Field of the Invention

The filling and manipulation of liquids/samples into the closed channel system has been realized by using centrifugal force with the help of the present invention. The microfluidic chip is rotated at a certain rotational speed and the liquid placed in the reservoir is filled into the closed channel, which is only the inlet. This principle has the potential to be applied to many studies in chemical analysis, cell analysis, diagnostics, biomedicine and health.

State of the Art (Prior Art)

Microfluidic systems have become a great advantage for bedside testing by working with small volumes of liquids. In particular, centrifugal microfluidic systems eliminate the need for structures such as pumps and active valves, eliminating off-chip sample processing and error- prone pipetting (Hugo et al., 2014).

Centrifugal platforms, called lab-on-disc, can provide processes with low-cost fabrication. However, in these studies, the channels and reservoirs are located in different layers and require a ventilation layer at the top layer so as to ensure fluid movement (Clime et al., 2019). In a pumping technique based on the interaction between capillary and centrifugal force, where the microfluidic platform can circulate the liquid back and forth along a microfluidic channel in the radial direction, the liquids can move inward along the channel according to the capillary action, with the reduction of the rotation speed of the platform (Garcia-Cordero et al. ,2010). Although it is a simple and effective method, only a small part of the fluid can be displaced towards the center in a lower reservoir in this method. Therefore, this method is suitable for mixing only a few miscible solutions or for recirculating analytes on a functional surface. A similar mechanism in fluid flow between two reservoirs connected via a microfluidic channel is achieved by the Euler inertial force generated by the sudden accelerations and decelerations of the microfluidic disc. The Euler force is produced by the angular velocity of the disc in the upward direction along the microfluidic channel. Since this force will always compete with the centrifugal force, it needs optimizations according to channel size and direction (Deng et al., 2014). A study that pumps fluids radially inward was carried out using a high-density fluid that, under the action of centrifugal force, would push a less-dense sample fluid toward the center of rotation (Kong et al., 2012). Immiscible liquids or air as the intermediate phase are needed to maintain sample integrity. Its unidirectional character and the need for additional fluid for pumping limit this approach. A presented passive pumping system incorporates a principle based on pneumatic compression of air by hydrostatic pressure generated in the fluid branch in an adjacent channel. In this study, the accumulated pneumatic energy is released by reducing the rotation speed of the platform and is used to pump the fluid back to the rotation center (Clime et al., 2019). Although the method is precise and reproducible, it requires the fabrication of additional compression chambers. In a centrifuge system using a siphon valve, the siphon structure can discharge fluids within a rotational speed range or cut off the flow by increasing the rotation frequency (Zhu et al., 2018). The intermittent siphon valve consists of an additional air hole at the top of the siphon. When the rotation is stopped, the siphon is fed, and when the disc starts to rotate, the liquid in the siphon moves away from the center if the rotation speed is high enough. Thus, air will enter the siphon through the air hole at the top of the siphon and cut off the liquid in the siphon channel. At lower rotational speed, it forms a meniscus at the gas-liquid interface near the vent. The liquid is conveyed from the charging chamber to the collection chamber via the siphon. These centrifugal systems involving siphon structures are complex to design and manufacture, and in this study, the disc is subjected to constant external forces as it rotates, and the liquid-gas interface will depend on stabilization at specific positions.. In a centrifugal disc that can separate serum from blood with a system compatible with hydrophilic and hydrophobic biomarkers, the cross-flow filtration method separates the serum and retains the amphiphilic biomarkers in the serum (Lenz et al., 2021). The device consists of chambers located one above the other, separated by a membrane, and there are four separation units on the disc with different functions. During cross-flow filtration, the sample passes tangentially through the filter by centrifugal force. Components smaller than the membrane pores pass through the filter as the pressure increases,, while larger components remain on the membrane surface. In a study providing hematocrit measurement in a closed channel on the polymer disc, an inlet chamber, an overflow channel with a hydrophobic valve and a two-layer capillary channel were formed on the disc (Riegger, Lutz et al, 2007). Centrifugally assisted capillary filling was achieved by using two-layer channels at different levels (upper level wider than lower-mid level) and special shaping of the closed channel tip. The capillary force assisted by centrifugation transports the blood into the closed capillary channel. When the channel reaches the capillary tip, it begins to fill in the opposite direction, as the hydrophobic valve is opened, the excess blood is filled into the reservoir here and the hematocrit value is measured with the sedimentation of the red blood cells. The fabrication process is tedious and costly, as this method only requires microfluidic chips with well-calculated specific channel cross-sectional area. In a study utilizing the air permeability of PDMS for filling liquid into channels, a gradient solution and microparticles were filled into channels under vacuum. (Oksuz & Tekin, 2021). In order for the microfluidic system to be centrifuged, the inlet blocking is prevented by this system and the separation of microparticles of different densities has been achieved. Although the system is an advantageous study for cell separation, it is not a suitable system for different liquid manipulations, and it takes a long time to load samples into the channels under vacuum.

Active elements require the application of external forces such as vacuum, magnetic, electrical and mechanical forces for fluid flow and control. The unidirectional character of fluid flows induced by centrifugal force leads to a fundamental limitation in the design of microfluidic circuits. In a study using an active pumping element, there is a pump system closed with deformable polymer layers on which permanent magnets are integrated (Haeberle & Zengerle, 2007). A constant flow of liquid begins by compressing the air in the deformable chambers by allowing the chambers to rotate in a constant magnetic field. Although active element centrifugal microfluidic systems offer interesting methods, their implementation requires sets to be mounted on a rotating platform and their control. In a centrifugal platform with the installation of electronic pumps and the use of electromechanical valves, the platform rotates at high speed, while generating air pressure in the pressure ports on the chip through a pneumatic connection (Clime et al., 2019). The resulting air pressure interacts with the circuit elements on the chip and performs functions such as valving, reverse pumping and bubble mixing. However, the cost required to produce such a complex platform is increasing.

The sample taken from the patient must be pre-treated and centrifuged in stages such as diagnosing many diseases and monitoring the disease process in the clinic. Microfluidic systems can demonstrate the centrifugal feature, which is an indispensable step in the clinic, with centrifugal microfluidic systems. In the case of passive pumping methods in lab-on-a- disc systems called lab-on-a-disc systems, fluid movement can be provided by chambers, ventilation holes, siphon structures, membranes or valves positioned in different layers. In addition to increasing cost and restricting fluid movement, these designs cannot guarantee efficient fluid transfer. Moreover, since the elements used for fluid manipulation are delicate, optimizing the method well requires additional compression or waste chambers so as to prevent the fluids from overflowing. Each chamber and layer added to microfluidic systems directly affects the cost and ease of use. In the case of active pumping systems, although studies using external mechanical, electrical or magnetic forces are methods that allow many complex manipulations and analysis, components that need to be mounted on a rotating platform limit the usability of the system. In addition, most centrifugal microfluidic systems in the prior art are designed in the form of a disc. This means that a rotating platform in the form of a disc reader, i.e. an additional device design, would be needed to add rotational speed to the designed system. In summary, the technical problem encountered in the prior art is the use of expensive and complicated fabrication and unstable structures and methods in centrifugal microfluidic systems for fluid manipulation and sample analysis. Moreover, it is necessary to purchase an additional device for the users.

Brief Description and Objectives of the Invention

The present invention relates to a microfluidic platform that fulfills the above-mentioned requirements, eliminates the disadvantages and brings some additional advantages.

In the present invention, all additional structures used in the technique were eliminated with the filling method presented by simplifying the production. In this way, it has been compatible with the centrifuge device in every laboratory. What makes the filling principle unique is that the hydraulic resistance changes according to the different characteristics (width, height, length) of each channel and accordingly the rotation speed (RPM) required for filling also changes. As the hydraulic resistance increases, the required rotation speed increases. With the centrifugal force applied, the pressure in the channel is increased and the channel is filled by discharging air through the channel into the sample reservoir.

Centrifugal microfluidic systems are known in the art as lab-on-disc or lab-on-cd, and they are systems that provide fluid manipulation by applying centrifugal force. In traditional microfluidic systems known in the literature, channel filling is provided by inlet and outlet holes. A closed channel must be used to apply centrifugal force to the channel, and active and passive valves, membranes, vents must be used for this to occur with the centrifugal microfluidic systems known in the art. In the present invention, for the first time, fluid manipulation was performed in a straight closed channel without using any additional structure (valve, siphon, membrane, etc.). In addition, an additional device (such as CD ROM or designed in this way) is required for the application of centrifugal force in lab-on-disc or lab-on-cd. Since the method and design presented with the invention are suitable for the centrifuge device, it does not need such devices. In this way, the field of application is also widened.

The primary object of the present invention is to develop a centrifugal microfluidic system that provides closed channel filling and analysis in a single channel with only centrifugal force, without the need for additional filling chambers, valves, membranes, siphons, etc. In the invention, placing the liquid in a reservoir and filling the same into the channel and performing the necessary operations are carried out entirely by centrifugal force.

All additional structures used in the art were eliminated with the inventive filling method. In this way, it is compatible with the centrifuge device in every laboratory. What makes the filling principle unique is that the hydraulic resistance changes according to the different characteristics (width, height, length) of each channel and accordingly the rotation speed (RPM) required for filling also changes. As the hydraulic resistance increases, the required rotation speed increases. The applied centrifugal force increases the pressure within the channel, and by enabling air outlet from the channel into the sample reservoir, filling is achieved.

The microfluidic system of the present invention is compatible with many biological analysis processes (blood sample analysis, DNA analysis, purification, separation, enrichment, single cell) many of which can be viewed with a microscope as well as analyzed by telephone. The invention offers the advantage of eliminating the main problems of the prior art, such as cost, complex design and the need for an additional rotating device, as well as offering a principle that is not available in the literature by offering the possibility of using a single inlet chamber in two directions (inlet and outlet) without the use of additional compression chambers, siphons and membrane structures mentioned in the literature. With this proposed method, the research aims to target a technique that can process low-volume (0.1 pL-500 pL) samples in the centrifuge device presented in the cell and diagnostic laboratories, provides short-term analysis, and has the potential to eliminate sample pretreatment. The channel design was developed according to the desired method and analysis. All that is needed to get results is to place the microfluidic chip in the centrifuge device. In this way, compared to other methods, the invention will provide a great advantage in terms of cost, while maintaining this advantage in terms of ease of use. Since the method is also programmable, it allows many analysis steps to be performed automatically in the centrifuge device. For the first time, the inventive product and the method used were used to fill a closed channel without the use of additional structures. What makes the filling principle unique is that the hydraulic resistance changes according to the different characteristics (width, height, length) of each channel and accordingly the rotation speed (RPM) required for filling also changes. As the hydraulic resistance increases, the required rotation speed increases. With the centrifugal force applied, the pressure in the channel is increased and filling is achieved by providing air outlet from the channel into the sample reservoir.. What makes the product unique is that it has become a product that is compatible with the centrifuge device, provides liquid manipulation and sample analysis depending on the filling principle. Centrifugal microfluidic systems are known in art as lab-on-disc or lab-on-cd, and are systems that provide fluid manipulation by applying centrifugal force. In traditional microfluidic systems known in the literature, channel filling is provided by inlet and outlet holes. In order to apply centrifugal force to the channel, a closed channel must be used, and with centrifugal microfluidic systems known in the art, active, passive valves, membranes, vents, special channel architectures must be used for this to occur. In the invention, for the first time, fluid manipulation was performed in a straight closed channel without using any additional structure (valve, siphon, membrane, etc.). Moreover, an additional device (such as CD-ROM or designed in this way) is required for the application of centrifugal force in the laboratory systems on disc and CD presented in the art. Since the method and design presented in the invention are suitable for the centrifuge device often found in the laboratory, such devices are not needed. In this way, the field of application is also widened.

The chip can be filled sequentially from different reservoirs. By using different channel sizes and centrifuge speeds, only liquid can be drawn from the desired reservoir to the chip. Thus, solutions for molecular analysis can be fed sequentially to the chip. By adjusting the channel size, the desired volume of liquid can be drawn from the reservoir. Thus, a precise amount of liquid can be used for analysis. With the invention, the amount of hematocrit and white blood cells can be determined and the separated plasma can be collected from the chip.

Definitions of Figures Explaining the Invention

Figure 1: Closed-channel microfluidic design (A) The parts of the microfluidic chip (top view). (B) Microfluidic chip formed using double-sided tape contains a closed-end filling channel and sample reservoir (side view). Figure 2: Centrifuge tube designed to place the microfluidic chip in the centrifuge device

Figure 3: Variation of filling speed of microfluidic chip depending on channel width and channel height. (A) Rotational speeds required to fill channels simultaneously with different channel widths and channel heights. (B) Relationship between hydraulic resistance depending on channel height and rotational speed. (C) The relationship of the hydraulic resistance depending on the channel width and the rotational speed.

Figure 4: Time-dependent filling profile of the channel depending on the channel length at a constant channel width and height.

Figure 5: Relationship between hydraulic resistance and filling speed according to channel length, keeping channel width and height constant.

Figure 6: Buffy coat and plasma regions formed by centrifugation of the whole blood sample in the microfluidic chip for 5, 10, and 15 minutes, respectively.

Figure 7: (A) The hematocrit value calculated at different rotation speeds. (B) The amount of cells remaining in the plasma after centrifugation.

Figure 8: Comparison of on-chip hematocrit values with measurements made with a microhematocrit tube.

Figure 9: Correlation between buffy coat thickness and white blood cell count.

Definitions of Elements/Parts/Pieces Forming the Invention

In order to better explain the microfluidic platform developed with this invention, the parts/pieces/elements in the figures prepared are given below.

1 : Sample Reservoir

2: Filling Channel

3 : Microfluidic Chip

4: Centrifuge Tube Detailed Description of the Invention

The present invention is a microfluidic platform for liquid filling characterized in that; it comprises

• Microfluidic chip (3) comprising at least one filling channel (2) with a closed end and straight cross-section and at least one sample reservoir (1) connected to the channel (2); and

• At least one centrifuge tube (4) designed to insert the microfluidic chip (3) into the centrifuge.

The rotation speed of the centrifuge device in which the microfluidic chip (3) is positioned according to the channel length (L), channel width (w) and channel height (h) of the filling channel (2) is adjustable or programmable. The microfluidic chip (3) in the invention consists of a flat and closed filling channel (2) with a length of 5-50 mm and different widths (0.1-5 mm) (Figure 1 (A)). The microfluidic chip (3) consists of a 0.15 mm thick double-sided tape, a 1 mm thick lamella on the bottom surface, a Polymethyl methacrylate (PMMA) layer on the top surface containing the sample reservoir (1), and a closed filling channel (Figure 1 (B)). The volume of the sample reservoir (1) is 1-100 pL.

The liquid filling method comprises the following process steps:

• Providing at least one microfluidic chip (3) containing at least one closed-end filling channel (2) with at least one sample reservoir (1) connected to the channel (2), wherein rotational speed on the centrifuge device is adjusted based on the channel length (L), channel width (w), and channel height (h) of the filling channel (2).

• Providing at least one centrifuge tube (4) designed to place the microfluidic chip (3) in the centrifuge device,

• Filling the liquid into at least one sample reservoir (1),

• Placing the microfluidic chip (3) into the centrifuge tube (4),

• Adjusting the rotation speed of the centrifuge device according to the channel length (L), channel width (w) and channel height (h) of the filling channel (2),

• Filling the determined liquid volumes from the sample reservoir (1) into the channel (2) by rotating the microfluidic chip (3) for a certain period at the set centrifugal speed, by providing air outflow through the channel (2) into the sample reservoir (1) via centrifugal force. The method of the present invention is used to programmatically fill different liquids from the desired sample reservoir (1) into the filling channel (2) by rotating the microfluidic chip (3) at different centrifuge speeds for specific times.

The microfluidic chip (3) is placed in the centrifuge device by placing the same in a specially designed centrifuge tube (4) after the liquid to be filled into the channel (2) is placed in the reservoir (Figure 2). The centrifuge tube (4) is a tube that the microfluidic chip fits tightly inside (3) and keeps the chip (3) stationary in the centrifugal device. Technically, it fits tightly into the centrifugal device and comprises at least one recess allowing the microfluidic chip (3) to fit inside and preventing the movement of the chip (3) during centrifugation.

Different liquid volumes can be filled into the channel (2) by rotating the microfluidic chip (3) at different centrifuge speeds (100-1000 rpm). Each channel (2) has different hydraulic resistance due to its characteristics (length, width, height). The increase in hydraulic resistance means that the channel (2) will need a higher rotational speed for filling. For this reason, each channel (2) was first rotated one by one starting from 100 rpm 5 min until 1000 rpm 5 min, the aim here is to find the rotational speed at which they fill according to the channel (2) characteristics. Different channel (2) sizes can fill at different rotational speeds (Figure 3A). This means that if different channel (2) widths connected to the reservoir are used on the chip (3), a programmable flow profile will be created at different rotational speeds. Thus, processes that require mixing of different liquids sequentially can be carried out in the centrifugal device with the proposed principle. In this invention, the filling of the closed channel (2) system is presented for the first time without the use of valve, membrane, siphon structures.

With the invention, it is observed that different channel (2) sizes can also be filled at different rotational speeds (Figure 3A). According to the results obtained, it is concluded that the rotational speed required for the liquid to fill the channel (2) decreases as the width of the channel (2) and the height of the channel (2) increase, and the hydraulic resistance is calculated with Equation 1 and the filling principle is revealed (Equation 1). Hydraulic resistance (R) is related to channel height (h), channel width (w), channel length (L), and fluid viscosity (p). By calculating the hydraulic resistance depending on the channel width and channel height, a correlation with the rotational speed required for filling is observed (Figure 3B, C). This means that if different channel (2) widths connected to the reservoirs on the chip (3) are used, a programmable flow profile will be generated at different rotational speeds. In this invention, the filling of the closed channel (2) system is presented for the first time without the use of valve, membrane, siphon structures.

R = - - — j - (Equation 1)

1-0,63 (-) h ³ w

With a constant channel width of 2 mm and a constant channel height of 150 pm, the timedependent volume profile with respect to channel length was analyzed (Figure 4). (Figure 4). The hydraulic resistance was calculated using Equation 1 and the relationship between the channel length and the rotation speed required for filling the channel is shown (Figure 5). Thus, the channel with low hydraulic resistance, i.e. less channel length, takes less time to reach 95% of its volume, while this time increases as the channel length increases.

Within the scope of the present invention, the hematocrit and white blood cell count routinely performed in the hematology unit were applied in the closed channel (2) system. 10 pL of whole blood sample was placed in the sample reservoir (1) and then centrifuged in the centrifuge device at different rotational speeds and times. In this way, plasma is separated from whole blood without using any additional processes or additional structures. The hematocrit value can be calculated from the ratio of the red blood cells collected in the lower part of the channel to the sample volume in the total channel (2) (Figure 8). Considering the result obtained, since the effects of rotational speeds on the calculation of hematocrit were the same, the amount of cells remaining in the plasma was counted and the optimum rotational speed and time were determined as 4000 rpm 10 minutes (Figures 7 A, B). In the clinic, for plasma separation, all the cells in the blood should be precipitated and no cells should remain in the plasma. For this reason, the number of cells remaining in the plasma was examined at rotational speeds, and the minimum amount of cells was found at 4000 rpm in 10 minutes. It was observed that there was no difference in cell numbers between 10 and 15 minutes.

From the whole blood sample, it was observed that the buffy coat region between the plasma separation and red blood cells and containing white blood cells and platelets was obtained in the invention (Figure 6). The amount of white blood cells in the blood can also be calculated by looking at the buffy coat thickness (Figure 9).

With the presented technique, for the first time, a closed channel was filled from a single reservoir by centrifugal force without the need for additional structures, and the hematocrit value and white blood cell amount, which are routine tests in the hematology unit, could be determined. This invention can be applied to various blood tests and has the potential to be used in studies such as separation, purification and enrichment by serving different molecular- level applications.

In the case of the filling principle, it is seen that the liquid forms a meniscus structure towards the channel surface in the direction of rotation of the chip. The meniscus structure moves along the channel and fills the liquid starting from the lower part of the channel, and the air in the channel is discharged in the opposite direction.

It is possible to fill samples from different reservoirs and mix different samples in the channel, respectively by making changes in the shape and positions of the channels on the designed system. Moreover, In addition, with the designed channel and the proposed principle, plasma separation from whole blood can be achieved and biomarkers (such as creatinine) in the separated plasma can be measured. To do this, the whole blood sample is placed in the sample reservoir and then centrifuged. In this way, while the red blood cells in the whole blood are collected at the bottom, a region known as the buffy coat is formed in the middle where the white blood cells are collected, and pure plasma remains at the top. The area covered by the red blood cells is measured, and the hematocrit value is found by proportioning the same to the entire sample area through the microfluidic chip image. Moreover, since white blood cells are collected in the middle area called the buffy coat, the number of white blood cells can be calculated from this area. For the measurement of biomarkers in the plasma, the blood sample is mixed with solutions preferably yielding colorimetric products upon reaction with biomarkers outside the chip, and the mixture is then introduced into the sample reservoir. Then, the mixture is taken into the closed channel by centrifugation and red blood cells and white blood cells are precipitated with high-speed centrifugation. The biomarkers in the plasma region above react with the solutions and reveal color in the channel with different intensities according to the concentration of the biomarker found. According to this color intensity, the biomarker concentration can be calculated. With this method, blood cells are separated from plasma in the same channel and the negative effect of blood cells on colorimetric measurements (such as background noise) is eliminated. The proposed invention has the potential to be used in cell separation, single-cell studies, and many molecular and clinical tests that require centrifugation. Moreover, the proposed system can process a very small amount of samples (0.1-2 pL), and the separated samples can also be examined under a microscope due to the transparent structure of the microfluidic chip. The separated samples can also be collected from the microfluidic chip and used in different analyzes.