated micropore array chip in Embodiment 1, the process of which is as follows: Figure 4 As shown, taking the sample introduction process of one of the micropore arrays and the corresponding integrated microfluidic channel in the chip as an example, the sample introduction process is divided into 8 parts, each labeled with a number on the left side of the respective pattern. The left side of the microfluidic channel is the sample inlet, the right side is the sample outlet, the blue pattern in the middle is the micropore array area, the light yellow part in the figure represents the sensitive element solution, and the arrows indicate the direction of liquid flow. Specifically, it includes the following steps:

[0038] S1. First, prepare the sensitive element solution (concentration 2.5 × 10⁻⁶). 5 The sample was shaken for 30 seconds (8 μL / sample) to ensure uniform distribution of the sensing elements, and then aspirated into a 20 μL pipette. The injection port was connected to the air pump via the 20 μL pipette and a 0.6 mm diameter rigid air tube. Gas was introduced into the micropore array at 8 mbar pressure. Figure 4 As shown in Part 2, after all 8 μL of the sensing element solution has entered the microfluidic channel and covered the micropore array area, the gas pressure is stopped, and the chip is left to stand for 90 seconds to allow the sensing element to settle. Figure 4 As shown in Part 3, at this point, a small number of sensitive elements have fallen into the array, while most of the remaining sensitive elements are located at the bottom of the microfluidic channel.

[0039] S2. After sedimentation, insert another pipette tube into the outlet to collect the sensing element solution flowing out of the microfluidic channel. Apply a pressure of 5 mbar to the inlet, such as... Figure 4 As shown in Part 4, the liquid flow velocity is relatively fast in the initial stage of applying gas pressure, but decreases significantly when the liquid reaches the micropore array at the edge. This is because a large number of sensing elements are clustered together, and the slower velocity at this point allows sensing elements already at the bottom to be carried into the array by the liquid. The sensing elements that have just fallen into the micropores will exert some resistance on the liquid. At a gas pressure of 5 mbar, the liquid passes through the entire micropore array in approximately 4 minutes. After the solution has completely passed through the micropore array area, the gas pressure is stopped at the injection port, and the solution settles for 90 seconds. Figure 4 As shown in Part 5.

[0040] S3. After settling, apply a pressure of 5 mbar to the sample outlet at the other end, such as... Figure 4 As shown in Part 6, the liquid is still allowed to pass through the micropore array relatively slowly. The applied air pressure is stopped just as the liquid edge at the sample outlet reaches the edge of the micropore array near the outlet. A 90-second settling period is then allowed to allow the sensing element to settle. Figure 4As shown in Part 7. By this time, a considerable number of sensitive elements have fallen into the micropores. The entire process takes approximately 10 minutes.

[0041] S4. Repeat the above injection as one oscillating injection. Figure 4 The procedure described in sections 2-7 involves a second oscillatory injection to improve the final filling rate. Two oscillatory injection cycles are performed, totaling 20 minutes, after which excess liquid is aspirated. Figure 4 As shown in Part 8, the entire injection process is now complete.

[0042] The chip was left in place, and the fill rate of the micropore array was characterized using fluorescence microscopy. A self-fluorescent sensing element was used to facilitate observation of the fill rate. Multiple experiments were conducted to demonstrate the stability of the sample introduction method. The results of one experiment are shown below. Figure 5 and Figure 6 As shown, Figure 5 These are images of the micropore array under fluorescence and bright field images from a confocal microscope. From top to bottom, they are filled images of the upper left, lower left, middle, upper right, and lower right. Figure 6 The results are obtained using statistical software. When traditional sample introduction methods are applied to microwell arrays, the overall fill rate is between 30% and 35%, with some areas exhibiting extremely low fill rates, resulting in overall instability. The results show that, through optimized sample introduction methods, the microfluidic channel integrated microwell array chip achieves an overall fill rate of 56%, with some areas reaching 67%. Compared to traditional sample introduction methods, this represents a significant improvement in both overall fill rate and fill stability, which is beneficial for meeting the detection needs of specific experimental and clinical studies.

Claims

1. A microfluidic channel integrated micowell array chip, characterized by, The chip includes a micropore array and a microfluidic channel; the microfluidic channel has a strictly distinct inlet and outlet at both ends; the micropore array is located near the outlet of the microfluidic channel. A single micropore array contains 220,000-225,000 micropore units, each with a diameter of 5.0-5.4 μm and a spacing of 4.5 μm between adjacent micropore units. The micropore units in adjacent rows within the micropore array are staggered.

2. The microfluidic channel integrated micowell array chip of claim 1, wherein, The chip has eight micropore arrays and microfluidic channels integrated with each micropore array.

3. The microfluidic channel integrated micowell array chip of claim 1, wherein, The micropore array is used to achieve physical isolation of individual sensitive elements; the microfluidic channel is used for transporting sensitive elements and assisting in filling sensitive elements.

4. The microfluidic channel integrated micropore array chip according to claim 1, characterized in that, The chip is fabricated using two methods: Method 1: Treat the surface of the microfluidic channel and the micropore array with oxygen plasma; set alignment marks on the microfluidic channel, align the alignment marks with the micropore array, and bond them together. After bonding, heat in an 80 ℃ oven for 2 hours to make the microfluidic channel and the micropore array firmly bonded. Method 2: The microfluidic channel and the microporous array chip are combined using a fixture; the fixture consists of two square plastic clamps, each with a thread at each of its four corners for connection; one of the clamps has a slot to expose the inlet and outlet; screws are used to tightly connect the microfluidic chip and the microporous array chip, thereby producing a microfluidic channel integrated microporous array chip.

5. The microfluidic channel integrated micropore array chip according to claim 4, characterized in that, The micropore array is fabricated using micro-nano fabrication technology, including the following steps: selecting a quartz substrate as the substrate for the micropore array, organically cleaning the quartz substrate, and performing oxygen plasma treatment on the surface; subsequently, spin-coating photoresist as an etching protective layer, and exposing and developing the micropore pattern; then etching the quartz substrate using an ICP etching process, selecting Freon and argon as working gases; after etching, immersing the quartz substrate in acetone and alcohol for ultrasonic cleaning, and drying it with nitrogen.

6. The microfluidic channel integrated micropore array chip according to claim 4, characterized in that, The height of the microfluidic channel ranges from 65 to 145 μm, the distance between the inlet and outlet is 29.0 to 29.2 mm, and the channel substrate is any one of COP, PC thermoplastic, or PDMS, Flexdym polymer materials; the microfluidic channel is prepared by soft lithography, precision machining or precision injection molding.

7. A periodic bidirectional oscillation sample introduction method based on the microfluidic channel integrated micropore array chip as described in claim 1, characterized in that, The sample introduction method includes the following steps: bidirectional oscillation, that is, the sensitive element solution is driven alternately in the direction of the inlet and outlet of the microfluidic channel, and this step is repeated periodically. Before each drive, the chip is allowed to stand still to make the sensitive element solution completely stable and allow the sensitive element to naturally sink to the bottom of the micropore array.

8. The sample introduction method according to claim 7, characterized in that, The specific process of completing a single bidirectional oscillation is as follows: S1. Shake the sensitive element solution for 30 s to distribute the sensitive element evenly, and then aspirate it into the pipette tube; connect the inlet of the microfluidic channel to the pipette tube and the rigid gas tube to connect to the gas pump, and introduce gas pressure of 8 mbar into the microfluidic channel. After all the sensitive element solution has entered the microfluidic channel and covered the micropore array area, stop the gas pressure and let the chip stand for 90 s to allow the sensitive element to settle to the bottom of the micropore array; S2. After sedimentation, insert another pipette into the sample outlet to contain the sensitive element solution flowing out of the microfluidic channel; When a pressure of 5 mbar is applied at the injection port, the liquid takes 4 minutes to pass through the entire micropore array. After the solution of the sensitive element has completely passed through the microporous array region, the applied gas pressure is stopped, and the solution settles for 90 seconds. S3. After sedimentation, apply 5 mbar air pressure to the other end of the sample outlet. Stop applying air pressure when the liquid edge of the sample outlet just reaches the edge of the micropore array near the sample outlet. Let it stand for 90 seconds to allow the sensitive element to settle. The time to complete a single bidirectional oscillation is 10 minutes.

9. The sample introduction method according to claim 8, characterized in that, Repeat the bidirectional oscillation operation to improve the final filling rate, then aspirate the excess liquid to complete the entire injection process.

10. The sample introduction method according to claim 8, characterized in that, The sensitive element solution concentration is set to 2.5 x 10 5 microliters; the air pump is connected to the chip through a 20 microliter pipette tube and a 0.6 mm diameter hard air tube.

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