Drug analysis platform based on lensless imaging technology

Abstract

The invention discloses a drug analysis platform based on a lensless imaging technology, the drug analysis platform combines the lensless imaging technology and a microfluidic technology, can complete efficient and rapid cell imaging and activity analysis, and realizes evaluation of dose-dependent cell reactions under various different drug concentrations on a small area. Therefore, low-error, low-cost and high-throughput drug screening work is completed.

Description

Technical Field

[0001] The invention relates to a drug analysis platform based on lens-free imaging technology, belonging to the technical field of microfluidic chips. Background Art

[0002] Lensless microscopic imaging technology refers to recording the projection of the sample above the imaging chip through an image sensor such as CCD or CMOS under the imaging chip without an optical lens. When cells are directly attached to the imaging area of ​​the lensless imaging chip for culture, the best quality cell projection can be obtained when the light source above the imaging area directly illuminates the cells. The image sensor directly below the imaging area records the cell projection generated above, and then transmits the image information through the electronic components and circuits in the surrounding area, and reconstructs the cell projection through digital imaging technology and mathematical calculations, and finally obtains a clear cell image. Although the imaging area is very small, this technology can clearly capture the image of tens of thousands of cells in the imaging area, and realize high-resolution and large-field microscopic imaging of living cells. Traditional live cell imaging technology mainly uses optical microscopes. Unlike direct projection and digital imaging, it refracts and focuses the light of the object through a lens and projects it onto the imaging plane to form an image of the object. It cannot have both high resolution and a large field of view, making it difficult to monitor large-scale dynamic reactions of cells and quantify cell viability, and it is not easy to capture dynamic changes such as cell growth and migration. Lens-free microscopy technology overcomes the disadvantages of traditional optical microscopes in living cell imaging through unique imaging technology. In the field of drug analysis, it analyzes cell viability by observing the growth and migration of living cells and counting the optical morphology and confluence of cells, thereby evaluating cellular drug responses.

[0003] As an analytical technology that can accurately control the flow of fluids, microfluidic chip technology has the advantages of miniaturization, high efficiency, rapidity, and low cost. It is widely used in the field of drug analysis. Among them, concentration gradient microfluidic chip is an important technology for drug analysis. This technology completes high-throughput drug screening by constructing drug concentration gradients to study dose-dependent cell responses under different drug concentrations. Traditional drug concentration gradient experiments are mainly completed on multi-well plates, but this method usually requires manual pipetting, which has the disadvantages of large workload, easy to produce errors, and high reagent consumption. The concentration gradient microfluidic chip can add different concentrations of drugs to different injection ports. After these drug fluids are separated and mixed layer by layer, they automatically form a precise and stable concentration gradient at the outlet. Therefore, the concentration gradient microfluidic chip is widely used in the construction of drug concentration gradients due to its advantages such as automatic pipetting, small errors, and low reagent consumption.

[0004] At present, most concentration gradient microfluidic chips are combined with traditional live cell imaging technology to evaluate dose-dependent cellular responses by monitoring cell viability, and screen out potential drugs for treating diseases and the optimal concentration of drugs based on the evaluation results. However, due to the limitations of traditional optical microscopy, this method often requires a large imaging area to provide enough cell samples to complete drug analysis. The experimental conditions are complex, data analysis is difficult, and efficient drug screening is difficult to achieve. Therefore, combining concentration gradient microfluidic chips with lensless microscopy technology is expected to complete the screening of several drugs on a smaller imaging area compared to traditional drug analysis platforms, greatly improving drug screening efficiency. However, the size of concentration gradient microfluidic chips is often larger than that of lensless imaging chips, making it difficult to match the packaging area of ​​lensless imaging chips. The commonly used concentration gradient microfluidic chips often adopt a "Christmas tree" structure, see Figure 1 , the structure is shaped like a Christmas tree, consisting of multiple layers of main channels from top to bottom, and each layer is connected by multiple branch channels. After the drugs of different concentrations are injected from the top layer, the flow is distributed to each branch in the main channels of each layer and mixed to produce a new concentration, and finally a concentration gradient is formed at the bottom layer. The tortuous structure of the branch channel is conducive to the full mixing of the drugs. In addition to occupying a large area, this structure also has the problem that the flow channels can only be concentrated on one side. There are a large number of tortuous flow channels, and the flow channel optimization is difficult, which makes it difficult to integrate with the lensless imaging chip. And because the imaging area of ​​the lensless imaging chip has different functions from the surrounding area, the materials and surface structures of the two are often different, making it difficult to achieve a close fit with the concentration gradient microfluidic chip. Therefore, how to combine the concentration gradient microfluidic chip with the lensless microscopic imaging technology to overcome the technical challenges of the drug analysis platform built based on the traditional living cell imaging technology has become an urgent problem to be solved in this technical field. Summary of the invention

[0005] Purpose of the invention: The first purpose of the present invention is to provide a concentration gradient microfluidic chip. The second purpose of the present invention is to provide a drug analysis platform suitable for the above-mentioned concentration gradient microfluidic chip without lens imaging technology.

[0006] Technical solution: The present invention provides a concentration gradient microfluidic chip, which is formed by bonding a flow layer and a connecting layer, wherein a first injection port and a second injection port are respectively provided at two ends of the flow layer, and a groove is provided on the lower surface of the flow layer, and a fluid channel is formed after the connecting layer and the flow layer are bonded; the fluid channel is connected to a through hole arranged at the center of the connecting layer and is arranged around the through hole, and there are at least two through holes, and the fluid channel includes different branch channels connected in parallel or / and in series so that the drug solution or diluent presents a concentration gradient when it reaches the outlets corresponding to different through holes from the first injection port or the second injection port.

[0007] Furthermore, the branch channel comprises flow channels of different lengths, and the flow channels of different lengths are respectively realized by different bending lengths and bending times.

[0008] Furthermore, the design formula of the fluid channel is as follows:

[0009]

[0010] Where Q is the flow rate in m 3 / s; Δp is the pressure drop, in Pa; R F is the flow resistance, in Pa·s / m 3 ;

[0011]

[0012] In the formula, Δp n is the pressure drop of each fluid channel, in Pa; R Fn is the flow resistance of each flow channel, in Pa·s / m 3 ;Q n is the flow rate of each fluid channel, in m 3 / s, m ​​is the number of outlets corresponding to the through hole;

[0013]

[0014] Where, L n is the length of each flow channel, in m.

[0015] Furthermore, two branch channels are branched out through the fluid channel on one side of the first injection port or the second injection port, respectively connecting through hole 1 and through hole 2; 1, 2, 3...m-2 branch channels are branched out on the other side of the first injection port or the second injection port, respectively connecting through holes 3, 4, 5...m-2, where m is the number of through holes.

[0016] Furthermore, assuming that the diluent enters from the first injection port and the drug enters from the second injection port, the diluent flows through L1+L4 to reach the outlet 1 corresponding to the through hole; the drug flows through L7+L6 to reach the outlet m corresponding to the through hole;

[0017] When the number of outlets corresponding to the through hole is m=4, the diluent flows through L8 and flows through L7+L A3 The liquids merge and then flow through L B3 +L E3 Then it reaches the outlet 3 corresponding to the through hole; the diluent flows through L8 and L7+L A3 The liquids merge and then flow through L B3 +L5 and then merge with the dilution liquid flowing through L1+L2, and then flow through L3 to reach the outlet 2 corresponding to the through hole;

[0018] When the number of outlets corresponding to the through hole is m=5, the diluent flows through L8 and flows through L7+L A4 +L A3 The liquids merge and then flow through L B3 +L D3 +L E3 Then it reaches the outlet 3 corresponding to the through hole; the diluent flows through L8 and L7+L A4 +L A3 The liquids merge and then flow through L B3 +L D3 +L5 and then merge with the dilution liquid flowing through L1+L2, and then flow through L3 to reach the outlet 2 corresponding to the through hole; the dilution liquid flows through L8 and merges with the dilution liquid flowing through L7+L A4 +L A3 The liquids merge and then flow through L B3 +L C3 With the flow through L7+L A4 +L B4 The liquids merge and then flow through L E4 Arrive at the exit 4 corresponding to the through hole;

[0019] When the number of outlets corresponding to the through hole is m=6, the diluent flows through L8 and flows through L7+L A5 +L A4 +L A3 The liquids merge and then flow through L B3 +L D3 +L E3 Then it reaches the outlet 3 corresponding to the through hole; the diluent flows through L8 and L7+L A5 +L A4 +L A3 The liquids merge and then flow through L B3 +L D3 +L5 and then merge with the dilution liquid flowing through L1+L2, and then flow through L3 to reach the outlet 2 corresponding to the through hole; the dilution liquid flows through L8 and merges with the dilution liquid flowing through L7+L A5 +L A4 +L A3 The liquids merge and then flow through L B3 +L C3 With the flow through L7+L A5 +L A4 +L B4 The liquids merge and then flow through L D4 +L E4 Arrives at outlet 4 corresponding to the through hole; the diluent flows through L8 and L7+L A5 +L A4 +L A3 The liquids merge and then flow through L B3 +L C3 With the flow through L7+L A5 +LA4 +L B4 The liquids merge and then flow through L D4 +L C4 With the flow through L7+L A5 +L B5 The liquids merge and then flow through L E5 Then it reaches the exit 5 corresponding to the through hole;

[0020] When the number of exports m>6, the same applies;

[0021] The relationship between the lengths of the various flow channels is as follows:

[0022] Q2L2+Q3L3=Q4L4

[0023] Q5L5+Q3L3=Q E3 L E3

[0024]

[0025] Q E3 L E3 +Q D3 L D3 +Q B3 L B3 +Q8L8=L1+L4

[0026]

[0027] Q A3 L A3 +Q C3 L C3 +Q B3 L B3 =Q B4 L B4

[0028] Q E3 L E3 +Q D3 L D3 =Q C3 L C3 +Q D4 L D4 +Q E4 L E4

[0029] Q E(m-2) L E(m-2) =Q C(m-2) L C(m-2) +Q E(m-1) L E(m-1)

[0030] Q An L An +QBn L Bn +Q Cn L Cn +Q Dn L Dn =Q B(n+1) L B(n+1) (n=4, ..., m-2)

[0031] Q En L En =Q Cn L Cn +Q D(n+1) L D(n+1) +Q E(n+1) L E(n+1) (n=4, ..., m-3)

[0032]

[0033] In the formula, s is the exit number, s = 3, 4, 5,...m-1.

[0034] Furthermore, the dimensions of the groove are 100 μm in width and 100 μm in height.

[0035] Furthermore, the connection layer and the flow layer are equal in size.

[0036] Furthermore, the number of the through holes is 4 to 9.

[0037] The present invention also provides a drug analysis platform based on lens-free imaging technology, which includes a lens-free imaging chip, a cell chamber layer, and the above-mentioned concentration gradient microfluidic chip, which are bonded and assembled in order from bottom to top; the lens-free imaging chip is provided with a lens-free imaging area; the cell chamber layer is provided with a through hole corresponding to the position of the connecting layer, and a cell culture chamber is formed after bonding, and the position of the cell culture chamber is within the range of the lens-free imaging area.

[0038] Furthermore, the cell chamber layer and the lens-free imaging region are equal in area and smaller than the size of the concentration gradient microfluidic chip.

[0039] The area design of the cell chamber layer should be slightly larger than the lens-free imaging area. If the area of ​​the cell chamber layer is smaller than the lens-free imaging area, it is impossible to maximize the use of the imaging area. If the area of ​​the cell chamber layer is too large, it may not be possible to fit due to the unevenness of the lens-free imaging area.

[0040] Furthermore, the dimensions of the cell chamber layer are 3 to 10 mm in length, 3 to 10 mm in width, and ≥1 mm in height; the dimensions of the flow layer and the connection layer are 29 to 37 mm in length, 23 to 27 mm in width, and ≥1 mm in height; and the diameter of the through holes on the connection layer and the cell chamber layer is 1 to 3 mm.

[0041] Furthermore, the height of the cell chamber layer is 1 to 5 mm; the height of the flow layer and the connection layer is 1 to 4 mm.

[0042] Furthermore, the cell chamber layer, the connection layer and the flow layer are all made of polydimethylsiloxane.

[0043] Furthermore, the cell culture chamber is used to establish neuronal cell lines, primary neurons and glial cell models.

[0044] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: the present invention provides a drug analysis platform based on lensless imaging technology with a compact structure and a light volume. By designing a connecting layer and a cell chamber layer, the flow layer and the lensless imaging chip are indirectly connected, which solves the difficulty that the concentration gradient microfluidic chip cannot be directly and tightly fitted with the lensless imaging chip due to the different materials and surface structures of the lensless imaging area and its peripheral chip area, and realizes the combination of the concentration gradient microfluidic chip and the lensless imaging technology; an annular fluid channel is designed on the periphery of the central area corresponding to the position of the bottom lensless imaging area on the flow layer to ensure that the fluid channel does not block the lensless imaging area, so that the fluid channel is evenly arranged, the flow channel area is greatly reduced, and the space in the peripheral packaging area of ​​the lensless imaging chip is saved, so that the flow channel can be changed according to the needs of drug analysis, and the length of each section of the flow channel is accurately calculated by the equivalent circuit method, so that the concentration gradient microfluidic chip can generate a stable concentration gradient; the through circular hole in the central area of ​​the platform allows the light source above the platform to be unobstructed while culturing cells, and directly illuminates the attached cells on the lensless imaging chip to complete projection imaging. The platform can apply different concentrations of drugs to cells in each chamber while monitoring cell growth and migration activities, and then analyze cell viability to evaluate dose-dependent cell responses. The platform integrates a concentration gradient microfluidic chip and a lensless imaging chip, and by designing an array of through holes, it maximizes the use of the imaging area of ​​the lensless imaging chip, so that the lensless imaging chip, which originally could only carry one concentration of drugs, can simultaneously analyze drugs of multiple concentrations, greatly improving the efficiency of drug screening, thereby completing low-cost, low-error, and high-throughput drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is a picture of the combination of a traditional concentration gradient microfluidic chip and a lens-free imaging chip;

[0046] Figure 2 This is a physical picture of the drug analysis platform based on lensless imaging technology;

[0047] Figure 3This is a front view of the drug analysis platform based on lensless imaging technology;

[0048] Figure 4 This is a top view of the drug analysis platform based on lensless imaging technology;

[0049] Figure 5 This is a top view of the lens-free imaging chip of the drug analysis platform based on the lens-free imaging technology;

[0050] Figure 6 This is a schematic diagram of the fluid channel structure of the drug analysis platform based on lensless imaging technology;

[0051] Figure 7 This is a schematic diagram of the four-chamber flow layer of the drug analysis platform based on lensless imaging technology;

[0052] Figure 8 This is a schematic diagram of the five-chamber flow layer of the drug analysis platform based on lensless imaging technology;

[0053] Fig. 9 This is a schematic diagram of the connection layer of the drug analysis platform based on lensless imaging technology;

[0054] Fig.10 This is a schematic diagram of the cell chamber layer of the drug analysis platform based on lens-free imaging technology;

[0055] Fig.11 This is a simulated diagram of the concentration gradient in the four chambers of the drug analysis platform based on lensless imaging technology;

[0056] Fig.12 This is a simulated diagram of the five-chamber concentration gradient of the drug analysis platform based on lensless imaging technology;

[0057] Fig.13 This is a fluorescent dye rendering of the fluid channel of the drug analysis platform based on lensless imaging technology;

[0058] Fig.14 It is a fluorescence dye spectrum diagram of the fluid channel of the drug analysis platform based on lensless imaging technology;

[0059] Fig.15 These are pictures of cells cultured in circular holes of the same size as the cell culture chamber of the drug analysis platform based on lensless imaging technology, where the upper picture shows HT22 cells and the lower picture shows BV2 cells. DETAILED DESCRIPTION

[0060] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0061] Example 1

[0062] The structure of the drug analysis platform in this example is referenced Figure 2 to Figure 10 . Figure 2 This is a physical picture of the drug analysis platform of the present invention. Taking four cell culture chambers and five cell culture chambers as examples, the drug platform is composed of a lensless imaging chip 1, a cell chamber layer 3, a connection layer 4 and a flow layer 5 in order from bottom to top, and they are bonded and assembled after aligning the geometric center. The cell chamber layer 3, the connection layer 4 and the flow layer 5 are all made of polydimethylsiloxane with light-transmitting properties. Among them, the size of the lensless imaging area 2 is equal to that of the cell chamber layer 3; the size of the connection layer 4 and the flow layer 5 is equal, and is larger than the size of the lensless imaging area 2.

[0063] The lens-free imaging chip 1 is provided with a lens-free imaging area 2 .

[0064] The connection layer 4 and the flow layer 5 are bonded to form a concentration gradient microfluidic chip. The first injection port and the second injection port are respectively provided at both ends of the flow layer 5. A groove is provided on the lower surface of the flow layer 5. After the connection layer 4 and the flow layer 5 are bonded, a fluid channel is formed. The size of the groove is 100 μm in width and 100 μm in height. A circular through hole is provided on the connection layer 4. The fluid channel is connected to the through hole set in the center of the connection layer and is set at the periphery of the through hole. The fluid channel includes different branch channels connected in parallel or / and in series so that the drug solution or diluent presents a concentration gradient when it reaches different through holes from the first injection port or the second injection port. The dimensions of the connection layer 4 and the flow layer 5 are both 29 mm in length, 23 mm in width, and 4 mm in height.

[0065] The dimensions of the cell chamber layer 3 are 10 mm in length, 10 mm in width and 5 mm in height. The cell chamber layer 3 is provided with through holes corresponding to the positions of the connection layer 4, which form a cell culture chamber 6 after bonding. The diameter of the cell culture chamber 6 is 3 mm.

[0066] The concentration gradient microfluidic chip of the present invention combines the characteristics of the lens-free imaging chip, designs the annular flow channel through the equivalent circuit method, and sets the lens-free imaging area in the center of the flow channel area. Under the condition of ensuring that the concentration gradient is formed at each outlet solution and the flow rate is the same, the flow channel is evenly arranged and the structure is compact.

[0067] The equivalent circuit method refers to equating the pressure drop, flow rate and flow resistance of each flow channel to the voltage, current and resistance in a closed circuit.

[0068] The relationship between the pressure drop of the flow channel, flow rate and flow resistance is as follows:

[0069]

[0070] Where Q is the flow rate in m 3 / s; Δp is the pressure drop, in Pa; R F is the flow resistance, in Pa·s / m3 .

[0071] According to Kirchhoff's law, the algebraic sum of the currents at each node in a closed circuit is 0, and the algebraic sum of the voltages in each loop is 0, so:

[0072]

[0073] In the formula, Δp n is the pressure drop of each flow channel, in Pa; R Fn is the flow resistance of each flow channel, in Pa·s / m 3 ;Q n is the flow rate of each flow channel, in m 3 / s.

[0074] When the channel width and height are unified, the flow resistance of the channel is proportional to the length of the channel, so:

[0075]

[0076] Where, L n is the length of each flow channel, in m.

[0077] When injecting drugs, it is necessary to ensure that each cell culture chamber is injected synchronously to ensure the same injection volume and generate the required linear concentration gradient, so that the drug concentration in each chamber is different, so as to achieve the purpose of high-throughput drug screening. Therefore, assuming that the initial drug flow rate of the two injection ports is the same, the initial drug flow rate required to be allocated to each flow channel is calculated according to the required results (including the injection speed and concentration of each chamber) to ensure that the two initial drugs of different concentrations are mixed at each node according to the preset flow ratio, and finally form a concentration gradient, and then control the height and width of each flow channel to be consistent, and calculate the length of each flow channel according to the flow size of each flow channel and Kirchhoff's law, so as to achieve the purpose of adjusting the flow resistance to control the flow.

[0078] like Figure 6 As shown, when the number of cell culture chambers is m, the relationship between the lengths of each flow channel is as follows:

[0079] Q2L2+Q3L3=Q4L4

[0080] Q5L5+Q3L3=Q E3 L E3

[0081]

[0082] Q E3 L E3 +Q D3 L D3 +Q B3 L B3+Q8L8=L1+L4

[0083]

[0084] Q A3 L A3 +Q C3 L C3 +Q B3 L B3 =Q B4 L B4

[0085] Q E3 L E3 +Q D3 L D3 =Q C3 L C3 +Q D4 L D4 +Q E4 L E4

[0086] Q E(m-2) L E(m-2) =Q C(m-2) L C(m-2) +Q E(m-1) L E(m-1)

[0087] Q An L An +Q Bn L Bn +Q Cn L Cn +Q Dn L Dn =Q B(n+1) L B(n+1) (n=4, ..., m-2)

[0088] Q En L En =Q Cn L Cn +Q D(n+1) L D(n+1) +Q E(n+1) L E(n+1) (n=4, ..., m-3)

[0089]

[0090] The following are the specific parameters of the flow layer of the four-cell culture chamber and the five-cell culture chamber. The fluid channel is designed within a rectangular range of 24 mm in length and 19 mm in width. The concentrations of the solutions at the upper and lower inlets are 0 and 1 mol / m, respectively. 3 .

[0091] See also Figure 7 , the length relationship of each flow channel of the four-chamber flow layer is as follows:

[0092]

[0093] The lengths of the various flow channels of this four-chamber flow layer example are shown in the following table:

[0094] Runner <![CDATA[L1]]> <![CDATA[L2]]> <![CDATA[L3]]> <![CDATA[L4]]> <![CDATA[L5]]> <![CDATA[L6]]> <![CDATA[L7]]> <![CDATA[L8]]> <![CDATA[L A3 ]]> <![CDATA[L B3 ]]> <![CDATA[L E3 ]]> Length / mm 23.3 3.9 10.5 12 26 43.5 17 17 5 10 23.5

[0095] See also Figure 8 , the length relationship of each flow channel of the five-chamber flow layer is as follows:

[0096]

[0097] The lengths of the flow channels of the five-chamber flow layer example are shown in the following table:

[0098]

[0099] Example 2

[0100] In order to evaluate the effectiveness of the fluid channel 8 constructed by the equivalent circuit method, the flow layer 5 was modeled using AutoCAD 2023 software, and the simulation experiment was carried out using COMSOL Multiphysics 6.0 software at a flow rate of 50 μL / min. The solution concentrations of the upper and lower inlets were 0 and 1 mol / m 3 . See Fig.11 and Fig.12 The results showed that the flow rates of the solutions in the four-cell culture chamber and the five-cell culture chamber were similar, and both could be sampled simultaneously. The concentrations of the four-cell culture chamber were 0, 0.33 mol / m 3 , 0.67mol / m 3 , 1mol / m 3 The concentrations of the five cell culture chambers were 0, 0.23 mol / m 3 , 0.51mol / m 3 , 0.76mol / m 3 , 1mol / m 3 , both form stable linear concentration gradients.

[0101] Specifically, in the four cell culture chambers, a solution with a concentration of 0 enters from the upper inlet (the first inlet), flows through L1+L4 and reaches outlet 1; a solution with a concentration of 0 enters from the upper inlet, flows through L8 and enters from the lower inlet (the second inlet) and flows through L7+L A3 The concentration is 1 mol / m 3 The solutions are combined and then passed through L B3 +L E3Arrives at outlet 3; the solution with a concentration of 0 enters from the upper inlet, flows through L8 and enters from the lower inlet (second inlet) and flows through L7+L A3 The concentration is 1 mol / m 3 The solutions merge and pass through L B3 Then it passes through L5 and merges with the solution with a concentration of 0 that passes through L1+L2, and then passes through L3 to reach outlet 2; the concentration is 1 mol / m 3 The solution enters from the lower inlet, flows through L7+L6 and reaches outlet 4.

[0102] In the five cell culture chambers, a solution with a concentration of 0 enters from the upper inlet (the first inlet), flows through L1+L4 and reaches outlet 1; a solution with a concentration of 0 enters from the upper inlet, flows through L8 and enters from the lower inlet (the second inlet), flows through L7+L A4 +L A3 The concentration is 1 mol / m 3 The solutions merge and flow through L B3 +L D3 +L E3 Then enter outlet 3; the solution with a concentration of 0 enters from the upper inlet, flows through L8 and enters from the lower inlet (second inlet) and flows through L7+L A4 +L A3 The concentration is 1 mol / m 3 The solutions merge and flow through L B3 +L D3 +L5, and then merge with the solution with a concentration of 0 that enters from the upper inlet (first inlet) and flows through L1+L2, and then flows through L3 to enter outlet 2; the solution with a concentration of 0 enters from the upper inlet and flows through L8, and merges with the solution that enters from the lower inlet (second inlet) and flows through L7+L A4 +L A3 The concentration is 1 mol / m 3 The solutions merge and flow through L B3 +L C3 Then, the flow from the lower inlet (second inlet) passes through L7+L A4 +L B4 The concentration is 1 mol / m 3 The solutions merge and then flow through L E4 Enter outlet 4; enter from the lower inlet (second inlet) with a concentration of 1 mol / m 3 The solution flows through L7+L6 and reaches outlet 5.

[0103] Example 3

[0104] The formation of the concentration gradient was further verified by measuring the UV-visible absorption spectrum of rhodamine 6G. First, a 0.15 mM rhodamine 6G solution was prepared, and then the solution and deionized water were injected into different injection ports 7 using a syringe pump. Fig.12 As shown (taking four cell culture chambers as an example), the absorbance of the solution in each cell culture chamber 6 is finally measured. The absorbance of the four cell culture chambers 6 at a wavelength of 527 nm is as follows: Fig.14 As shown, the results show that the absorbance of the solution in each chamber changes linearly, which is consistent with the experimental results of the simulation.

[0105] Example 4

[0106] To prove that the cell culture chamber 6 with a diameter of 3 mm can meet the needs of cell culture, HT22 cells and BV2 cells, which are commonly used for drug screening of neurodegenerative diseases, were selected and cultured in a circular hole with a diameter of 3 mm based on polydimethylsiloxane for 24 h, with a cell density of 1×10 5 The results showed that both cells could grow normally in the round wells.

[0107] Example 5

[0108] A lensless imaging-based drug analysis platform with four chambers, each with a cell imaging area of ​​0.07065 cm 2 Under the condition of 400 times the resolution of traditional optical microscopy, the field of view of live cell imaging is increased by 35.325 times compared with traditional drug analysis platforms. By integrating concentration gradient microfluidic chip technology and lens-free imaging technology, high-throughput cell imaging is achieved, the error of quantifying cell viability is reduced, and the efficiency of drug screening is improved.

Claims

1. A concentration gradient microfluidic chip, characterized in that: The concentration gradient microfluidic chip is formed by bonding a flow layer and a connection layer, wherein a first injection port and a second injection port are respectively provided at two ends of the flow layer, and a groove is provided on the lower surface of the flow layer, so that a fluid channel is formed after the connection layer and the flow layer are bonded; the fluid channel is connected to a through hole provided at the center of the connection layer and is provided at the periphery of the through hole, and there are at least two through holes, and the fluid channel includes different branch channels connected in parallel or / and in series so that a drug solution or a diluent presents a concentration gradient when it reaches outlets corresponding to different through holes from the first injection port or the second injection port.

2. The concentration gradient microfluidic chip according to claim 1, characterized in that: The branch channel comprises flow channels of different lengths, and the flow channels of different lengths are respectively realized by different bending lengths and bending times.

3. The concentration gradient microfluidic chip according to claim 1, characterized in that: The design formula of the fluid channel is as follows: Where Q is the flow rate in m 3 / s; Δp is the pressure drop, in Pa; R F is the flow resistance, in Pa·s / m 3 ; In the formula, Δp n is the pressure drop of each flow channel, in Pa; R Fn is the flow resistance of each fluid channel, in Pa·s / m 3 ;Q n is the flow rate of each fluid channel, in m 3 / s, m ​​is the number of outlets corresponding to the through hole; Where, L n is the length of each flow channel, in m.

4. The concentration gradient microfluidic chip according to claim 1, characterized in that: Two branch channels are branched out from one side of the first injection port or the second injection port through the fluid channel, respectively connecting through hole 1 and through hole 2; 1, 2, 3...m-2 branch channels are branched out from the other side of the first injection port or the second injection port, respectively connecting through holes 3, 4, 5...m-2, where m is the number of through holes.

5. The concentration gradient microfluidic chip according to claim 1, characterized in that: The connection layer is equal in size to the flow layer.

6. A drug analysis platform based on lensless imaging technology, characterized in that: The drug analysis platform comprises a lens-free imaging chip, a cell chamber layer, and the concentration gradient microfluidic chip according to claim 1, which are bonded and assembled in order from bottom to top; the lens-free imaging chip is provided with a lens-free imaging area; the cell chamber layer is provided with a through hole corresponding to the position of the connection layer, and a cell culture chamber is formed after bonding, and the position of the cell culture chamber is within the range of the lens-free imaging area.

7. The drug analysis platform based on lensless imaging technology according to claim 6, characterized in that: The cell chamber layer and the lens-free imaging region are equal in area and smaller than the size of the concentration gradient microfluidic chip.

8. The drug analysis platform based on lensless imaging technology according to claim 6, characterized in that: The dimensions of the cell chamber layer are 3-10 mm in length, 3-10 mm in width, and ≥1 mm in height; the dimensions of the flow layer and the connection layer are 29-37 mm in length, 23-27 mm in width, and ≥1 mm in height; and the diameter of the through holes on the connection layer and the cell chamber layer is 1-3 mm.

9. The drug analysis platform based on lensless imaging technology according to claim 6, characterized in that: The cell chamber layer, the connection layer and the flow layer are all made of polydimethylsiloxane.

10. The drug analysis platform based on lensless imaging technology according to claim 6, characterized in that: The cell culture chamber is used to establish neuron cell lines, primary neurons and glial cell models.

Images