Technical Field of The Invention

The invention relates to an integrated acoustophoretic microfluidic device (EAMC) in which integrated two or more step operations to be applied to micro and/or biological particles can be performed in a single microfluidic system using the acoustophoresis method in at least two of these.

State of the Art

Microfluidic systems are technologies that allow biological and chemical processes to be performed at lower cost, faster and often more sensitively, generally in a laboratory environment. In many applications performed in microfluidic systems, it is necessary to control the movements of bioparticles within microchannels. These systems are commonly employed in life sciences such as chemistry, biology, and medicine, and are also called micro total analysis systems (pTAS) or lab-on-a-chip(LOC). LOC systems are microfluidic platforms that can integrate complex chemical management and analysis systems on a single millimetre or centimetre chip and interact with electronic and optical sensing systems. These microfluidic chips enable the examination and detailed analysis of physical interactions, chemical reactions and biological events with a chip of few millimetres or centimetres. In addition, microfluidic technology reduces sample and chemical agent consumption, shortens test time and reduces overall expenses for these processes. Providing that minute amount of sample suffice and many laboratory protocols can be combined on a single chip of only few square centimetres, microfluidic technologies are viable alternative to conventional laboratory techniques. Microfluidic systems have been widely implemented in many applications such as blood cell separation, pathogen/toxin detection, biochemical measurements, chemical synthesis, genetic analysis, drug screening, electrochromatography, and organ-on-chip. Microfluidic chips can be fabricated out of using silicon, glass or polymer materials (polydimethylsiloxane/PDMS, polymethyl methacrylate/PMMA etc.) by micro-fabrication techniques.

In the literature, many different methods including hydrodynamic, electrokinetic, acoustic, magnetic, and optical have been proposed for manipulation of micro and biological particles in microchannels [1], These methods have several advantages and disadvantages. Especially in analyses in clinical applications, it is important that different processes are integrated on the same microfluidic chip with a capability of processing large number of biological particles. Therefore, throughput (number of particles processed per unit time) of the system needs to be high to be able to complete the analysis in reasonable time. In this regard, acoustophoresis, which uses acoustic waves in the manipulation of micro and biological particles, is one of viable options among different particle manipulation techniques. This method relies on the force induced on the particles with acoustic pressure due to the formation of standing waves within the microchannel via the generated acoustic waves in the microfluidics chip. This acoustic radiation force pushes the particles from the channel walls towards the centre. The magnitude of these forces depends on on size, density and compressibility of the particles. Since the force depends on size and type of particles, particles can be moved to different positions. The wavelength and amplitude of the acoustic wave depend on the geometric properties of the microfluidic chip.

The chip material is an important factor in the transmission of acoustic waves. In the literature, silicon and glass chips are frequently used since their acoustic damping is low. When acoustophoretic microfluidic devices are designed for a targeted operation (particle separation or focusing, etc.), although the chip thickness and width can be in the extent of millimetres (mm), the length can be near tens of centimetres (cm) for the success of the operation. Therefore, when two or more operations are combined on the same microfluidic system, the size of the chip may not be reasonable. Furthermore, since microfluidic chips out of materials with low acoustic damping transmit acoustic waves very well, the same acoustic frequency needs to be empoyed in all units for acoustophoretic chip with multiple units. Not using the same frequency for all units may cause interference and negatively affect the operation of the acoustophoretic device.

In the state of the art, co-operation of different manipulation techniques has been demonstrated in many different combinations. For example, the combination of acoustic forces and dielectric forces [2], optical forces and acoustic or dielectric forces [3, 4], magnetic forces and dielectric forces [5], and hydrodynamic and dielectric forces [6] is already present for different applications.

In recent years, acoustic waves have been implemented frequently to generate force for separation, focusing and capturing of micro-scale particles, and have been shown to be successful and advantageous in many applications [7-13], P. Ohlsson et al. [14], who perform more than one acoustic operation on separate chips, used two separate acoustic chips for enriching and capturing processes using acoustic waves. However, this system has disadvantages which are as follows; (i) the risk of sedimentation and sticking due to the long fluidic pathway of the samples which is transported between the chips by a plastic tubing; (ii) the complication of the imaging process and a crowded environment due to separate chips and plastic tubing connections. In addition, when it is desired to perform acoustic operations at different frequencies on a single chip, either frequency multiples or frequencies that are far from each other are selected or the length of the chip is extended [15-17], The main reason for this is to prevent the interference of acoustic waves with different frequencies and any possible disturbance for the desired operation.

In the literature, an acoustophoretic device for a single operation were fabricated with a length of 5-20 cm, width of 5-20 cm and height of 1 -2 mm [12-17], These devices are generally made of materials with low acoustic damping. Such materials with low acoustic damping are generally hard, brittle and thin materials (glass, silicon, fused silica, quartz, silicon carbide, etc.). There exists acoustophoretic devices which can handle multiple operations in serial architecture where long chips have to be arranged one after the other to reduce the acoustic wave transmission between the stations where different operations were performed [14], In this case, laboratory prototypes became small in width and thickness compared to their length, which can reach 50 cm, and adifficult to be packaged, transported and commercialized without any mechanical damage. In addition, long channels in one direction may cause some problems such as collapse and sticking.

Inadequacy and drawbacks of the solutions in the state of the art for performing integrated two or more step operations on micro and/or biological particles in microfluidic systems, necessitates the current development which enables the integration of two or more processes on micro and/or biological particles in a single chip.

Brief Description and Aims of the Invention

The present invention describes an acoustophoretic microfluidic device with a multilayer structure. With the device subject to the invention, two or more processes on micro and/or biological particles can be integrated in a single system.

An object of the invention invention is to carry out integrated two or more step operations to be applied to micro and/or biological particles in a single device. In the device that is the subject of the invention, at least two processes can be performed in an integrated manner by means of at least two layers which enables acoustophoretic manipulations and an insulation layer between these layers. By means of the insulation layer made of acoustically damped material, the interference of acoustic waves at different frequencies is avoided which minimizes disturbance on each chip during operation.

The most important aim of the invention is to perform more than one manipulation technique in a single microfluidic device. At least two different bioparticle manipulation techniques and an acoustic bioparticle manipulation technique can be applied simultaneously in the device that is the subject of the invention. In the microfluidic device having a layered structure that is the subject of the invention, by the presence of polymer material that will act as an acoustic insulator between the layers, multiple microfluidic units which operate at different frequencies can be placed in different layers of a single device.

Another aim of the invention is to minimize the problems such as collapse, sticking and possible damage during transportation that occur in the state of the art in case of applying more than one operation to micro and/or biological particles. By means of the layered structure of the device that is the subject of the invention, the crowded test environment is simplified, and there is no longer any need to extend the chip length for multiple operations, and hence, the problems such as collapse, sticking, and possible damage during transportation caused by the chip length are minimized. Description of Drawings

Figure 1: Sectional view of the two-layer EMAC.

Figure 2: The average acoustic pressure values formed in the separation and enrichment units; A) EAMC with polymer layer, B) EAMC without polymer layer.

Definition of Elements/ZParts Composing the Invention

The parts and pieces in the figure are numbered and the equivalent of each number is given below:

1. Chip

2. Cover

3. Microchannel

4. Piezoelectric actuator

5. Inlet

6. Outlet

7. Flow path hole

8. Insulation layer

Detailed Description of the Invention

The invention relates to an integrated acoustophoretic microfluidic device (EAMC) in which two or more micro and/or biological particle manipulation operations at least two of which are acoustic manipulation can be performed in an integrated manner in a single microfluidic system.

Integrated acoustophoretic microfluidic device (EAMC) which is the subject of the invention comprises at least two layers capable of acoustophoretic manipulation and an insulating layer (8) between the layers capable of acoustophoretic manipulation, at least one inlet (5) on the outer layers of the device that allows the sample to be loaded into the device and at least one outlet (6) that allows the sample to be removed from the device, and at least one flow path hole (7) to allow passage between layers. Each layer in which the aforementioned acoustophoretic manipulation can be performed comprises a chip (1 ) containing a microchannel (3) for the flow of liquids containing micro and/or biological (cell, bacteria, virus, etc.) particles and is made of low acoustic damping material which has been fabricated by chemical etching and/or mechanical processing, a cover (2) made of a material with low acoustic damping, forming the microchannel structure by covering the flow path in the microchannel (3) with one side open inside the chip (1 ), and is also used to reflect acoustic waves in the liquid, and a piezoelectric actuator (4) that generates acoustic waves used to separate micro and/or biological particles.

Figure 1 shows the definitive view of the two-layer acoustophoretic microfluidic device (EAMC), which is an embodiment of the invention. In each layer, the chip (1 ), the cover (2) and the piezoelectric actuator (4) do not need to be in the order shown in Figure 1 , these three components can be arranged in a different order as needed. For example, the chip (1 ) can be on the top, the cover (2) on the bottom, and vice versa.

In the invention, the low acoustic damping material of the chip (1 ) may be glass, silicon, fused silica, quartz or silicon carbide. In an embodiment of the invention, the cover (2) is bonded to the chip. This bonding process can be carried out with chemical adhesives (cyanacrylate, etc.) or, for some chip materials, the surface can be activated by plasma treatment and attached to the glass. Liquid containing micro and/or biological (cell, bacteria, virus, etc.) particles flows in the microchannel (3) between the chip (1 ) and the cover (2), and acoustic waves travel inside the chip (1 ) and affect the particles in the microchannel (3). The piezoelectric actuator (4) may be bonded on the chip (1 ) or the cover (2) with chemical adhesives according to geometrical needs, or it can be placed on the chip without bonding instead by use of transmitting fluid (ultrasonic gel, etc.) that provides acoustic wave transmission between the chip/cover and the piezoelectric actuator. Micro and/or biological particles are loaded into the device from the inlet (5) and taken from at least one outlet (6). The flow path (7) is formed by creating holes on both the chip and the insulation layer for the flow of the liquid containing the particles between the layers. The insulation layer (8) is made of polymer materials (epoxy, double-sided tape, polydimethylsiloxane (PDMS) etc.).

Different operations can be performed on each of the layers in the device. Some of these processes can be defined as directing the particles to a position in the channel, separation, enrichment (concentration), purification, mixing, lysis by cavitation, etc. For efficient processes, different channel widths hence different frequencies are required since the resonance frequency of the acoustic waves depends on the channel width. However, since each layer is made of acoustically low damping materials, acoustic waves in one layer can travel to other layers in the device having a multi-layer structure, and this may adversely affect the operations in the other layers that are driven at different frequencies. For this reason, an insulation layer (8) consisting of a material with high acoustic damping is employed between each layer in order to insulate acoustic waves. The number of layers can be increased as needed, depending on the needs of the application. Each layer is positioned on top of each other on the vertical axis (z-axis). By means of the layered structure of the invention, a device with better aspect ratio, which is more resistant to mechanical damage due to increased thickness, decreased length and more suitable for safe production, packaging and transportation is achieved.

In one embodiment of the invention, the device may include at least one electrical/magnetic/optical/hydrodynamic manipulation layer, in addition to at least two layers capable of acoustophoretic manipulation. Electrodes can be placed in the microchannel in the layer capable of electrical manipulation. In the layer capable of magnetic manipulation, magnets can be placed on the outside of the microchannel or on the outside of the device. The light source can be placed on the outside of the microchannel or the outside of the device in the optical manipulation layer. The layer that can perform hydrodynamic manipulation may have a specially designed microchannel architecture with some obstacles, narrowing and/or expanding regions placed at strategic locations.

The device that is the subject to the invention is obtained by mounting two or more microfluidic chips (1 ) on the vertical axis (z-axis), and the flow created inside is transferred between the chips (1 ). In this device, operations to be performed on each chip with different work descriptions (directing particles/cells to a certain part of the channel, separating them from one another, increasing their concentration, purifying, fragmenting them, etc.) can be performed in an integrated and simultaneously in layered architecture. There is an insulation layer (8) between each chip (1 ) layer in order to prevent mutual transmission and mixing of acoustic waves of different amplitudes and frequencies used in each layer. By means of this insulation layer, an acoustophoretic device is obtained, the form factor of which is suitable for a commercial product, allowing packaging, transportation and modular production.

The length of each layer is in the range of 5-20 cm, the width is in the range of 1 -10 cm. The height of each chip is 0.5-2 mm, the height of each cover is 0.5-2 mm. By means of these dimensions, problems such as collapse, adhesion, transportation etc. caused by the length of the device are minimized.

In the invention, the insulation layer (8) which is made of a material with high acoustic damping is placed between the layers and the effects the tranmission of acoustic waves between the layers has been examined with a numerical simulation approach. The method used in the numerical simulation is the finite element method. The numerically modelled system consists of two layers. Particle separation in the first layer (top chip, upper layer, separation unit in Figure 1 ) and increasing the concentration of separated particles in the second layer (bottom chip, lower layer, concentration unit in Figure 1 ) is aimed. The piezoelectric actuator (4) used for the separation unit in the upper layer was operated at a frequency range of 600-740 kHz under a potential difference of 60 V. In order for the separation to be successful, the value of the acoustic pressure and force created by the piezoelectric actuator (4) in the microchannel must be high in the upper separation unit (first/upper layer). However, the acoustic force formed in the upper layer being transmitted to the lower layer (concentration unit) at a high level is not desired. Because the concentration unit is driven at a different frequency by the second piezoelectric actuator (4) (piezoelectric actuator (4) in the lower layer in Figure 1 ), and acoustic waves from the upper layer can create a disruptive effect on the concentration process. Therefore, the ratio of the average amplitude of acoustic pressure and force generated in the channel in the lower layer (concentration) by the upper piezoelectric actuator to the amplitude of the average acoustic pressure and force in the upper layer (separation unit) is desired to be a low value. The fact that the acoustic pressures formed in the lower layer are lower compared to the upper layer shows that the polymer layer in between reduces the acoustic transmission between the layers. The acoustic simulations of the integrated acoustophoretic microfluidic device that is the subject of the invention were carried out for two situations with and without an insulating layer (8) made of a polymer between the layers made of silicon. The numerical simulations on this two-layer unit were made for a system with and without an insulating layer (8) in between made of damping polymer, and the results are given at different frequencies in Table 1. Theoretically, since the acoustic damping of the polymer layer is high and the wavelengths are short in the polymer layer, the acoustic waves penetrating the polymer material slowdown in the polymer layer and are exposed to high damping. This requires a decrease in the amplitudes of the acoustic waves that pass to the lower layer. Table 1 shows that the theory and numerical simulation overlap. In the device with an insulating layer in between, the average acoustic pressure force ratios were lower than the system without layers at all frequencies. It is seen that the transmission at different frequencies is blocked by the damping layer by half, up to thousands of times at different values. In addition, numerical simulation results for the polymer layer system show higher acoustic pressure in the upper channel compared to the system without polymer layer. It is thought that the reason for this is that the polymer layer reduces the acoustic energy flow to the lower layer and as a result, the pressures in the upper layer increase. Numerical simulations have shown that it is necessary to put insulating material with high damping between the layers so that different processes in different layers do not affect each other's operating performance.

Table 1 : The ratio of the average pressure and force values in the enrichment unit to the values in the separation unit in cases where the insulation layer made of polymer material is present or not. REFERENCES

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