Patent Publication Number: US-10766031-B2

Title: Microfluidics based analyzer and method for fluid control thereof

Description:
TECHNICAL FIELD 
     At least one embodiment of the present disclosure provides a microfluidics based analyzer and a method for fluid control thereof, more particularly to a microfluidics based analyzer having a microfluidic flow controlling design and an operational method thereof. 
     DESCRIPTION OF THE RELATED ART 
     In the conventional analysis methods, the enzyme-linked immunosorbent assay (ELISA) has been widely adopted in medicine, pharmacy, biotechnology, food industry, and environmental testing due to the attributes, such as high specificity, fast, sensitive, low costs, and capable of performing tests simultaneously on a large number of sample. 
     In the conventional ELISA, the operations are mainly performed on the 96-well microtiters plate. The operations may include an incubation process, a cleaning process, a coloring reaction process, and a detection process. It may take 4 to 6 hours for users to finish all the processes. During each of the processes, the users require to use a large amount of cleaning solution to dilute the residual reagent and drain the reaction chamber after adding the reagent for reaction, so as to reduce detection errors caused by contamination of the reagents in the previous and subsequent steps. For testing a large number of samples, the tedious and highly repeatable steps and actions described above may place a heavy burden on the users and may cause human errors. 
     To solve the above problem, James Lee et al. proposed the concept of enzyme-linked immunoassay (CD ELISA) on a microfluidic disc platform in early 2000. The CD ELISA may control the processes and steps of ELISA by controlling the rotation speed of the microfluidic disk platform. The users only need to inject the reagents required in each step into each temporary storage chamber on the microfluidic disk, and then select different rotation speeds to release different reagents in sequence, so as to automatically perform the processes, such as the incubation process, the cleaning process, the coloring reaction process, and the detection process of the ELISA. In addition, in the microfluidic system, the reagent volume requirement is small and the surface area of the reaction is large, thus the process of the ELISA may be accelerated. As such, the detection time of the CD ELISA can be shortened within 1 to 2 hours. 
     However, the CD ELISA has defects. During the step of injecting cleaning solution into the mixing chamber to replace the liquid in the reaction chamber, the cleaning solution may be mixed in the mixing chamber, causing residual reagents in some reaction chambers. Therefore, the cleaning step requires a large volume of cleaning solution and the mixing chamber requires to be rinsed several times to reduce the amount of the remaining reagent and to eliminate the influence resulting from the residual reagents on the detection signals. Moreover, the available space on the microfluidic disc is limited. If the cleaning solution occupies too much space, it may reduce the total number of single-chip inspections and reduce the economic benefits. 
     Therefore, a microfluidic design capable of improving the cleaning efficiency and reducing the storage space of the cleaning solution is provided. The microfluidic design may increase detection sensitivity, and increase the number of detections on the disc. 
     SUMMARY 
     The present disclosure provides a microfluidics based analyzer and a method for fluid control thereof, having a simple operational process and a high cleaning efficiency. Specifically, adopting the microfluidic disc of the present disclosure, the residual liquid in the reaction chamber may be effectively drained, thereby improving the cleaning efficiency and reducing the amount of the cleaning solution. In addition, adopting the method of the liquid flow control through the rotational speed, the reagent may be controlled by the rotational speed, so as to perform an incubation process and a cleaning process. In some examples, the method may only require to control the two stages of the motor, i.e., the high rotational speed and the low rotational speed, to complete all of the inspection steps. 
     In one aspect, the present disclosure relates to a microfluidic-based analyzer, including: a drive module; a microfluidic disc detachably configured on the drive module, wherein the microfluidic disc includes: at least one injection chamber; at least one microfluidic structure, including: a mixing chamber connecting to the at least one injection chamber; a waste chamber; and a capillary, including: a first access connected to the mixing chamber, wherein the first access is configured on a first radius; a second access connected to the waste chamber, wherein the second access is configured on a second radius; and a turning section connected to the first access and the second access, wherein the turning section is configured on a third radius; wherein the first radius is less than the second radius, and the third radius is less than the first radius. 
     In another aspect, the present disclosure relates to a microfluidic controlling method of a microfluidic-based analyzer, including: providing the microfluidic-based analyzer described in above; injecting a liquid into the microfluidic structure; operating the drive module at a high rotational speed to control the liquid to flow into the mixing chamber, wherein a rotational speed of the drive module comprises a critical rotational speed, a first rotational speed, and a second rotational speed, the first rotational speed is less than the critical rotational speed, and the second rotational speed is greater than the critical rotational speed; operating the drive module at a low rotational speed, wherein the drive module rotates at the first rotational speed and controls the liquid to flow into the second access by a capillary phenomenon; and operating the drive module at the high rotational speed, wherein the drive module rotates at the second rotational speed, the drive module controls the liquid to penetrate the second access and to enter the waste chamber until the liquid in the mixing chamber is completely drained. 
     In one example, the rotational speed of the drive module is greater than the critical rotational speed. The drive module only requires a two-stage rotational speed, one is greater than the critical rotational speed of the second access, and the other one is less than the critical rotational speed of the second access. 
     In one example, the rotational speed of the drive module is switched to selectively retain the agent in the mixing chamber or to drain the agent to the waste chamber. 
     In one example, adopting the microfluidic disc, the residual liquid in the reaction chamber may be effectively drained, thereby improving the cleaning efficiency and reducing the amount of the cleaning solution. As such, the microfluidic-based analyzer may maintain an accuracy without spending a large amount of the cleaning fluid. 
     The method for fluid control has a simple operational process. In addition to biochemical testing and medical testing, the method can also be used in areas such as chemical testing, water quality testing, environmental testing, food testing, and defense industries. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1A  is a schematic view illustrating a microfluidic-based analyzer in accordance with one embodiment of the present disclosure. 
         FIG. 1B  is a schematic view illustrating a microfluidic-based analyzer in accordance with one embodiment of the present disclosure. 
         FIG. 2  is a schematic view illustrating a microfluidic disc in accordance with one embodiment of the present disclosure. 
         FIG. 3  is a schematic view illustrating a microfluidic structure in accordance with one embodiment of the present disclosure. 
         FIG. 4  is a flowchart illustrating a method of operating a microfluidic-based analyzer in accordance with one embodiment of the present disclosure. 
         FIG. 5  is a schematic view illustrating a microfluidic structure in accordance with one embodiment of the present disclosure. 
         FIG. 6A  to  FIG. 6F  are diagrams illustrating a method of operating a microfluidic-based analyzer in accordance with one embodiment of the present disclosure. 
         FIG. 7  is a diagram illustrating a method of operating a microfluidic-based analyzer in accordance with one embodiment of the present disclosure. 
         FIG. 8A  to  FIG. 8G  are diagrams illustrating a method of operating a microfluidic-based analyzer in accordance with another embodiment of the present disclosure. 
     
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     At least one embodiment of the present disclosure provides a microfluidics based analyzer and a method for fluid control thereof, more particularly to a microfluidics-based analyzer having a microfluidic flow control design and an operational method thereof. 
       FIG. 1A  is a schematic view illustrating a microfluidic-based analyzer in accordance with one embodiment of the present disclosure and  FIG. 1B  is a schematic view illustrating a microfluidic-based analyzer in accordance with one embodiment of the present disclosure. The microfluidic-based analyzer may include a drive module  10  and a microfluidic disc  20 . The drive module  10  is configured to drive and control the microfluidic disc  20  to rotate. The microfluidic disc  20  is detachably configured on the drive module  10 . The microfluidic disc  20  has a rotating center  21  and a rim  22 . A variety of tests may be conducted on the microfluidic disc  20 . As shown in  FIG. 1B , the microfluidic disc  20  may include at least one microfluidic structure  50 . 
     In one example, as shown in  FIG. 1A , the drive module  10  may be a centrifuge or a rotary motor. When drive module  10  is operating, the microfluidic disc  20  may be controlled to rotate. The microfluidic disc  20  may be a symmetrical disc of circle, square, or polygonal. The microfluidic disc  20  may be made of polyethylene, polyvinyl alcohol, polypropylene, polystyrene, polycarbonate, polymethyl methacrylate, polydimethylsiloxane, silicon dioxide, or a combination thereof. 
     As shown in  FIGS. 1A and 1B , the microfluidic-based analyzer may further include a detector  30 . The detector  30  connects to the drive module  10 . The drive module  10  is configured to control the microfluidic disc to rotate in accordance with results detected by the microfluidic-based analyzer. For example, the detector  30  may be a spectrophotometer, a colorimeter, a turbidimeter, thermometer, a pH meter, an ohmmeter, a colonometer, an image sensor, or a combination thereof. 
     Referring to  FIG. 2 ,  FIG. 2  is a schematic view illustrating a microfluidic disc in accordance with one embodiment of the present disclosure. The microfluidic disc  20  may include an injection chamber  40  and a plurality of the microfluidic structures  50 . The injection chamber  40  is configured on the rotating center of the microfluidic disc  20 , and the injection chamber  40  connects to the other elements of the microfluidic structures  50  via individual microfluidic valves  570  of each of the microfluidic structures  50 . When liquid is injected into the injection chamber  40 , one single liquid may be dispensed into the microfluidic structure  50 , and multiple tests may be performed simultaneously. Specifically, after the liquid enters the microfluidic structures  50 , the liquid may flow into the microfluidic valve  570 , a mixing chamber  520 , a capillary  540 , and a waste chamber  530  in sequence. The microfluidic structures  50  may include a plurality of ventilation holes  42  configured to reduce resistance resulting from air pressure when the liquid moves in the microfluidic structure  50 . For example, the ventilation hole  42  may be configured on a temporary storage  510 , the mixing chamber  520 , and waste chamber  530 . The temporary storage chamber  510  may be arranged according to different detection requirements and is configured to temporarily store other reagents to be injected to the mixing chamber  520 . It is noted that not all of the embodiments of the present disclosure require the arrangement of the temporary storage chamber  510 . As shown in  FIG. 2 , a height of the ventilation hole  42  of the waste chamber  530  extending toward the rotating center of the microfluidic disc  20  is higher than a position of an overflow channel  550 . That is, the ventilation hole  42  of the waste chamber  530  is closer to the rotating center of the microfluidic disc  20  than the overflow channel  550 . 
     In one example, as shown in  FIG. 2 , the microfluidic disc  20  may include a plurality of independent microfluidic structures  50 . Each of the microfluidic structures  50  connects to one or more of the injection chambers  40 . As such, the different liquids may be injected into each of the microfluidic structures  50 , and the same test or the different tests may be performed (referring to  FIG. 8A to 8G ). In another example, the microfluidic structures  50  may be designed as a group. For example, eight of the microfluidic structures  50  on the microfluidic disc  20 , which is depending on the requirement, may be designed to be as each two of the microfluidic structures  50  share one injection chamber  40 , and each of the injection chambers  40  includes a splitter configured to equally distribute the liquid. The splitter may be of a triangle or a petaloid-shaped. As such, four pairs of the microfluidic structures  50  may be formed on the microfluidic disc  20 . When the liquid is injected into one of the injection chambers  40 , the liquid may flow through the splitter of the injection chamber  40 , and the liquid may be equally distributed. The equally distributed liquid may flow into the two microfluidic structures  50 , and two different tests may be performed simultaneously. 
     As shown in  FIG. 2 , one kind of the liquid, such as sample, buffer solution, wash buffer, reagent, and solvent, may be injected into the injection chamber  40 . In one example, the injected liquid may be magnetic bead solution, which may include at least one magnetic bead, which is in a stationary phase, and solution, which is in a flowing phase. In another example, the injected liquid may be a color development reagent, which may only include the solution which is in the flowing phase. 
     The microfluidic valve  570  shown in  FIG. 2  is configured to prevent the solution from flowing into the mixing chamber  520  in advance at predetermined situations. For example, when the drive module  10 , as shown in  FIG. 1B , of the microfluidic-based analyzer is operating, the liquid may stay at the microfluidic valve due to confrontation between surface tension and centrifugal force. If the rotational speed of the drive module  10  is increased and the centrifugal force is greater than the surface tension of the liquid, the liquid may flow through the microfluidic valve  570  and flow into the mixing chamber  520 . 
       FIG. 3  is a schematic view illustrating a microfluidic structure in accordance with one embodiment of the present disclosure. The microfluidic structure  50  shown in  FIG. 3  may include the mixing chamber  520 , a capillary  540 ′, a waste chamber  530 ′, and the overflow channel  550 . A width of the capillary  540 ′ is less than a width of the overflow channel  550 . As shown in  FIG. 3 , the overflow channel  550  is configured to quantify the liquid. A level of liquid surface in the mixing chamber  520  may be controlled by the centrifugal force, and the level of the liquid surface in the capillary  540 ′ and the mixing chamber  520 , resulting from a connected tube effect when a gravity force simulated by the centrifugal force is conducted, may be controlled, so as to quantify the liquid. 
     A connection portion of the capillary  540 ′ and the mixing chamber  520  is configured to be as a first access  541 . A connection portion of the capillary  540 ′ and the waste chamber  530 ′ is configured to be as a second access  543 . The capillary may include a turning section  545  configured between the first access  541  and the second access  543 . A connection portion of the overflow channel  550  and the mixing chamber  520  is configured to be as a third access  551 . A connection portion of the overflow channel  550  and the waste chamber  530 ′ is configured to be as a fourth access  553 . 
     The microfluidic structure shown in  FIG. 3  is configured on the circular microfluidic disc  20  shown in  FIG. 1A . Referring to  FIG. 3 , a first radius R 1 , a second radius R 2 , a third radius R 3 , and a fourth radius R 4  are based on a starting point from the rotating center  21  of the microfluidic disc  20 . The first access  541  is configured on the first radius R 1 . The second access  543  is configured on the second radius R 2 . The turning section  545  is configured on the third radius R 3 . The third access  551  is configured on the fourth radius R 4 . The fourth access  553  is configured on the second radius R 2 . 
     A difference between the first radius R 1  and the second radius R 2  may affect a value of a critical rotational speed ω c . The critical rotational speed ω c  is generated by the drive module  10 , and is configured to rotate the microfluidic disc  20 . The critical rotational speed ω c  may determine a threshold value of the surface tension that the liquid temporarily stored in the capillary  540 ′ may break before the liquid flows into the waste chamber  530 ′. 
     To understand the operational principle of the critical rotational speed ω c , the detail will be described in below accompanying with  FIG. 4 ,  FIG. 5 , and  FIG. 6A  to  FIG. 6F . 
       FIG. 4  is a flowchart illustrating a method of operating a microfluidic-based analyzer in accordance with one embodiment of the present disclosure. The method includes: providing the microfluidic-based analyzer shown in  FIG. 2 ; injecting the liquid into the microfluidic structure, operating the drive module at a high rotational speed to control the liquid to flow into the mixing chamber, wherein a rotational speed of the drive module includes a critical rotational speed ω c , a first rotational speed, and a second rotational speed, the first rotational speed is less than the critical rotational speed ω c , and the second rotational speed is greater than the critical rotational speed ω c ; operating the drive module at a low rotational speed, wherein the drive module rotates at the first rotational speed and controls the liquid to flow into the second access by a capillary phenomenon; and operating the drive module at the high rotational speed, wherein the drive module rotates at the second rotational speed, and drive module controls the liquid to flow through the second access and to enter the waste chamber until the liquid in the mixing chamber is completely drained. 
     In one example, the second rotational speed may include a plurality of driving rotation speeds. The driving rotation speeds are all greater than the critical rotational speed ω c  shown in  FIG. 8A  to  FIG. 8G . Similarly, the first rotation speed may arbitrarily be changed according to the embodiment and detection content, and the present disclosure is not limited thereto. 
     Referring to  FIG. 5 ,  FIG. 5  is a schematic view illustrating a microfluidic structure in accordance with one embodiment of the present disclosure. The microfluidic structure may include a mixing chamber  520 ′, the capillary  540 ′, and a waste chamber  530 ″. Two ends of the capillary  540 ′ respectively connect to the mixing chamber  520 ′ and the waste chamber  530 ″.  FIG. 5  illustrates a portion of the microfluidic structure shown in  FIG. 1B . Configuration of the mixing chamber  520 ′, the capillary  540 ′, and the waste chamber  530 ″ is similar to configuration of the mixing chamber  520 , the capillary  540 , and the waste chamber  530 ′ shown on  FIG. 3 . In addition, the mixing chamber  520  shown in  FIG. 5  may further connect to other elements of the microfluidic disc  20 . 
       FIG. 6A  to  FIG. 6F  are diagrams illustrating a method of operating a microfluidic-based analyzer in accordance with one embodiment of the present disclosure.  FIG. 6A  to  FIG. 6F  illustrate operational process of the microfluidic structure, and motions and distribution of the liquid inside the microfluidic structure. When first liquid  60  is injected into the microstructure shown in  FIG. 5  by a high centrifugal force, the distribution of the liquid is shown in  FIG. 6A . Comparing the structures shown in  FIG. 6A  and  FIG. 3 , it can be seen that both of the structures include the capillary  540 ′, the turning section  545 , the first radius R 1 , and the second radius R 2 . The differences between the structures shown in  FIG. 6A  and  FIG. 3  reside in that the structure shown in  FIG. 3  includes the overflow channel  550 . For the experiments that have been conducted by a liquid quantification process, the overflow channel  550  is a selective structure configured to cooperate with the injection chamber and the centrifugal force. 
     In one example, as shown in  FIG. 6A , the first liquid  60  may include the stationary phase  61  and the flowing phase  63 . The stationary phase may be magnetic beads, and the flowing phase may be the solution. The flowing phase  63  in the mixing chamber  520 ′ and the capillary  540 ′ may have the same level due to the connected tube effect resulted from the gravity force simulated by the centrifugal force. The turning section  545  of the capillary  540 ′ configured on the third radius R 3  is configured to form the connected tube effect. 
     As shown in  FIG. 6B , when the drive module decelerates the rotational speed, the centrifugal force may be reduced, and the flowing phase  63  of the first liquid  60  may flow into and fill up with the capillary  540 ′ due to the capillary phenomenon. That is, a capillary force is greater than the gravity force simulated by the centrifugal force. The first liquid  60  may stay at an intersection between the capillary  540 ′ and the waste chamber  530 ′ due to the surface tension, i.e., the first liquid  60  may stay at a position of the second access  543  shown in  FIG. 3 . 
     Referring to  FIG. 6C , the drive module accelerates the rotational speed again to break the surface tension at the second access  543  by the centrifugal force. The flowing phase  63  in the capillary  540 ′ may flow into the waste chamber  530 ″. The centrifugal force of the flowing phase  63  at the second access  543  shown in  FIG. 3  may be obtained by the formula below.
 
Δ P   c =ρω 2   ΔR R     (1)
 
     The centrifugal force of the flowing phase  63 , which is configured to break the surface tension at the second access  543  and is obtained by the formula above, is the centrifugal force that must be capable of breaking the surface tension. In other words, not all embodiments require such great centrifugal force to break the surface tension. 
     In the formula (1), “ρ” indicates a liquid density of the flowing phase  63 . “ω” indicates the rotational speed. “ΔR” indicates a height difference of the first radius R 1  and the second radius R 2 . “ R ” indicates an average radius of the capillary  540 ′. “ΔR” is defined as the height difference of the first radius R 1  and the second radius R 2  due to the gravity force is simulated by the centrifugal force. In one example, the height difference indicates a radius difference between the first radius R 1  and the second radius R 2  based on the starting point from the rotating center of the microfluidic disc  20 . 
     Therefore, when the flowing phase  63  in the capillary  540 ′ breaks the surface tension and flows into the waste chamber  530 ″ by the gravity force simulated by the centrifugal force, the flowing phase  63  in the mixing chamber  520 ′ may be controlled to flow into the waste chamber  530 ″ continuously until the first liquid  63  in the mixing chamber  520 ′ and the capillary  540 ′ is completely drained to the waste chamber  530 ″ by a stress, such as a siphon effect. 
     A pressure difference of the surface tension of the flowing phase  63  may be obtained by the formula below. 
     
       
         
           
             
               
                 
                   
                     Δ 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     
                       P 
                       s 
                     
                   
                   = 
                   
                     
                       C 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       γ 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       sin 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       θ 
                     
                     A 
                   
                 
               
               
                 
                   ( 
                   2 
                   ) 
                 
               
             
           
         
       
     
     In formula (2), “C” indicates a surface tension constant which may be adjusted according to different flowing phases  63 . “γ” indicates the surface tension. “θ” indicates a contact angle of the flowing phase resulting from the liquid surface bended by the surface tension at the second access  543 . “A” indicates a cross-sectional area of the second access  543 . Therefore, according to the formula (1) and formula (2), a formula of the critical rotational speed ω c  may be obtained by the formula as below. 
     
       
         
           
             
               
                 
                   
                     ω 
                     c 
                   
                   = 
                   
                     60 
                     ⁢ 
                     
                       
                         ( 
                         
                           
                             γ 
                             ⁢ 
                             
                                 
                             
                             ⁢ 
                             sin 
                             ⁢ 
                             
                                 
                             
                             ⁢ 
                             θ 
                           
                           
                             
                               π 
                               2 
                             
                             ⁢ 
                             
                               d 
                               H 
                             
                             ⁢ 
                             ρΔ 
                             ⁢ 
                             
                                 
                             
                             ⁢ 
                             R 
                             ⁢ 
                             
                               R 
                               _ 
                             
                           
                         
                         ) 
                       
                       0.5 
                     
                   
                 
               
               
                 
                   ( 
                   3 
                   ) 
                 
               
             
           
         
       
     
     In formula (3), “d H ” may change according to a height and a width of the second access  543 , and “d H ” may be obtained by the formula below. 
     
       
         
           
             
               
                 
                   
                     d 
                     H 
                   
                   = 
                   
                     
                       2 
                       ⁢ 
                       
                           
                       
                       ⁢ 
                       WH 
                     
                     
                       ( 
                       
                         W 
                         + 
                         H 
                       
                       ) 
                     
                   
                 
               
               
                 
                   ( 
                   4 
                   ) 
                 
               
             
           
         
       
     
     In formula (4), “W” indicates the width of the second access  543 . “H” indicates the height, which is a parameter to form an interface of liquid and gas. 
     As shown in  FIG. 6D , a second liquid  65  is injected into the mixing chamber  520 ′. Similar to the first liquid  60 , the second liquid  65  in the mixing chamber  520 ′ and the capillary  540  may have the same level when being conducted by the high centrifugal force. As shown in  FIG. 6E , when the drive module decelerates the rotational speed, the centrifugal force may be reduced, and the second liquid  65  may flow into and fill up with the capillary  540 ′. The second liquid  65  may stay at the second access due to the surface tension. 
     As shown in  FIG. 6F , when the drive module accelerates the rotational speed again, the surface tension at the second access may be broken by the high centrifugal force, and the second liquid  65  in the capillary  540 ′ may flow into the waste chamber  530 ″. The second liquid  65  in the mixing chamber  520 ′ may continuously be drained to the waste chamber  530 ″ by the syphon effect until the second liquid  65  in the mixing chamber  520 ′ and the capillary  540 ′ is completely empty. In the above process, the stationary phase  61  may be retained in the mixing chamber  520 ′ by an external force. 
     The rotational speed of the drive module  10  shown in  FIG. 6C  and  FIG. 6F  is greater than the critical rotational speed ω c . Such that the flowing phase  63  may break the surface tension and flow into the waste chamber  530 ″. In one example, a wall of the capillary  540 ′ may be made of polymethyl methacrylate (PMMA) and an oxygen plasma may be conducted on a portion of the PMMA to perform a surface hydrophilic treatment. 
       FIG. 7  is a diagram illustrating a method of operating a microfluidic-based analyzer in accordance with one embodiment of the present disclosure. The microfluidic structures shown in  FIG. 6C  and  FIG. 7  are similar. The difference between the microfluidic structures shown in  FIG. 6C  and  FIG. 7  resides in that the rotational speed of the drive module shown in  FIG. 7  is less than the critical rotational speed ω. As shown in  FIG. 7 , the rotational speed of the drive module is less than the critical rotational speed ω, and the pressure difference resulting from the centrifugal force is too small to completely drain the flowing phase  63  in the mixing chamber  520 ′. Thus, the flowing phase  63  may fill up with the capillary  540  due to the capillary phenomenon. After the second liquid  63  flows into the mixing chamber  520 ′ in the following steps, the second liquid  65  may be drained to the waste chamber  530 ″ since the second liquid  65  is in contact with the flowing phase  63 , and the second liquid  65  may not be retained in the mixing chamber  520 ′. 
     At least one embodiment of the present disclosure adopts the microfluidic-based analyzer shown in  FIG. 1A  to cooperate with the microfluidic disc shown in  FIG. 2  to perform the enzyme-linked immunosorbent assay (ELISA). First, 1 μl of the magnetic bead solution, 10 μl of detection antibody, and 20 μl of antigen are injected into the mixing chamber  520  of the microfluidic disc  20 . The microfluidic disc  20  is configured on the drive module  10 , and the drive module  10  is activated to accelerate the rotational speed to 4000 revolutions per minute (RPM). When the magnetic bead solution, the detection antibody, and the antigen are mixed to form the first liquid, the drive module may decelerate the rotational speed to 10 RPM and maintain the rotational speed for 30 minutes. As such, the magnetic bead solution, the detection antibody, and the antigen may fully react and form a bond. Due to the centrifugal force is not enough to simulate the gravity force and inhibit the capillary phenomenon, the flowing phase of the first liquid may flow into the capillary  540  by the capillary force. After the reaction is completed, the drive module  10  may accelerate the rotational speed to 4000 RPM again. At the high rotational speeds, which are continuously maintained without interruption, the flowing phase in the mixing chamber  520  may be drained to the waste chamber  530  due to the pressure difference resulting from the gravity force simulated by the centrifugal force, and only the stationary phase, such as the magnetic beads, may stay in the mixing chamber  520 . When it is determined that the flowing phase in the mixing chamber  520  is completely drained, 320 μl of the wash buffer may be injected into the injection chamber  40 , and the drive module  10  may be activated again to accelerate the rotational speed to 4000 RPM. In this step, injecting the wash buffer after determining the flowing phase in the mixing chamber  520  is completely drained is to prevent the wash buffer from contacting with the flowing phase and being drained to the waste chamber  530  before the mixing chamber is cleaned. The wash buffer may be distributed to each of the mixing chamber  520  from each of the microfluidic structures. After the wash buffer has been distributed, the drive module  10  decelerates the rotational speed to 10 RPM to clean the stationary phase in the mixing chamber  520 . A portion of the wash buffer may flow into the capillary  540  due to the centrifugal force is not enough to inhibit the capillary phenomenon. 
     After cleaning the stationary phase in the mixing chamber  520 , the drive module  10  may accelerate the rotational speed again to 4000 RPM. The wash buffer in the mixing chamber  520  may be controlled to drain to the waste chamber  530  by the pressure difference resulting from the centrifugal force, and only the stationary phase, such as the magnetic beans, may stay in the mixing chamber  520 . Then, 48 μl of the color development reagent may be injected into the injection chamber  520 , and the drive module  10  may be activated to accelerate the rotational speed to 4000 RPM. In this step, the color development reagent may be distributed to each of the mixing chamber  520  from each of the microfluidic structure  50 . After the color development reagent is distributed, the drive module  10  decelerates the rotational speed to 10 RPM and maintains the rotational speed for 15 minutes. As such, the color development reagent may fully react with the stationary phase in the mixing chamber  520 . Reaction results may be detected after the coloring process is completed. 
     Referring to  FIG. 8A  to  FIG. 8G ,  FIG. 8A  to  FIG. 8G  are diagrams illustrating a method of operating a microfluidic-based analyzer in accordance with another embodiment of the present disclosure. In one example, the test described in the present disclosure may adopt an enzyme-linked immune sorbent assay. As shown in  FIG. 8A , the mixing chamber  520  in configured to connect to three injection chambers  40   a ,  40   b , and  40   c . The injection chamber  40   b  and the injection chamber  40   c  respectively connect to the mixing chamber  520  via the arrow-shaped microfluidic valves  570 . In one example, the injection chambers  40   a ,  40   b , and  40   c  may respectively include an injection hole  41   a ,  41   b , and  41   c  in sequence. In another example, the microfluidic valve  570  may be of spherical or beaded-shaped, and the present disclosure is not limited thereto. 
     As shown in  FIG. 8B , the stationary phase  61  and a flowing phase  63   a  are injected into the injection hole  41   a . In one example, the stationary phase  61  may be 1 μl of the magnetic bead having a surface with capture antibodies, and the flowing phase  63   a  may be a solution of 10 μl of the detection antibodies and 20 μl of the antigens. A flowing phase  63   b  and a flowing phase  63   c  are injected into the injection holes  41   b  and  41   c  in sequence. For example, the flowing phase  63   b  may be 40 μl of the wash buffer, and the flowing phase  63   c  may be 10 μl of the color development reagent. 
     In one example, the critical rotational speed w may be 850 RPM. After the microfluidic disc  20  is configured on the drive module  10  and the drive module  10  is activated to accelerate to the second rotational speed, i.e., 1000 RPM, the connected tube effect may be generated on the flowing phase  63   a  due to the gravity force simulated by the centrifugal force causing by the second rotational speed. 
     The first rotational speed, which is less than the critical rotational speed ω, is maintained for 30 minutes. As such, the stationary phase  61  and the flowing phase  63   a  may be fully mixed and bonded. The flowing phase  63   a  may fill up with the capillary  540 . After the reaction is completed, the rotational speed may be adjusted to the second rotational speed, i.e., 1000 RPM, to generate the syphon effect on the flowing phase  63   a  of the capillary  540  by the gravity force simulated by the centrifugal force. As shown in  FIG. 8D , the flowing phase  63   a  in the mixing chamber  520  may be completely drained to the waste chamber  530   a.    
     As shown in  FIG. 8E , after the flowing phase  63   a  in the mixing chamber  520  is completely drained, the microfluidic disc  20  may be accelerated to an another second rotational speed, i.e., 2000 RPM, and the flowing phase  63   b  in the injection chamber  40   b  may flow through the microfluidic valve  570  and flow into the mixing chamber  520 . As shown in  FIG. 8A , when the mixing chamber  520  is fully filled up, the overflow channel  550  is configured to perform a quantification process on the flowing phase  63   b , i.e., the wash buffer. A remaining flowing phase  63   b  may flow into a waste chamber  530   b . In another example, the waste chamber  530   a  and the waste chamber  530   b  may be a connected structure, and the present disclosure is not limited thereto. 
     After the flowing phase  63   b  is quantified, the drive module maintains the first rotational speed, Such that the capillary  540  may be filled up with the flowing phase  63   b  by the capillary force. After cleaning the mixing chamber  520 , the rotational speed may be accelerated to the second rotational speed, i.e., 1000 RPM, again. As shown in  FIG. 8F , the flowing phase  63   b  may be completely drained to the waste chamber  530   a.    
     After the flowing phase  63   b  is completely drained to the waste chamber  530   a , the drive module may accelerate the rotational speed to a highest second rotational speed, i.e., 3000 RPM. As shown in  FIG. 8G , the flowing phase  63   c  in the injection chamber  40   c  may flow through the microfluidic valve  570  and flow into the mixing chamber  520 . After 15 minutes of reaction, due to the flowing phase  63   c  is the color development reagent, the detection module  30  may detect the reaction results. 
     The above description is merely the embodiments in the present disclosure, the claim is not limited to the description thereby. The equivalent structure or changing of the process of the content of the description and the figures, or to implement to other technical field directly or indirectly should be included in the claim. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present disclosure without creative efforts shall fall within the protection scope of the present disclosure.