A cascade Fermat spiral microfluidic mixer for cancer cell detection and detection method thereof

Abstract

The present invention discloses a cascade Fermat spiral microfluidic mixer for cancer cell detection and a detection method thereof. The mixer comprises an upper detection layer, wherein the lower side of the upper detection layer is connected to a lower detection layer, a plurality of mixing channels arranged at intervals are provided on the downward side of the upper detection layer, a plurality of first liquid storage tanks are arranged on the downward side of the upper detection layer at one end of the front mixing channel, a plurality of second liquid storage tanks are provided on the downward side of the upper detection layer between two adjacent mixing channels, a plurality of liquid inlet holes corresponding to the first liquid storage tanks and the second liquid storage tanks are opened on the upper detection layer, liquid storage pools are formed between the first liquid storage tanks and the lower detection layer, and between the second liquid storage tanks and the lower detection layer. The present invention can realize the detection of multiple cancer cells.

Description

Technical Field

[0001] The present invention relates to the technical field of cancer cell detection, in particular to a cascade Fermat spiral microfluid mixer for cancer cell detection and a detection method thereof. Background Art

[0002] As cancer has become one of the most serious threats to human health, highly sensitive cancer cell detection is crucial for improving the accuracy of early clinical diagnosis. Existing microfluidic chips involve complex fluid movement between multiple layers of the chip. These chips, while integrating only traditional cancer cell detection methods, do not reduce detection time and exhibit low sensitivity. Furthermore, the chips are bulky, require complex operation steps, and require a high degree of specialized technology, making them difficult to use as point-of-care (POCT) devices. Summary of the Invention

[0003] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0004] In view of the above and / or existing problems in existing body fluid detection, the present invention is proposed.

[0005] Therefore, the object of the present invention is to provide a cascade Fermat spiral microfluidic mixer for cancer cell detection and a detection method thereof, which can achieve a wide range of cancer cell detection with short detection time and high sensitivity.

[0006] To solve the above technical problems, the present invention provides the following technical solutions: a cascade Fermat spiral microfluidic mixer for cancer cell detection and a detection method thereof, comprising:

[0007] An upper detection layer, wherein a plurality of mixing channels are provided on the downward side of the upper detection layer at intervals, a plurality of first liquid storage tanks are arranged on the downward side of the upper detection layer at one end of the front mixing channel, and a plurality of second liquid storage tanks are provided on the downward side of the upper detection layer between two adjacent mixing channels, and a plurality of liquid inlet holes corresponding to the first liquid storage tanks and the second liquid storage tanks are opened on the upper detection layer;

[0008] The lower detection layer is connected to the lower side of the upper detection layer. Liquid storage pools are formed between the first liquid storage tank and the lower detection layer, and between the second liquid storage tank and the lower detection layer.

[0009] As a preferred solution of the cascade Fermat spiral microfluidic mixer for cancer cell detection described in the present invention, wherein: a closed port is provided on the reaction ring on the side where the reaction liquid storage tank is arranged opposite to the injection tube, and an injection cavity is formed between the closed port and the inner wall of the reaction ring at one end away from the reaction liquid storage tank, and a closed portion is provided at the end of the injection tube away from the dispersion injection seat, and the closed portion can just block the closed port, and when the first inlet of the injection tube is in the corresponding first outlet, the second inlet is in the injection cavity.

[0010] As a preferred solution of the cascade Fermat spiral microfluidic mixer for cancer cell detection according to the present invention, it further comprises a solution collecting component, which is arranged in the lower detection layer.

[0011] As a preferred solution of the cascade Fermat spiral microfluidic mixer for cancer cell detection described in the present invention, the solution in the second liquid storage tank can flow controllably to the next mixing channel.

[0012] As a preferred solution of the cascade Fermat spiral microfluidic mixer for cancer cell detection described in the present invention, wherein: an upper connecting hole is opened on the upper detection layer between two adjacent mixing channels, and a lower connecting hole corresponding to the upper connecting hole is opened on the lower detection layer. The upper detection layer and the lower detection layer are respectively connected to control valves via the upper connecting hole and the lower connecting hole, and the control valve controls the on-off between the two adjacent mixing channels and the corresponding second liquid storage tank.

[0013] As a preferred solution of the cascade Fermat spiral microfluidic mixer for cancer cell detection described in the present invention, the control valve is provided with a first connecting channel and a second connecting channel spaced apart from bottom to top, the first connecting channel connects the two adjacent mixing channels, one end of the second connecting channel is aligned with the liquid outlet end of the second liquid storage tank, and the other two ends of the second connecting channel are respectively aligned with the two adjacent mixing channels.

[0014] As a preferred embodiment of the cascade Fermat spiral microfluidic mixer for cancer cell detection described in the present invention, three first liquid storage tanks are arranged on the downward side of the upper detection layer at one end of the front mixing channel, a second liquid storage tank is provided on the downward side of the upper detection layer between the two front mixing channels, and a second liquid storage tank is provided on the downward side of the upper detection layer between the two rear mixing channels.

[0015] As a preferred solution of the cascade Fermat spiral microfluidic mixer for cancer cell detection described in the present invention, the mixing channel is a Fermat spiral microchannel, the two front mixing channels are the same size, with a width of 400±10μm and a depth of 400±10μm, and the rear mixing channel has a width of 600±12μm and a depth of 400±10μm.

[0016] A method for detecting cancer cells using a cascade Fermat spiral microfluidic mixer for cancer cell detection comprises the following steps:

[0017] Inject H2O2 solution, Amplex Red and Cu into the first three liquid storage tanks respectively. 2+ solution, injecting AS1411-AuNPs into the middle second liquid storage tank, and injecting cancer cells into the second liquid storage tank at the back;

[0018] The solution in each first liquid storage tank enters the first mixing channel, so that the H2O2 solution, Amplex Red and Cu 2+ The solutions are evenly mixed, producing obvious fluorescence, and the solution after the first mixing is ready to enter the second mixing channel;

[0019] Press down the first control valve to connect the first mixing channel and the second mixing channel, and the solution after the first mixing enters the second mixing channel. Continue to press down the first control valve to connect the first and second liquid storage tanks with the second mixing channel, and press the AS1411-AuNPs in the first and second liquid storage tanks into the second mixing microchannel, so that the AS1411-AuNPs are evenly mixed with the first three, resulting in obvious quenching phenomenon. The solution after the second mixing is ready to enter the third mixing channel.

[0020] Press down the second control valve to connect the second mixing channel and the third mixing channel, and the solution after the second mixing enters the third mixing channel;

[0021] Continue to press down the second control valve to connect the second second liquid storage tank and the third mixing channel, press the cancer cells in the second second liquid storage tank into the third mixing channel, so that the cancer cells and the first four are evenly mixed, producing fluorescence recovery phenomenon, and the mixed solution enters the solution collection component.

[0022] As a preferred embodiment of the detection method of the present invention, the concentration of the H2O2 solution is 5mmol / L to 10mmol / L, the concentration of Amplex Red is 500nmol / L to 0.5μmol / L, and the Cu 2+ The concentration of the solution is 0.4 μmol / L to 2 μmol / L, and the concentration of AS1411-AuNPs is 0.3 mmol / L to 2.4 mmol / L.

[0023] As a preferred embodiment of the detection method of the present invention, the concentration of the H2O2 solution is 8 mmol / L, the concentration of Amplex Red is 0.1 μmol / L, the concentration of the Cu2+ solution is 0.5 μmol / L, and the concentration of AS1411-AuNPs is 1.26 mmol / L.

[0024] The beneficial effects of the present invention are as follows: by controlling the valve to select the designated reagent, the sample and the detection reagent are evenly mixed through the mixing microchannel, the mixing effect is good, and after the mixing is even, a fluorescence-quenching-recovery phenomenon is generated, which is converted into a marker concentration, with a short detection time, simple operation and high detection sensitivity; the detection of multiple cancer cells in a wide concentration range is achieved. Different cancer cells often require different detection methods for detection. The combined setting of the detection chip and the detection method in the present invention can achieve the detection of multiple cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0026] Figure 1 The three-dimensional structure of the present invention Figure 1 .

[0027] Figure 2 The three-dimensional structure of the present invention Figure 2 .

[0028] Figure 3 The three-dimensional structure of the upper detection layer in the present invention Figure 1 .

[0029] Figure 4 This is a three-dimensional structural diagram after the upper detection layer is hidden in the present invention.

[0030] Figure 5 for Figure 4 A partial enlarged view of point A in the middle.

[0031] Figure 6 This is a three-dimensional structural diagram of the lower detection layer of the present invention after the first connecting portion is hidden.

[0032] Figure 7 for Figure 6 A partial enlarged view of point B in the middle.

[0033] Figure 8 It is a three-dimensional structural diagram of the first connecting part in the present invention.

[0034] Figure 9 for Figure 8 A partial enlarged view of point C in the middle.

[0035] Figure 10 It is a three-dimensional structural diagram of the second connecting part in the present invention.

[0036] Figure 11 for Figure 10 A partial enlarged view of point D in the middle.

[0037] Figure 12 The three-dimensional structure of the upper detection layer in the present invention Figure 2 .

[0038] Figure 13 It is a three-dimensional structural diagram of the control valve in the present invention.

[0039] Figure 14 This is a fluorescence color rendering coefficient diagram of cancer cells at different concentrations under the left limit concentration system in the present invention.

[0040] Figure 15 This is a graph of the fluorescence color rendering coefficients of cancer cells at different concentrations under the right limit concentration system of the present invention.

[0041] Figure 16 is the optimal concentration fluorescence spectrum detection result of the present invention, wherein, Figure 16 (A) As the concentration of MCF-7 cells increased from 0 to 1.0*10 7 , Fluorescence spectrum of Amplex Red at 570nm-650nm; Figure 16 (B) The fluorescence intensity of Amplex Red at 584 nm changes linearly with the concentration of MCF-7 cells.

[0042] Figure 17 This is a graph showing the results of the detection of four types of cells in the present invention.

[0043] In the figure, 100 control valve, 101 first connecting channel, 102 second connecting channel, 200 upper detection layer, 201 liquid inlet hole, 202 second liquid storage tank, 203 mixing channel, 204 first liquid storage tank, 205 upper connecting hole, 206 drainage sink hole, 207 liquid outlet channel, 300 lower detection layer, 301 first detection part, 301a outer upper rotating semi-sink hole, 301b second support shaft, 301c through hole, 301d upper connecting sink, 301e upper sliding port, 301f upper collecting sink, 301g upper partition, 301h inner upper rotating semi-sink hole, 301i upper movable sink ...f upper collecting sink, 301g upper partition, 301h inner upper rotating semi-sink hole, 301i upper movable sink, 301f upper collecting sink, 301f upper collecting sink, 301f upper collecting sink, 301g upper partition, 301h inner upper rotating semi-sink hole, 301i upper movable sink, 301f upper collecting sink, 301f upper collecting sink, 301f upper collecting sink, 301f upper collecting sink, 301f upper collecting sink, 301f upper collecting sink, 301f upper collecting sink, 301f upper collecting sink, 301f upper collecting sink, 301f 02 second detection part, 302a lower partition, 302b lower collecting trough, 302c first support shaft, 302d lower sliding port, 302e lower connecting trough, 302f outer lower rotating semi-sunk hole, 302g lower moving trough, 302h inner lower rotating semi-sunk hole, 303 sliding trough, 304 moving port, 305 collecting trough, 306 lower connecting hole, 400 solution collection assembly, 401 transmission screw, 402 collection tube, 402a collection part, 402b liquid inlet part, 402b-1 liquid inlet hole, 403 transmission belt, 404 rotating wheel, 405 baffle, 406 moving block, 406a push rod. DETAILED DESCRIPTION

[0044] Before describing the technical solution of the present invention, the terms used herein are defined as follows:

[0045] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0046] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0047] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0048] Example 1

[0049] Reference Figures 1 to 13 This is the first embodiment of the present invention. This embodiment provides a cascade Fermat spiral microfluidic mixer for cancer cell detection, which can realize simultaneous detection of multiple body fluids with high detection efficiency.

[0050] A cascade Fermat spiral microfluidic mixer for cancer cell detection includes an upper detection layer 200, the lower side of which is connected to a lower detection layer 300, a downward side of the upper detection layer 200 is provided with a plurality of spaced mixing channels 203, a downward side of the upper detection layer 200 at one end of the front mixing channel 203 is arranged with a plurality of first liquid storage tanks 204, a downward side of the upper detection layer 200 between two adjacent mixing channels 203 is provided with a plurality of second liquid storage tanks 202, the upper detection layer 200 is provided with a plurality of liquid inlet holes 201 corresponding to the first liquid storage tanks 204 and the second liquid storage tanks 202, and liquid storage pools are formed between the first liquid storage tanks 204 and the lower detection layer 300, and between the second liquid storage tanks 202 and the lower detection layer 300.

[0051] In order to further improve the uniformity of solution mixing, the mixing channel 203 is a Fermat spiral microchannel. The two front mixing channels 203 are of the same size, with a width of 400±10μm and a depth of 400±10μm. The width of the rear mixing channel 203 is 600±12μm and a depth of 400±10μm. The Fermat spiral channel parameter equation includes:

[0052] ;

[0053] Among them, t is the parameter of the Fermat spiral equation, the center of the Fermat spiral equation is the coordinate origin 0, x is the horizontal coordinate, and y is the vertical coordinate.

[0054] Furthermore, a solution collecting component 400 for collecting solution is provided in the lower detection layer 300 .

[0055] Furthermore, the solution in the second liquid storage tank 202 can flow controllably to the next mixing channel 203. An upper connecting hole 205 is opened on the upper detection layer 200 between the two adjacent mixing channels 203, and a lower connecting hole 306 corresponding to the upper connecting hole 205 is opened on the lower detection layer 300. The upper detection layer 200 and the lower detection layer 300 are respectively connected to the control valve 100 through the upper connecting hole 205 and the lower connecting hole 306. The control valve 100 controls the on-off between the two adjacent mixing channels 203 and the corresponding second liquid storage tank 202. The control valve 100 is provided with a first connecting channel 101 and a second connecting channel 102 from bottom to top. The first connecting channel 101 connects the two adjacent mixing channels 203. One end of the second connecting channel 102 can be aligned with the liquid outlet end of the second liquid storage tank 202, and the other two ends of the second connecting channel 102 are respectively aligned with the two adjacent mixing channels 203.

[0056] Furthermore, three first liquid storage tanks 204 are arranged on the downward side of the upper detection layer 200 at one end of the front mixing channel 203, a second liquid storage tank 202 is provided on the downward side of the upper detection layer 200 between the two front mixing channels 203, and a second liquid storage tank 202 is provided on the downward side of the upper detection layer 200 between the two rear mixing channels 203. The liquid storage tanks formed between the three front first liquid storage tanks 204 and the lower detection layer 300 are the first liquid storage tank, the second liquid storage tank and the third liquid storage tank respectively, the liquid storage tank formed between the middle second liquid storage tank 202 and the lower detection layer 300 is the fourth liquid storage tank, and the liquid storage tank formed between the end second liquid storage tank 202 and the lower detection layer 300 is the fifth liquid storage tank.

[0057] The present invention realizes the solution mixing reaction according to the experimental sequence by setting the control valve 100. First, the solutions in the first liquid reservoir, the second liquid reservoir and the third liquid reservoir are mixed in the first mixing channel 203. The first control valve 100 is controlled to be pressed down to connect the first mixing channel 203 and the second mixing channel 203. The solution after the first mixing enters the second mixing channel 203. The first control valve 100 is pressed down again, and the solution in the fourth liquid reservoir enters the second mixing channel 203 and mixes with the first three mixed solutions. After the mixture is uniform, the second control valve 100 is pressed down to connect the second mixing channel 203. The third mixing channel 203 is connected, and the four evenly mixed solutions enter the third mixing channel 203. The second control valve 100 is pressed down again to connect the fifth liquid reservoir and the third mixing channel 203. Cancer cells enter the third mixing channel 203 and react with the four evenly mixed solutions. The evenly mixed solutions in the third mixing channel 203 are driven by positive pressure to connect the peristaltic pump at the corresponding liquid inlet 201 and flow into the solution collection component 400, where the solutions are collected. The present invention is simple to operate. When detecting different cancer cells, it is only necessary to replace the cancer cells in the fifth liquid reservoir, which is convenient for detection and can detect multiple cancer cells.

[0058] Example 2

[0059] Reference Figure 14 , which is the second embodiment of the present invention. The difference from the second embodiment is that this embodiment provides a method for detecting cancer cells using a microfluidic mixer. This embodiment can realize the detection of cancer cells, is easy to operate, and has high detection efficiency.

[0060] The method for detecting cancer cells using a microfluidic mixer comprises the following steps:

[0061] 5mmol / L H2O2 solution was injected into the first reservoir, 500nmol / L Amplex Red was injected into the second reservoir, and 0.4μmol / L Cu was injected into the third reservoir. 2+ solution, injecting AS1411-AuNPs with a concentration of 0.3 mmol / L into the fourth reservoir, and injecting cancer cells into the fifth reservoir;

[0062] The solutions in the first liquid reservoir, the second liquid reservoir and the third liquid reservoir enter the first mixing channel 203, so that the H2O2 solution, Amplex Red and Cu 2+ The solutions are evenly mixed, producing obvious fluorescence, and the solution after the first mixing is ready to enter the second mixing channel 203;

[0063] The first control valve 100 is pressed down to connect the first mixing channel 203 with the second mixing channel 203, and the solution after the first mixing enters the second mixing channel 203. The first control valve 100 is further pressed down to connect the fourth liquid reservoir with the second mixing channel 203, and the AS1411-AuNPs in the fourth liquid reservoir are pressed into the second mixing microchannel, so that the AS1411-AuNPs are evenly mixed with the first three, resulting in obvious quenching. The solution after the second mixing is ready to enter the third mixing channel 203.

[0064] Press down the second control valve 100 to connect the second mixing channel 203 and the third mixing channel 203, and the solution after the second mixing enters the third mixing channel 203;

[0065] The second control valve 100 is further pressed down to connect the fifth liquid reservoir and the third mixing channel 203. The cancer cells in the fifth liquid reservoir are pressed into the third mixing channel 203, so that the cancer cells and the first four liquid reservoirs are evenly mixed, causing fluorescence recovery. The mixed solution enters the solution collection assembly 400.

[0066] The mixed solution collected in the solution collection component 400 is subjected to fluorescence detection, referring to Figure 14 The horizontal axis is the logarithmic function value corresponding to the cancer cell concentration, and the vertical axis is the fluorescence value. It can be seen from the figure that 10 2 ——10 7 Cells at a concentration of 10 cells / mL were added to the reservoir for measurement, and the fluorescence color development coefficient was as low as 0.2 and as high as 4, with a nonlinear correlation.

[0067] Among them, Amplex Red is a fluorescent red dye or 10-acetyl-3,7-dihydroxyphenazine, and AS1411-AuNPs are gold nanoparticles labeled with the aptamer AS1411.

[0068] Example 3

[0069] Reference Figure 15 , which is the third embodiment of the present invention. The difference from Example 1 and Example 2 is that this embodiment provides a method for detecting cancer cells using a microfluidic mixer. This embodiment can realize the detection of cancer cells, is easy to operate, and has high detection efficiency.

[0070] The method for detecting cancer cells using a microfluidic mixer comprises the following steps:

[0071] The first reservoir was injected with 10 mmol / L H2O2 solution, the second reservoir was injected with 0.5 μmol / L Amplex Red, and the third reservoir was injected with 2 μmol / L Cu. 2+ solution, injecting AS1411-AuNPs with a concentration of 2.4 mmol / L into the fourth reservoir, and injecting cancer cells into the fifth reservoir;

[0072] The solutions in the first liquid reservoir, the second liquid reservoir and the third liquid reservoir enter the first mixing channel 203, so that the H2O2 solution, Amplex Red and Cu 2+ The solutions are evenly mixed, producing obvious fluorescence, and the solution after the first mixing is ready to enter the second mixing channel 203;

[0073] The first control valve 100 is pressed down to connect the first mixing channel 203 with the second mixing channel 203, and the solution after the first mixing enters the second mixing channel 203. The first control valve 100 is further pressed down to connect the fourth liquid reservoir with the second mixing channel 203, and the AS1411-AuNPs in the fourth liquid reservoir are pressed into the second mixing microchannel, so that the AS1411-AuNPs are evenly mixed with the first three, resulting in obvious quenching. The solution after the second mixing is ready to enter the third mixing channel 203.

[0074] Press down the second control valve 100 to connect the second mixing channel 203 and the third mixing channel 203, and the solution after the second mixing enters the third mixing channel 203;

[0075] Continue to press down the second control valve 100 to connect the fifth liquid reservoir and the third mixing channel 203, press the cancer cells in the fifth liquid reservoir into the third mixing channel 203, and evenly mix the cancer cells with the first four, generating fluorescence recovery. The mixed solution enters the solution collection component 400.

[0076] The mixed solution collected in the solution collection component 400 is subjected to fluorescence detection, referring to Figure 15The horizontal axis is the logarithmic function value corresponding to the cancer cell concentration, and the vertical axis is the fluorescence value. It can be seen from the figure that 10 2 ——10 7 Cells at a concentration of 10 cells / mL were added to the reservoir for measurement, and the fluorescence color development coefficient was as low as 0.2 and as high as 3.5, with a nonlinear correlation.

[0077] Example 4

[0078] Reference Figure 17 , which is the fourth embodiment of the present invention. The difference from Examples 1 to 3 is that this embodiment provides a method for detecting cancer cells using a microfluidic mixer, and this embodiment can detect multiple cancer cells in a wide concentration range.

[0079] The method for detecting cancer cells using a microfluidic mixer comprises the following steps:

[0080] Take four prepared microfluidic mixers and inject H2O with a concentration of 1.2 mol / L into the first reservoir. 2 O 2 solution, Amplex Red with a concentration of 15µmol / L was injected into the second reservoir, and Cu with a concentration of 75µmol / L was injected into the third reservoir. 2+ solution, and 12.6 nmol / L AS1411-AuNPs were injected into the fourth reservoir. The volume of the above four solutions was 100 μL. Normal cells L-02, cancer cells HepG2, MCG-7, and HeLa cells were injected into the fifth reservoirs of the four microfluidic mixers respectively;

[0081] The solutions in the first liquid reservoir, the second liquid reservoir and the third liquid reservoir enter the first mixing channel 203, so that the H2O2 solution, Amplex Red and Cu 2+ The solutions are evenly mixed, producing obvious fluorescence, and the solution after the first mixing is ready to enter the second mixing channel 203;

[0082] The first control valve 100 is pressed down to connect the first mixing channel 203 with the second mixing channel 203, and the solution after the first mixing enters the second mixing channel 203. The first control valve 100 is further pressed down to connect the fourth liquid reservoir with the second mixing channel 203, and the AS1411-AuNPs in the fourth liquid reservoir are pressed into the second mixing microchannel, so that the AS1411-AuNPs are evenly mixed with the first three, resulting in obvious quenching. The solution after the second mixing is ready to enter the third mixing channel 203.

[0083] Press down the second control valve 100 to connect the second mixing channel 203 and the third mixing channel 203, and the solution after the second mixing enters the third mixing channel 203;

[0084] Continue to press down the second control valve 100 to connect the fifth liquid reservoir and the third mixing channel 203, press the cells in the fifth liquid reservoir into the third mixing channel 203, and evenly mix the cells with the first four. The mixed solution enters the solution collection component 400.

[0085] Detect the fluorescence phenomenon of the solution in the solution collection component 400, such as Figure 17 As shown, Figure 17 The horizontal axis in A is af, which is the concentration of MCF-7 cancer cells 0-106 cells / mL, and gk, which is the concentration of L-02 normal cells 10 2 -10 6 cells / mL, the vertical axis is the fluorescence value, Figure 17 As can be seen in A, the reaction system of the present invention can well distinguish normal cells from cancer cells; Figure 17 In B, the horizontal axis is the fluorescence value of different cells L-02, HepG2, and HeLa, and the vertical axis is the fluorescence value. Figure 17 As can be seen from Figure B, the reaction system of the present invention can well distinguish various cells; it can be seen that different cells and cancer cells have quite different results in a given reaction system and microfluidic chip, which shows that the present invention can achieve the distinction between different cells.

[0086] Example 5

[0087] Reference Figure 16 , which is the fifth embodiment of the present invention. The difference from Examples 1 to 4 is that this embodiment provides a method for detecting cancer cells using a microfluidic mixer, and this embodiment can detect multiple cancer cells in a wide concentration range.

[0088] The method for detecting cancer cells using a microfluidic mixer comprises the following steps:

[0089] Inject H with a concentration of 1.2 mol / L into the first reservoir. 2 O 2 solution, Amplex Red with a concentration of 15µmol / L was injected into the second reservoir, and Cu with a concentration of 75µmol / L was injected into the third reservoir. 2+ solution, 12.6 nmol / L AS1411-AuNPs were injected into the fourth reservoir, the volume of the above four solutions was 100 μL, MCF-7 cells were injected into the fifth reservoir, and the concentration of MCF-7 cells was changed by 10 2 -10 7 cells / mL;

[0090] The solutions in the first liquid reservoir, the second liquid reservoir and the third liquid reservoir enter the first mixing channel 203, so that the H2O2 solution, Amplex Red and Cu 2+ The solutions are evenly mixed, producing obvious fluorescence, and the solution after the first mixing is ready to enter the second mixing channel 203;

[0091] The first control valve 100 is pressed down to connect the first mixing channel 203 with the second mixing channel 203, and the solution after the first mixing enters the second mixing channel 203. The first control valve 100 is further pressed down to connect the fourth liquid reservoir with the second mixing channel 203, and the AS1411-AuNPs in the fourth liquid reservoir are pressed into the second mixing microchannel, so that the AS1411-AuNPs are evenly mixed with the first three, resulting in obvious quenching. The solution after the second mixing is ready to enter the third mixing channel 203.

[0092] Press down the second control valve 100 to connect the second mixing channel 203 and the third mixing channel 203, and the solution after the second mixing enters the third mixing channel 203;

[0093] Continue to press down the second control valve 100 to connect the fifth liquid reservoir and the third mixing channel 203, press the cells in the fifth liquid reservoir into the third mixing channel 203, and evenly mix the cells with the first four. The mixed solution enters the solution collection component 400.

[0094] Detect the fluorescence phenomenon of the solution in the solution collection component 400, such as Figure 16 As shown, Figure 16 The abscissa in A is the wavelength, and the ordinate is the fluorescence intensity; Figure 16 In Figure B, the horizontal axis is the logarithm of the cell concentration, and the vertical axis is the fluorescence intensity after removing the background value. It can be seen from the figure that the present invention can detect the range from 10 2 to 10 7 The detection limit is as low as 17 cells / mL. The present invention can realize the detection of a wide range of cell concentrations with high sensitivity. The change of fluorescence intensity is linearly correlated with the concentration of MCF-7.

[0095] Example 6

[0096] Referring to the figure, this is the sixth embodiment of the present invention. The difference from Examples 1 to 5 is that this embodiment provides a cascade Fermat spiral microfluidic mixer for cancer cell detection. This embodiment further realizes the collection of the solution after uniform mixing, making it convenient to take out the collected solution.

[0097] A cascade Fermat spiral microfluidic mixer for cancer cell detection, wherein a solution collection component 400 includes a collection tube 402, a lower detection layer 300 includes a first detection portion 301, a second detection portion 302 is connected to the lower side of the first detection portion 301, two lower connecting sinks 302e are provided on the upper side of the second detection portion 302 and are spaced apart in the left and right directions, two first support shafts 302c are provided in the lower connecting sinks 302e and are spaced apart in the length direction, a lower partition 302a is provided on the second detection portion 302 between the two lower connecting sinks 302e, an inner lower rotating semi-sunk hole 302h is provided on the upper side of the lower partition 302a, and the second detection portions 302a on the front and rear sides are provided with a plurality of lower connecting sinks. The part 302 is respectively provided with a lower collecting trough 302b and a lower moving trough 302g which are connected to the outside. The side of the second detection part 302 away from the lower moving trough 302g is provided with an outer lower rotating semi-sunk hole 302f which is coaxial with the lower rotating semi-sunk hole. The second detection part 302 is rotatably connected to the transmission screw 401 through the outer rotating semi-sunk hole and the inner lower rotating semi-sunk hole 302h. The transmission screw 401 in the lower moving trough 302g is threadedly connected to the moving block 406. The two first support shafts 302c in the lower connecting trough 302e are rotatably connected to the rotating wheels 404. A transmission belt 403 is connected between the two rotating wheels 404. The collection pipe 402 is just slidably connected in the lower collecting trough 302b.

[0098] Furthermore, two upper connecting grooves 301d are provided on the downward side of the first detection part 301, and two second support shafts 301b are provided in the length direction. The second support shaft 301b and the corresponding first support shaft 302c are coaxial. An upper partition 301g is provided on the first detection part 301 between the two upper connecting grooves 301d, and an inner upper rotating semi-sunk hole 301h is provided on the downward side of the upper partition 301g. The first detection part 301 on the front and rear sides of the upper partition 301g is respectively provided with an externally connected upper collecting groove 301f and an upper movable groove 301i, and the inner upper rotating semi-sunk hole 301h and the inner lower rotating semi-sunk hole 302h form a first circular hole. , the first detection part 301 is provided with an outer upper rotating semi-sunk hole 301a on the lower side away from one end of the upper collecting trough 301f, the outer lower rotating semi-sunk hole 302f and the outer upper rotating semi-sunk hole 301a form a second circular hole, and the upper connecting trough 301d is provided with two second support shafts 301b spaced apart in the length direction, and a moving port 304 is provided on the first detection part 301 on the upper side of the upper collecting trough 301f, and the collection pipe 402 includes a collecting part 402a and a liquid guide part 402b which are integrated, and the liquid guide part 402b is fixed on the upper side of the collecting part 402a, and a liquid guide hole 402b-1 is provided on the liquid guide part 402b, and the liquid guide hole 402b-1 is connected to the inner cavity of the collecting part 402a, and the upper partition 301g is away from the upper movable trough 30 The first detection part 301 at the left and right ends of one side of 1i is provided with an upper sliding port 301e connected to the upper connecting trough 301d on the downward side, and the second detection part 302 at the left and right ends of the lower partition 302a away from the lower movable trough 302g is provided with a lower sliding port 302d connected to the lower connecting trough 302e on the upward side. When the first detection part 301 is fixed on the second detection part 302, a collection trough 305 is formed between the upper collecting trough 301f, the lower collecting trough 302b and the lower detection layer 300, and the collecting part 402a can just slide along the collection trough 305. The upper end of the rotating wheel 404 is rotatably connected to the corresponding second support shaft 301b, and the upper sliding port 301e and the corresponding lower sliding port 302d form a sliding Slot 303, baffle 405 can just pass through the sliding slot 303 and fit on the outside of the collection tube 402, the inner end of the transmission screw 401 is rotatably connected to the first circular hole, and the outer end of the transmission screw 401 is rotatably connected to the lower detection layer 300 through the second circular hole. A baffle 405 is provided on one end of the transmission belt 403 facing the upper collection trough 301f. When the baffle 405 turns to the side of the transmission belt 403 away from the sliding slot 303, a space for the baffle 405 to pass through is left between the transmission belt 403 and the upper connecting trough 301d or the lower connecting trough 302e. The transmission belt 403 is connected to the moving block 406 on one side relative to the upper movable trough 301i. The moving block 406 is provided with two push rods 406a on the side facing the collection tube 402.The upper partition 301g has two through-holes 301c corresponding to the push rods 406a. The push rods 406a can slide along the corresponding through-holes 301c. In the initial state, the inner side of the collection tube 402 is attached to the side of the upper partition 301g away from the upper movable trough 301i. The push rods 406a are attached to the inner side of the collection tube 402, and the baffle 405 is attached to the outward side of the collection tube 402.

[0099] Furthermore, a liquid outlet channel 207 is provided on the upper detection layer 200 at the liquid outlet end of the mixing channel 203 at the end, and a drainage sink hole 206 is opened on the upper detection layer 200 at the end of the liquid outlet channel 207 away from the mixing channel 203. In the initial state, the drainage sink hole 206 is aligned with the liquid outlet hole 402b-1, and the liquid outlet hole 402b-1 covers the drainage sink hole 206.

[0100] A driving motor is installed on the outside of the lower detection layer 300, and the driving motor is connected to the transmission screw 401 on the outside of the lower detection layer 300, and the output shaft of the driving motor is plugged into the transmission screw 401 outside the lower detection layer 300; the solution mixed evenly through the third mixing channel 203 enters the collection tube 402 through the liquid outlet channel 207, the drainage sink hole 206 and the liquid drainage hole 402b-1 in sequence under positive pressure drive. After the solution collection is completed, the driving motor is activated, and the driving motor drives the transmission screw 401 to transmit, and the transmission screw 401 drives the transmission belt 403 to move. The transmission screw 401 drives the push rod 406a to push the collection tube 402 to move, and the transmission belt 403 drives the baffle 405 to move. The baffle 405 and the push rod 406a synchronously drive the collection tube 402 to slide outward. The setting of the baffle 405 ensures that the collection tube 402 moves outward stably. When the collection tube 402 slides close to the outside, the collection tube 402 can be taken out, which is convenient for further measurement of the cancer cell concentration.

[0101] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A cascade Fermat spiral microfluidic mixer for cancer cell detection, characterized by: These include, An upper detection layer (200), wherein three mixing channels (203) are provided on a downward side of the upper detection layer (200) at intervals, three first liquid storage tanks (204) are arranged on a downward side of the upper detection layer (200) at one end of the front mixing channel (203), a second liquid storage tank (202) is provided on a downward side of the upper detection layer (200) between the two front mixing channels (203), and a second liquid storage tank (202) is provided on a downward side of the upper detection layer (200) between the two rear mixing channels (203), and a plurality of liquid inlet holes (201) corresponding to the first liquid storage tanks (204) and the second liquid storage tanks (202) are opened on the upper detection layer (200), and the solution in the second liquid storage tank (202) can flow controllably to the next mixing channel (203); a lower detection layer (300), the lower detection layer (300) being connected to the lower side of the upper detection layer (200), and liquid reservoirs being formed between the first liquid storage tank (204) and the lower detection layer (300), and between the second liquid storage tank (202) and the lower detection layer (300); A solution collecting component (400), wherein the solution collecting component (400) is disposed in the lower detection layer (300); Inject H2O2 solution, Amplex Red and Cu into the first three liquid storage tanks (204) respectively. 2+ solution, and injecting AS1411-AuNPs into the second liquid storage tank (202) in the middle.

2. The cascade Fermat spiral microfluidic mixer for cancer cell detection according to claim 1, wherein: An upper connection hole (205) is formed on the upper detection layer (200) between two adjacent mixing channels (203), and a lower connection hole (306) corresponding to the upper connection hole (205) is formed on the lower detection layer (300). The upper detection layer (200) and the lower detection layer (300) are connected to a control valve (100) via the upper connection hole (205) and the lower connection hole (306), respectively. The control valve (100) controls the connection and disconnection between the two adjacent mixing channels (203) and the corresponding second liquid storage tank (202).

3. The cascade Fermat spiral microfluidic mixer for cancer cell detection according to claim 2, wherein: The control valve (100) is provided with a first connecting channel (101) and a second connecting channel (102) spaced apart from each other from bottom to top. The first connecting channel (101) connects two adjacent mixing channels (203). One end of the second connecting channel (102) is aligned with the liquid outlet end of the second liquid storage tank (202), and the other two ends of the second connecting channel (102) are aligned with the two adjacent mixing channels (203).

4. The cascade Fermat spiral microfluidic mixer for cancer cell detection according to claim 1 or 2, characterized in that: The mixing channel (203) is a Fermat spiral microchannel. The two front mixing channels (203) are of the same size, with a width of 400±10μm and a depth of 400±10μm. The rear mixing channel (203) has a width of 600±12μm and a depth of 400±10μm.

5. The cascade Fermat spiral microfluidic mixer for cancer cell detection according to claim 1 or 2, characterized in that: The concentration of the H2O2 solution is 5 mmol / L to 10 mmol / L, the concentration of Amplex Red is 500 nmol / L to 0.5 μmol / L, and the Cu 2+ The concentration of the solution is 0.4 μmol / L~2 μmol / L, and the concentration of AS1411-AuNPs is 0.3 mmol / L~2.4 mmol / L.

6. The cascade Fermat spiral microfluidic mixer for cancer cell detection according to claim 5, characterized in that: The concentration of the H2O2 solution was 8 mmol / L, the concentration of Amplex Red was 0.1 μmol / L, the concentration of the Cu2+ solution was 0.5 μmol / L, and the concentration of AS1411-AuNPs was 1.26 mmol / L.

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