Centrifugal microfluidic control systems and method for configuring the same

The disclosure provides a centrifugal microfluidics control system and a method configuring the same. The system may comprise a centrifugal tube; a centrifugal unit for accommodating the centrifugal tube and providing a centrifugal force to the centrifugal tube; a control unit fixed in bottom of the centrifugal tube; and a microfluidic supporting unit coupled to the control unit in the centrifugal tube. The control unit may change an orientation of the microfluidic supporting unit to change a direction of the centrifugal force applied to the microfluidic supporting unit.

TECHNICAL FIELD

The present disclosure generally relates to a centrifugal microfluidic control system, more particularly, to a centrifugal microfluidic control system, which is capable providing more than two freedoms for fluidic manipulation. In addition, the present disclosure further relates to a method for configuring the centrifugal microfluidic control system to enable the system to provide more than two freedoms for fluidic manipulation.

BACKGROUND

In recent years, centrifugal microfluidic or lab-on-a-disc (LOAD) system which aims at integrating tedious benchtop assays into one chip plays a more and more important role in point-of-care diagnostics, drug discovery and food analysis. However, on one hand, as the direction of the centrifugal force on the chip is always radially outward, the freedom of fluid manipulation in the LOAD system is limited. Besides, because the radius limitations and the flow direction are always away from the rotation center, large-scale assays integration on the disc-shaped chip is also difficult.

To add a new degree of freedom for fluidic manipulation, many systems have been developed in centrifugal microfluidic field. These systems comprise multi-manipulation strategies induced LOAD platforms such as active pressure pump or optical manipulation induced LOAD systems, chip orientation changeable LOAD system such as speed actuated inertial mechanical structure induced LOAD system or articulated centrifugal platform and so on. However, almost all these methods make the system more expensive and complicated, which increases the market entry barrier and increases the difficulty of point-of-care. Furthermore, the rotation system in the LOAD system is cumbersome compared with the disc-shaped chip, which sacrifices the portability of LOAD systems. Also, there is no unified specification in the design of these rotation systems, which also increases the market entry barrier.

To solve these problems, a lab-tube system and a lab on DVDs system have been suggested, which are more universal and portable. However, these lab-tube or lab on DVDs systems limited the freedom of the fluidic manipulation, which in turn increases the difficulty of implementation of bioassays in this system.

SUMMARY

The present disclosure provides a centrifugal microfluidic control system, which integrates a microfluidic supporting chip into a centrifugal tube to add more freedoms for fluidic manipulation, and makes the implementation of centrifugal microfluidic control easier. In addition, the commercial centrifuge may be used to provide a pumping force, which makes the system more universal and portable and decreases the market entering barrier.

On the other hand, the present disclosure enables 3D centrifugal microfluidic manipulation to be implemented. 3D centrifugal microfluidic control is very useful as different steps in different layers will not only make the integrations of the whole assay possible but also avoid the cross contamination between different steps. It gives more space for large-scale integration and may be a possible solution for addressing the limitation of space in centrifugal microfluidic tube.

The basic principle of this system is to integrate microfluidic supporting chip into a centrifugal tube. In this system, for example, the commercial available centrifuge may be used to provide the pumping force, which makes the system more universal and portable. Besides, as this chip in a tube system is completely closed, the safety of processing of infectious samples can be ensured, even outside a biological safety laboratory. In addition, in this platform, a small wireless controlled stepper motor may be introduced into the tube to change the orientation of the chip in real time and add more freedom for fluidic manipulation. This brings the concept of the 3D centrifugal microfluidic control.

According to an aspect of the present disclosure, a centrifugal microfluidic control system may comprise a centrifugal tube, a centrifugal unit, a control unit and a microfluidic supporting unit. The centrifugal unit may accommodate the centrifugal tube and provide a centrifugal force to the centrifugal tube. The control unit is fixed in bottom of the centrifugal tube. The microfluidic supporting unit is coupled to the control unit in the centrifugal tube. The control unit may change an orientation of the microfluidic supporting unit to change a direction of the centrifugal force applied to the microfluidic supporting unit.

According to an embodiment of the disclosure, the microfluidic supporting unit may comprise a microfluidic supporting chip or microfluidic supporting cube. The orientation of the microfluidic supporting chip or cube may be changed to change a direction of the centrifugal force applied thereto. As an example, the microfluidic supporting chip or cube may be made of polydimethylsiloxane, glass, plastic, silicon, polymer and the like.

According to an embodiment of the disclosure, the centrifugal unit may be a centrifuge widely used in the lab and hospital. The centrifugal unit may comprise a holder with a space for receiving the centrifugal tube; and an actuator configured to actuate the centrifugal unit to provide the centrifugal force to the microfluidic supporting chip.

According to an embodiment of the disclosure, the system may further comprise a communication unit for communicating signals to the control unit. The control unit may change the orientation of the microfluidic supporting unit in response to the signals from the communication unit. The control unit may comprise a wirelessly controlled stepper motor fixed in the bottom of the centrifugal unit. The microfluidic supporting chip then can be inserted on the stepper motor.

According to an embodiment of the disclosure, the microfluidic supporting chip may comprise one microfluidic supporting layer in a 2D application and comprise two or more microfluidic supporting layers in a 3D application. Each of the microfluidic supporting layers can comprise a plurality of chambers for accommodating samples and/or reagents and at least one passage for allowing the samples and/or reagents to flow between the chambers. The microfluidic supporting layers can comprise a filtering membrane or barrier membrane for removing undesired impurities or containing the desire component. In the case that the microfluidic supporting chip comprises two or more layers, at least one channel is provided between adjacent microfluidic supporting layers to fluidly communicate the adjacent layers such that different steps can be operated in different layers.

According to an further aspect of the present disclosure, a method for configuring a centrifugal microfluidic control system may comprise accommodating a centrifugal tube in a centrifugal unit for providing a centrifugal force to the centrifugal tube; fixing a control unit into a bottom of the centrifugal tube; coupling a microfluidic supporting unit to the control unit in the centrifugal tube, and changing, by the control unit, an orientation of the microfluidic supporting unit to change a direction of the centrifugal force applied to the microfluidic supporting unit.

According to an embodiment of the disclosure, the microfluidic supporting unit may comprise a microfluidic supporting chip with two or more microfluidic supporting layers. The method may further comprise placing the microfluidic supporting chip into the centrifugal tube. The microfluidic supporting chip is therefore coupled to the control unit.

According to an embodiment of the disclosure, the centrifugal unit may be a centrifuge widely used in lab and hospitals and can comprise a holder and an actuator. The method may further comprise a step of receiving the centrifugal tube into the holder and actuating the actuator to provide the centrifugal force to the microfluidic supporting chip.

According to an embodiment of the disclosure, the method may further comprise a step of communicating signals to the control unit by a communication unit, and changing the orientation of the microfluidic unit in response to the signals from the communication unit.

According to an embodiment of the disclosure, the method may further comprise providing samples to be treated and/or proper reagents on one of the microfluidic supporting layers; transporting an intermediate product obtained from the samples and/or reagents to an adjacent one of the microfluidic supporting layers; and providing other reagents on said adjacent one of the microfluidic supporting layers.

According to an embodiment of the disclosure, the method can further comprise forming a plurality of chambers on each of the microfluidic supporting layers for accommodating the samples and/or reagents; and providing at least one passage between the plurality of chambers for allowing the samples and/or reagents to flow therebetween.

According to an embodiment of the disclosure, the adjacent microfluidic supporting layers can be configured with at least one channel therebetween to fluidly communicate the adjacent microfluidic supporting layers to perform different step in different layers.

DETAILED DESCRIPTIONS

Reference will now be made in detail to some specific embodiments of the disclosure including the best modes contemplated by the inventors for carrying out the disclosure. Examples of these specific embodiments are illustrated in the accompanying drawings. While the disclosure is described in conjunction with these specific embodiments, it will be understood that it is not intended to limit the disclosure to the described embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the disclosure as defined by the appended claims. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present disclosure. The present disclosure may be practiced without some or all of these specific details. In other instances, well-known process operations have not been described in detail in order not to unnecessarily obscure the present disclosure.

FIG. 1illustrates a schematic structure of a centrifugal tube according to an embodiment of the present disclosure, in which the centrifuge for providing centrifugal field is not shown. The centrifuge may be a commercial centrifuge widely used in biology labs or hospitals, and thus increase the universality and applicability of the centrifugal microfluidic control system of the present disclosure. In addition, the platform of the present disclosure is based on tubes, therefore solves a technical issues that standard of the platform of the centrifugal microfluidic control is not uniform.

As shown inFIG. 1, centrifugal microfluidic control system may comprise a centrifugal tube1, a centrifugal unit (not shown), a microfluidic supporting unit2and a control unit3. The centrifugal unit may be a commercial available centrifuge that is widely used in biology labs or hospitals, and here will not describe it in detail. The centrifugal unit may be used to accommodate the centrifugal tube1and provide a centrifugal force to the centrifugal tube1. The centrifugal tube may be any of plastic centrifuge tube, glass centrifugal tube, steel centrifugal tube or any other centrifugal tube that may be available. In the present disclosure, since the microfluidic supporting unit2is actuated by a motor in a tube, the present disclosure may be seemed as a motor asslysisd chip in a tube (MACT) system.

The microfluidic supporting unit2is coupled to an upper side of the control unit3and the control unit3may be placed in the centrifugal tube1. In a 2D centrifugal microfluidic control, the microfluidic supporting unit2may comprise a microfluidic supporting chip or a microfluidic supporting cube with one microfluidic supporting layer, and may comprise a microfluidic supporting chip with two or more microfluidic supporting layers for performing different steps in different layers in a 3D centrifugal microfluidic control. For example, the microfluidic supporting chip or cube may be made of polydimethylsiloxane, glass, plastic, silicon, polymer and the like. However, the microfluidic supporting chip may also be made of any other proper material. When the microfluidic supporting unit2comprises two or more microfluidic supporting layers, as the direction of centrifugal force can be perpendicular to the surface of the microfluidic supporting unit2(i.e. microfluidic supporting chip), centrifugal force can drive the fluid from the upper layer into the next layer, which brings the concept of the 3D centrifugal microfluidic control.

When the centrifugal microfluidic control system operates, a microfluidic supporting unit2is placed into a holder of a centrifugal unit which provides a space for receiving the centrifugal tube. Then an actuator of the centrifugal unit actuates the centrifugal tube to provide a centrifugal force thereto.

To make the function of the centrifugal microfluidic control system more clarity, two specific disclosures, human blood plasma separation and bacterium plasmid DNA extraction which are very often used samples, are described as below. However, it should be understand that the above mentioned applications are only examples and not limit the scope of the present disclosure.

The control unit3is fixed in a bottom of the centrifugal tube1for providing driving force to drive the microfluidic supporting unit2to rotate. Therefore, an orientation of the microfluidic supporting unit2can be changed and thus a direction of the centrifugal force applied onto the microfluidic supporting unit2is changed.

In an embodiment of the present disclosure, the control unit3may be a wirelessly controlled stepper motor. Here, the wirelessly controlled stepper motor in the centrifugal tube1may be controlled by a wireless control module (not shown) in real time. The wireless control module may send a control command to cause the stepper motor to rotate via a communication unit (not shown). As the stepper motor rotating, the centrifugal field applied on the microfluidic supporting unit2will be changed to make the implementation of often used operations such as bidirectional flow and inward pumping very easy.

As the control unit3may change the orientation of the microfluidic supporting unit2in real time, the direction of centrifugal force may also be perpendicular to the surface of the microfluidic supporting unit2. As a consequence, the fluid manipulation between different layers in multilayer microfluidic supporting unit2becomes possible. Therefore, a 3D centrifugal microfluidic control will be achieved.

FIGS. 2(a)-2(b)show an exemplary 2D application of the centrifugal microfluidic control system according to an embodiment of the present disclosure. As shown inFIGS. 2(a)-2(b), the microfluidic supporting unit2may comprise chambers201and202, and a passage206for allowing the samples and/or reagents to flow between the chambers.

In an embodiment of the present disclosure, a blood separation process is selected as an example of a 2D centrifugal microfluidic application based on this system.FIG. 2(a)illustrates that blood is fully filled in the passage and is divided into two layers based on density difference; andFIG. 2(b)illustrates that the blood plasma is got separated from the whole human blood.

In the embodiment of blood plasma separation, firstly, certain volume of human blood pretreated with the anticoagulant EDTA (Becton, Dickinson and Company) is injected into the chip. Here, 5 μl human blood pretreated with the anticoagulant EDTA is selected as an example to explain the principle of 2D centrifugal microfluidics application. However, other kind of samples may also be used and not limit to human blood. In addition, any volume of samples may be selected. Then, as the state shown inFIG. 2(a), the blood is fully filled into the passage206and is divided into two layers203and204due to the density difference in the centrifugal field. For the two layers203and204, the supernatant203is plasma and the bottom layer204is white blood cells, red blood cells, and platelets. Then, the stepper motor is rotated 180° (FIG. 2(b)), the blood plasma is driven into the reserved chamber202and the separation is realized naturally. Experimental result illustrates that about 2.5 μl plasma is separated from the 5 μl human blood (without being diluted) in only 1 minute (1500 rpm) and no red cell is observed in the plasma. This separation method is effective and high repeatable.

In a 3D application, a microfluidic supporting chip with two or more microfluidic supporting layers will be used for performing different steps in different layers. As an example, in an embodiment of the present disclosure, two microfluidic supporting layers are selected to show the 3D application of the present centrifugal microfluidic control system. However, one should understand more than two microfluidic supporting layers will be applicable in other embodiments.

FIGS. 3(a)-3(l)show a whole process of an exemplary 3D application of the centrifugal microfluidic control system according to an embodiment of the present disclosure. As shown inFIG. 3(a)-3(l), the microfluidic supporting unit2comprises a microfluidic supporting chip having two microfluidic supporting layers21and22. There are a plurality of chambers and passages provided in each of the microfluidic supporting layers21and22. In addition, there is a channel (not shown) provided between the two microfluidic supporting layers21and22to fluidly communicate the adjacent layers.

As an example of 3D application of the present disclosure, bacterium plasmid DNA extraction is selected to explain the 3D control principle of the centrifugal microfluidic control system. In the embodiment of bacterium plasmid DNA extraction, the system in MACT based format mimicked a benchtop work. The microfluidic supporting chip used for DNA extraction may comprise two microfluidic supporting layers21and22: the first layer21may be used for bacteria lysis and the second layer22may be used for DNA purification. Between the first layer21and the second layer22, there is a channel vertical to the surface of the microfluidic supporting chip2throughout to fluidly communicate the adjacent layers.

As an example, plasmid DNA extraction buffer may be obtained from a DNA extraction kit. In this embodiment, there are four buffers being used, i.e. resuspension buffer composed of Tris buffer with RNase for removal of RNA contamination (buffer 1), lysis buffer composed of alkaline and detergent (buffer 2), neutralization buffer composed of acidic acetate buffer (buffer 3) and wash buffer composed of 70% ethanol (buffer 4). Chemically competent BacteriaE. coliDH5(alpha) (Life Technologies) may be transformed with plasmid DNA from Synthetic Biology Part Registry's Repository biobrick BBa_E0040, which contains the coding gene of green fluorescent protein derived from jellyflysisAequeora victoriawild-type GFP, in the synthetic biology plasmid vector pSB1A2, under 42 Degrees Celsius 1 min heat shock followed by overnight incubation in LB agar plate with 50 μg/mL ampicillin. A single colony may be picked and inoculated into liquid LB medium sparked with 50 μg/mL ampicillin and growth for 12 hours before plasmid DNA extraction.

The process of plasmid DNA extraction from bacteria based on MACT system will be described as below referring toFIGS. 3(a)-3(l).

The process begins with Step1, in which certain volume of a sample, proper volume of a lysis buffer and proper volume a neutralization buffer may be injected into the corresponding chambers301,302, and303on the first layer of the microfluidic supporting chip, respectively, as shown inFIG. 3(a). For purpose of explanation, here 15 μl sample, 15 μl lysis buffer and 21 μl neutralization buffer are selected as an example and the initial orientation of the microfluidic supporting chip may be determined as that shown inFIG. 3(a). It should be understood other proper volume of sample, lysis buffer and neutralization buffer may also be used without going beyond the scope of the present disclosure.

As shown inFIG. 3(b), in Step2, the sample and lysis buffer may flow into a mixing chamber304by gravity and the centrifugal force F for mixing at a proper rotation speed, for example, 5-50 rpm. It should be appreciated that the rotation speed of the control unit (motor)3may be different for different application.

The process proceeds to Step3, and the direction of the centrifugal force F is changed which is shown inFIG. 3(c). In this step, the orientation of the microfluidic supporting chip2may be changed by 90° alternately, for example at 30 rpm between the states ofFIGS. 3(b) and 3(c)for some times (such as 5 times) to make the mixing sufficiently.

FIG. 3(d)illustrates that in Step4, the orientation of the microfluidic supporting chip changes by 90°, such that the direction of the centrifugal force F is changed. In this Step, the neutralization buffer is injected into the mixing chamber304. As the orientation of the microfluidic supporting chip is changed at a rotate speed sufficient to cause the neutralization buffer contained in the chamber303to flow into the mixing chamber304, and thus the neutralization buffer is also driven into the mixing chamber304.

Then the process proceeds to Step5as shown inFIG. 3(e), and the direction of the centrifugal force F is changed. In this step, the orientation of the microfluidic supporting chip2may be changed by 90° alternately in a proper rotate speed (e.g. 30 rpm) between the states ofFIGS. 3(d) and 3(e)for some times (e.g. 5-10 times) to make the mixing sufficient. In this step, the rotate speed will not cause the mixed fluid flowing out of the mixing chamber304. As a consequence, protein-genomic DNA aggregate may be formed in this step.

In Step6, after the sample, the lysis buffer and the neutralization buffer are fully mixed, as shown inFIG. 3(f), the orientation of the microfluidic supporting chip2is changed by 180° relative to that shown inFIG. 3(d)such that the direction of the centrifugal force F is changed and all of the above mixed fluid may be driven into a buffer chamber305at a proper rotate speed which is sufficient to cause the mixed fluid to flow out of the mixing chamber304, for example, at 60 rpm in this embodiment.

FIG. 3(g)illustrates an operation of Step7. As shown inFIG. 3 (g)the microfluidic supporting chip2is driven to rotate to be perpendicular to the direction of the centrifugal force F. Therefore, all of the fluid may be driven into a second layer with the help of the centrifugal force at proper rotate speed (e.g. 50 rpm) via the channel between the two layers, while the insoluble factor including protein-genomic DNA aggregate may be removed by a paper filter320. As shown inFIG. 3(h), in this embodiment, the second microfluidic supporting layer22may comprise a chamber321for accommodating an elution buffer, a chamber322for accommodating a wash buffer, a waste chamber323for accommodating undesired fluid, and a yielding chamber324for receiving the plasmid DNA.

Then in Step8, the process begins to proceed in the second microfluidic supporting layer22shown inFIG. 3(h). In this step, the orientation of the microfluidic supporting chip2is changed by 90° relative to that in Step7. As shown inFIG. 3(h), the second layer22may also comprise a plurality of chambers and passages for allowing communication between the chambers. In addition, the second microfluidic supporting layer22may further comprise a barrier membrane330for containing plasmid DNA. The barrier membrane330may for example be a lab-made silica gel membrane. In addition, the barrier membrane330may be any other material that is able to contain the plasmid DNA and allow other components to flow through. As the process proceeds, the mixed fluid without impurities in this step may be driven into a waste chamber323at a proper rotate speed (e.g. 100 rpm), while plasmid DNA may be bound to the barrier membrane330.

FIG. 3(i)illustrates an operation of Step9for processing the plasmid DNA contained in the barrier membrane330. During the operation of Step9, a volume of wash buffer and corresponding volume of elution buffer may be injected into the corresponding chambers321and322in the second layer22, respectively. As an example, 44 μl wash buffer and 10 μl elution buffer may be injected into the corresponding chamber321and322.

Then the process proceeds to Step10shown inFIG. 3(j). In Step10, the wash buffer may be driven into the waste chamber323at proper rotate speed (e.g. 100 rpm) due to the centrifugal force F, and thus cause the barrier membrane330to contain the plasmid DNA to be washed.

As shown inFIG. 3(k), the orientation of the microfluidic supporting chip2then is changed by 180° relative to that in Step10such that the direction of the centrifugal force F is changed. In this step11, the elution buffer may be driven to soak the barrier membrane for a period time at a proper rotate speed, for example at 50 rpm and for 1 minute.

As shown inFIG. 3(l), in the orientation of Step11, the elution buffer carrying the plasmid DNA may be driven into the yielding chamber324at proper rotate speed, for example, at 200 rpm. Then the process for extracting the plasmid DNA is completed.

In this plasmid DNA extraction assay, the insoluble factor including protein-genomic DNA aggregate may be removed by the filter paper320when the reagent flows through it. Plasmid DNA may be first bound to the barrier membrane330which may be prepared for example by adding 2 μL of 100 mg/mL (optimized silica amount,) silica gel (Sigma-Aldrich, pore size 60 Å) in between two polycarbonate membrane (Sterlitech, pore size 0.01 μm). Then, the wash buffer may flow through the barrier membrane to get the DNA washed.

The detail structure and principle of the present disclosure have been described in different exemplary embodiments. The new system solves the problem that there is no uniform standard of the centrifugal microfluidics control platform. The present system is based on a tube, which means that the centrifuge widely used in a biological lab or hospital may be used to construct the centrifugal microfluidics control platform, thereby the applicability and universality is increased largely.

As described in the above, the current system is able to change the orientation of the microfluidics supporting chip in real time, such that the direction of the centrifugal force applied onto the chip may be changed. That is to say, 3D centrifugal microfluidics control becomes possible. In this aspect, this system may overcome the limited manipulation freedom of the common centrifugal microfluidics control platform. Thus application scope of the system is increased.