WO2021242179A1

WO2021242179A1 - Particle manipulation

Info

Publication number: WO2021242179A1
Application number: PCT/SG2021/050307
Authority: WO
Inventors: Ye AI; Yinning ZHOU; Zhichao MA
Original assignee: Singapore University of Technology and Design
Current assignee: Singapore University of Technology and Design
Priority date: 2020-05-29
Filing date: 2021-05-31
Publication date: 2021-12-02
Anticipated expiration: 2022-11-29

Abstract

The present invention relates to the manipulation and, more particularly, to the manipulation of micro-particles or objects (e.g. biological cells, polymer beads, multi-cellular organisms etc.) through a bulk vibration-based method. In an aspect of the present invention, there is provided a microfluidic device for manipulating a particle in a fluid suspension, the device comprising: (a) a chamber defined in a first substrate, at least one micro-structure disposed in the chamber; (b) an inlet in fluid communication with the chamber disposed at one end of the chamber, the inlet for receiving the fluid suspension; (c) an outlet in fluid communication with the chamber disposed at an end opposite the inlet, the outlet for discharging the fluid suspension; and (d) a second substrate having a surface, the first substrate is disposed on the surface of the second substrate; wherein at least one ultrasonic wave generator is disposed adjacent the second substrate to generate and deliver ultrasonic waves to the chamber.

Description

PARTICLE MANIPULATION

The present invention relates to the manipulation and, more particularly, to the manipulation of micro-particles or objects (e.g. biological cells, polymer beads, multi-cellular organisms etc.) through a bulk vibration-based method.

Contactless and precise manipulation of micro-components have developed rapidly over the past several decades, which play the influential role in biomedical application and pharmaceutical field. Artificial micro-components possess advantages of great stability, friendly preparation process and being capable of batch manufacturing, thus poses very promising future in the lab-on-a-chip field in microscale with function of particle/cell manipulation, pumping, mixing, propulsion and potential component of micro-robotic devices. In addition to the great existing works, further enhancement is still needed for simultaneously and uniformly manipulation of multiple micro-components, and also easier versatile fabrication means.

To conduct the mechanical motion of micro-components, it can be roughly classified as two categories according to the energy source: one is driven by the external actuation, such as through acoustic, electric, magnetic, optical field, and outside forces like hydrodynamic force and bacterial movement. Another type stimulus is relying on the chemical energy conversion inside the microfluidic system, which can obtain self-sustaining energy from the surrounding environment and exhibit micro-components self-propulsion capability. Compared with the other external fields driven, micro-components manipulated through acoustic actuation normally can work for all aqueous solutions without any special requirements, i.e., no specific need for solution with conductivity or magnetic compatibility. Not to mention that for micro components motivated by chemical energy, where the solution concentration gradient or active metal reacted with acid or water is necessary.

During recent several decades, microfluidic platform displays great potential solutions on biomedical and clinical task. Among which precisely manipulation of particles with micro or nano-sized have obtained great attention due to their extensive application. To achieve the accurate particle positioning, researchers typically adopt external fields introduction, such as electric, optic, magnetic and acoustic field. Compared to the others, acoustofluidics exhibit advantages of precise contactless particle manipulation, working in both liquids and gases with great biocompatibility. In order to manipulate particle with desired mode, the introduced acoustic field have to be triggered with proper transducer, i.e., appropriate spatially phase and wavelength. Specifically, for particle control down to the submicron scale, transducer frequency normally needs to achieve dozens or hundreds of MHz to afford enough acoustic radiation force (ARF), or ensure proper acoustic beam-induced microstreaming formation to manipulate tiny target. Dominated by ARF, Wu et al. have reported the 500 nm particles separated from 240 nm particles with tilted angle standing surface acoustic wave (SSAW) at interdigital transducers (IDTs) frequency of 33.13 MHz. Later Wu et al. continued to adopt the non-tilted SSAW to achieve separation of extracellular vesicles (exosomes, ~150 nm) and lipoproteins (~500 nm) with IDT frequency of 20 MHz. In virtue of acoustic microstreaming, Collins et al. demonstrated the continuous various submicron particles (100 ~ 500 nm) focusing with focused IDT under travelling surface acoustic wave (TSAW) at frequency of 633 MHz. Fakhfouri et al. have explored the micro/submicron particles (100 nm ~ 6 pm) behaviors with TSAW at straight IDT frequency among 110 ~ 260 MHz, revealing the particle streaming, patterning and drifting behavior transition by tuning the particle size, acoustic frequency and power. Tayebi et al. successfully achieved the multiple single 300 nm particles trapping into 500 nm cavities under TSAW with straight IDT at frequency of 196 MHz.

Considering the high cost of IDT manufacturing, cumbersome acoustofluidics chip fabrication and cleaning procedures, Zhao et al. utilized the disposable microchip by bonding the microchannel upon a hard thin polydimethylvinylsiloxane (PDMS) layer, which first investigated by Ma et al., in order to replace the permanent bonding with expensive IDT. Similarly, he achieved 110 nm particles separated from 1 pm particles at IDT frequency of 33.13 MHz. Furthermore, researchers are keep trying to reduce the experiments cost and simplify the whole microchip pre-preparation procedures on particle manipulation with submicron level. Guo et al. have chosen the removable nonuniform focused acoustic vortex transducer to manipulate the 400 nm droplets at the central frequency of 660 kHz. Bachman et al. adopted the double commercial buzzers (patched-transducer) sticked on the substrate to provide the vibration and further controlled 20 pm particles with low frequency within 4 ~ 6 kHz. Hayakawa et al. have utilized the three dimensions bulk vibration-induced asymmetric flow to manipulate the mouse oocyte (~50 pm) with ultra-low frequency of 100 ~ 200 Hz, and explored various pillar forms influence on pillar-around flow and possible applications.

The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Any document referred to herein is hereby incorporated by reference in its entirety.

The present invention relates to a device and method for particle manipulation, and characterization that would be useful for numerous applications. In an embodiment, the present invention includes the concentration, focusing, and/or characterisation of particles such as cells and/or microorganisms, and microrobotics manipulation.

Precisely controllable transport and rotation of microparticles and cells has great potential to enable new capabilities for single-cell level analysis. In this application, we present a versatile ultrasonic microstreaming based manipulation that enables active and precise control of transport and rotation of individual microscale particles and biological cells in a microfluidic device. Two different types of ultrasonic microstreaming flow patterns can be produced by oscillating embedded microstructures in circular and rectilinear vibration modes, which have been validated by both numerical simulation and experimental observation. We have further showcased the ability to transport individual microparticles along the outlines of complex alphabet letters, demonstrating its versatility and simplicity of single-particle level manipulation with a bulk vibration.

In a first aspect of the present invention, there is provided a microfluidic device for manipulating a particle in a fluid suspension, the device comprising: (a) a chamber defined in a first substrate, at least one micro-structure disposed in the chamber; (b) an inlet in fluid communication with the chamber disposed at one end of the chamber, the inlet for receiving the fluid suspension; (c) an outlet in fluid communication with the chamber disposed at an end opposite the inlet, the outlet for discharging the fluid suspension; and (d) a second substrate having a surface, the first substrate is disposed on the surface of the second substrate; wherein at least one ultrasonic wave generator is disposed adjacent the second substrate to generate and deliver ultrasonic waves to the chamber.

In various embodiments, the second substrate has a substantially rectangular shape having top and bottom surfaces and four sides, the ultrasonic wave generator is disposed on the bottom surface opposite the first substrate to generate and deliver ultrasonic waves along an X-axis and/or Y-axis to the chamber.

Alternatively, in other embodiments, the second substrate has a substantially rectangular shape having top and bottom surfaces and four sides, and , the ultrasonic wave generator may be placed adjacent a side of the second substrate and/or top or bottom surfaces of the second substrate. In various embodiments, a ultrasonic wave generator is disposed on each of two adjacent sides of the second substrate to generate and deliver ultrasonic waves along an X-axis and/or Y-axis into the chamber.

By "ultrasonic wave generator", it is meant to include any device that transforms electrical energy to sound energy to generate and cause these vibrations. This generator can vibrate with any input frequencies (within certain range). In particular, any device to produce ultrasonic waves to manipulate the micro-particles as will be described in this application in detail below. Examples include any agitator, vibrator, vibration stage, transducer etc.

In various embodiments, a plurality of micro-structures are disposed in the chamber, the plurality of the micro-structures are equally spaced apart. The plurality of micro-structures form a grid array.

Each micro-structure may be of any shape and size. The term "micro-structures" is used interchangeably with "pillars", "micro-pillars", "micropillar array" etc. and in intended to include any protrusion or obstacle disposed in the chamber of the microfluidic device. The term "chamber" may also include "channel", "microfluidic channel" etc. In various embodiments, each of the micro-structure is substantially cylindrical in shape. Alternatively, they may be cuboid in shape. By "grid array", it is meant to include any uniform alignment of the micro-structures in a grid like fashion, i.e. they are equally spaced to form a grid. In alternative embodiments, the micro structures may be arranged in any way desired to achieve the objectives set out by the present invention. For example, they may be arranged to form triangular, quadrilateral, pentagon shapes etc. In other alternative embodiments, the micro-structures may be arranged in a camber fashion where they are tilted to face an imaginary centre of a circle to form a curved outline while still maintaining their equal distance from each other.

In various embodiments, the distance between each micro-structure in the array is about 10 - 500 pm. The grid array may be 7 mm x 1 mm.

In various embodiments, the diameter and height of each micro-structure is 10-300 pm.

In various embodiments, the micro-structure is a rotor and each rotor comprises a hub and at least one rotatable propeller blade attached to the hub. As is similar in structure to a wheel having a hub and spokes, each rotor has the blade connected to the hub. There can be any number of suitable blades in order to achieve the objectives of the present invention.

In various embodiments, the propeller blade of each rotor is curved in a clockwise or counter clockwise direction and tapers at an end opposite the rotor. This shape of the propeller resembles that of a ninja's shuriken. The angle between the vibration direction and the rotor virtual axis (connecting the midpoints of two main rotor arms / propellers). This 2-propeller rotor will conduct CCW rotation when its initial position and vibration direction form an angle in the range: 27.5° < Q < 150°; and a CW rotation will occur when the angle is in the range: 0° < Q < 27.5° and 150° < Q < 180°.

In various embodiments, the rotor is adapted to rotate in a counter clock-wise direction when a frequency signal input of the ultrasonic wave generator is in an X direction, and the rotor is adapted to rotate in a clock-wise direction when the frequency signal input of the ultrasonic wave generator is in a Y direction. In various embodiments, the ultrasonic wave generator may be a piezoelectric patched- transducer patch or a vibration stage.

In various embodiments, the transducer is adapted to generate and deliver ultrasonic waves having a frequency less than or equal to 1000 kHz. The transducer may be adapted to generate and deliver ultrasonic waves having a frequency is 10-100 kHz.

In various embodiments, the first and second substrates are made of a material suitable for conducting an acoustic wave. The material may be any one selected from the group comprising: PDMS, glass, polymethylmethacrylate (PMMA), and polycarbonate (PC).

In various embodiments, the second substrate is 1mm thick.

In another aspect of the invention, there is provided a method for manipulating a particle in a fluid suspension using the microfluidic device of the first aspect of the invention described above. Hence, the technical features described for the microfluidic device will be applicable when describing the method of the invention. In particular, the method comprises: (a) providing a chamber defined in a first substrate, at least one micro-structure disposed in the chamber; (b) providing an inlet in fluid communication with the chamber disposed at one end of the chamber, the inlet for receiving the fluid suspension; (c) providing an outlet in fluid communication with the chamber disposed at an end opposite the inlet, the outlet for discharging the fluid suspension; and (d) providing a second substrate having a surface, the first substrate is disposed on the surface of the second substrate; and (e) manipulating the particle in the chamber by introducing the fluid suspension to the chamber via the inlet and by providing at least one ultrasonic wave generator disposed adjacent the second substrate to generate and deliver ultrasonic waves to the chamber.

In various embodiments, the second substrate has a substantially rectangular shape having top and bottom surfaces and four sides, and a transducer is disposed on each of two adjacent sides of the second substrate to generate and deliver ultrasonic waves along an X-axis and/or Y-axis into the chamber. In various embodiments, the particle is suspended in a suspension of a non-Newtonian viscoelastic fluid. The fluid may contain polyethylene oxide. In various embodiments, the concentration of the fluid polyethylene oxide (PEO) is between 0.01 to 5 wt%.

In various embodiments, the first and second substrates may be any suitable rectangular-like blocks. The micro-structures are embedded within the first substrate and are disposed within the chamber of the first substrate. The fluid suspension enters the chamber and would come into contact with the micro-structures that are fabricated inside the microfluidic channel or chamber. Only when these micro-structures vibrate in contact with fluids, they are able to generate fluid flows around them to manipulation nearby microparticles. As such, the micro particles in the fluid suspension do not necessarily come into contact with the micro structures for manipulation. The vibration of the micro-structures will generate fluid flows around them. Depending on the vibration mode as will be described later (e.g., circular and rectilinear vibration modes), the fluid flow fields can be different. Microparticles nearby will be dragged by the fluid flow for manipulation. In addition, by adding PEO into the aqueous solution, these particles are subjected to additional elastic lift force that can concentrate them near the micro-structures.

In various embodiments, the ultrasonic wave generator is adapted to generate and deliver ultrasonic waves having a frequency less than or equal to 1000 kHz.

In various embodiments, a plurality of micro-structures are disposed in the chamber, the plurality of the micro-structures are equally spaced apart. The plurality of micro-structures may form a grid array.

In various embodiments, each of the micro-structure is substantially cylindrical in shape.

In various embodiments, the distance between each micro-structure in the array is about 10- 500 pm.

In various embodiments, the diameter and height of each micro-structure is 10-300 pm. In various embodiments, the micro-structure is a rotor and each rotor comprises a hub and at least one rotatable propeller blade attached to the hub.

In various embodiments, the propeller blade of each rotor is curved in a clockwise or counter clockwise direction and tapers at an end opposite the rotor hub.

In various embodiments, the rotor is adapted to rotate in a counter clock-wise direction when a frequency signal input of the ultrasonic wave generator is in an X direction, and the rotor is adapted to rotate in a clock-wise direction when the frequency signal input of the ultrasonic wave generator is in a Y direction.

In various embodiments, the transducer is a piezoelectric patched-transducer.

In order that the present invention may be fully understood and readily put into practical effect, there shall now be described by way of non-limitative examples only preferred embodiments of the present invention, the description being with reference to the accompanying illustrative figures.

In the Figures:

Fig. 1. Schematic and experimental map shown the bulk vibration-based submicron particle manipulation in microfluidics (a) Schematic of the bulk vibration-based submicron particle manipulation in microfluidics setup. The vibration stage can manipulate the microchip movement both in X and Y directions. Patterns shown inside the red box are representative design of micropillar array (b) shown the microstreaming and submicron particle focusing. Double black arrow represents the vibration direction (c) Microscope image shows one pillar from pillar array in bright field. (d)~(f) show the 800 nm fluorescent particle behavior under inactive or active vibration mode at frequency of 800 Hz within different viscoelastic fluids. Scale bar is 40 pm.

Fig. 2. Numerical simulation of streaming situation among pillar array within various concentrations of viscoelastic fluids. (a)~(d) Numerical simulation of the fluid flow velocity profile. The colormap shows the fluid velocity and the black arrows show flow direction presents the pillar diameter and d₂ means the pillar center point distance. (e)~(h) Simulated first normal stress difference [N ) gradient among pillar array within PEO concentrations from 0 wt% to 0.15 wt%. The colormaps show the magnitude of N₁ . White two-way arrow represents the vibration direction. Scale bar is 40 pm.

Fig. 3. Fluorescence microscopic images of particle behavior within various PEO concentrations and maximum flow velocity comparison. (a)~(d) 800 nm particle behavior within 0 wt% ~ 0.15 wt% PEO at frequency of 800 Hz. Scale bar is 40 pm. (e)~(g) shows the simulated and experimented bulk vibration-based microstreaming and maximum flow velocity comparison within 0 wt% PEO. White double arrow represents the vibration direction.

Fig. 4. Fluorescence microscopic images of differently sized particles behavior within 0.1 wt% PEO at vibration frequency of 800 Hz: (a) 3 pm; (b) 1 pm; (c) 800 nm and (d) 500 nm. White double arrow represents the vibration direction. Scale bar is 40 pm.

Fig. 5. (a) Photograph of one fabricated microfluidic micro-rotors device (b) 3D design drawing of micro-rotors with various arm numbers (c) Schematic of the controlled vibration- induced actuation of microfluidic multiple micro-rotors setup. The vibration stage can manipulate the glass stage movement both in X and Y directions. Patterns shown inside the red box are representative design of multiple micro-rotors (can range from 1- to 6-arm) (d) Side view of the 2-arm micro-rotor. The diameter of the stator body is 100 pm. The distance between two endpoints of rotor is 360 pm and the diameter of the inner circle inside the rotor is 110 pm. The height for stator and rotor is 85 pm and 65 pm, respectively (e) Schematic of the controlled microstreaming induced by vibration actuation around the micro rotor shown with fluorescent microscopy image. Diameter of green fluorescent microparticle is 1 pm. Scaler bar is 100 pm.

Fig. 6. (a) Fluorescent microscopy images of 2-arm micro-rotor behavior controlled by vibration stage. Micro-rotor (2-arm) conducts counterclockwise rotation when frequency signal input from X direction (b) Micro-rotor (2-arm) conducts clockwise rotation when frequency signal input from Y direction (c) Micro-rotor (2-arm) conducts no rotation (achieving balance state) when frequency signals input both from X and Y directions. Diameter of green fluorescent microparticle is 1 pm. Scaler bar is 100 pm. (d) Micro-rotors with 1- to 6- arm fabricated through two-photon polymerization. Scaler bar is 100 pm. (e) Relationship between angular speed and micro-rotor arm numbers under input signal frequency of 800Hz. Error bars are calculated from three or more repeated experiments.

Fig. 7. Schematic of the ultrasonic microstreaming device with a microstructure array embedded in a microfluidic chamber. Two PZT transducers are firmly affixed onto the glass substrate for generating ultrasonic waves to induce the vibration of the microstructures. The circular and rectilinear vibration mode can produce a closed-loop circulating flow (a) and four symmetric vortices with respect to the center of a microcylinder (b), respectively.

Fig. 8. A closed-loop circulating ultrasonic streaming flow around a microcylinder in a circular vibration mode (a) Numerical simulation of the circulating ultrasonic streaming flow (b) Experimental observation of a neighboring 5 pm fluorescent microparticle circulating about the cylinder dragged by the streaming flow. The microscopic image is obtained by superimposing 10 frames recorded every 0.2 s. The scale bars are 20 pm. (c) Numerical simulation (solid line) and experimental measurement (red dots) of the streaming flow velocity as a function of the distance from the cylindrical center.

Fig. 9. Ultrasonic streaming flow around a microcylinder in a rectilinear vibration mode (a) Numerical simulation of four symmetric vortices with respect to the cylindrical center (b) Experimental observation of a circulating motion of a single 5 pm microparticle within one of the vortices. The microscopic image is obtained by superimposing 15 frames recorded every 0.2 s. The scale bars are 20 pm.

Fig. 10. Ultrasonic microstreaming produced by a double-slit microstructure in circular and rectilinear vibration modes (a) Numerical simulation of a closed-loop circulating streaming flow around the double-slit microstructure in a circular vibration mode (b) The closed-loop streaming flow circulates neighbouring microparticle around the double-slid microstructure. The field of view refers to the dashed rectangle in (a) (c) Numerical simulation of multiple circulating vortices surrounding the double-slit microstructure in a rectilinear vibration mode (d) Microparticle trap and circulation inside one of the vortices (e) A ~15 pm MCF-7 cell trapped within the vortex and rotated about itself. The field of view refers to the dashed rectangle in (c). The scale bars are 20 pm.

Fig. 11. Particle transport with ultrasonic streaming along complex trajectories defined by four alphabet letters. Numerical simulation of the closed-loop circulating streaming flow around the four letters, S (a), U (b), T (c) and D (d), in a circular vibration mode. Superimposed trajectories of 5 pm fluorescent microspheres moving around the four letters, S (e), U (f), T (g) and D (h), in a circular vibration mode. The scale bars are 50 pm.

Fig. 12(a) and (b) are schematic drawings of the microfluidic device according to an embodiment of the invention.

This invention presents the target (particle/cell/micro-components) with micro/submicron- scale manipulation through bulk vibration-based method. Precise particles and cells manipulation with micro/nanoscale is a crucial subject in the microfluidics community because of their great application in clinical and biomedical fields. Specifically, submicron particle manipulation with acoustic field typically have to involve the transducer with very high frequency (~MHz). Considering the high cost of interdigital transducer (IDT) manufacturing, cumbersome acoustofluidics chip fabrication and cleaning procedures, researchers are keep trying to reduce the experiments cost and simplify the whole microchip pre-preparation procedures on particle manipulation with micro/submicron level. Thus, bulk vibration-based manipulation induced with piezoelectric vibration stage or patched- transducer provide the great solutions. Compared with traditional IDT, which are typically limited by localized impact region due to their initial design, bulk vibration-induced manipulation can achieve batches of simultaneous and uniform particle manipulation. Also, the vibration stage and disposable patched-transducer allow the freely exchanging of microchips and flexible frequency tuning, thus greatly save the potential cost in real application. In this invention, for the first time, we demonstrate the submicron particle patterning and focusing and micro-rotor actuation with bulk vibration-based manipulation using ultra-low frequency (800 Hz), also the precise single microscale particle/cell control with patched-transducer. The developed particle/cell/micro-components manipulation method exhibits both simplicity and uniformity with low-cost nature, significantly broaden the target control way and potential applications in various biomedical fields.

The device of the present invention performing the method of the invention may be a portable device for simultaneously and uniformly micro or submicron scale target manipulation applicated in biomedical related fields. The invention utilizes acoustic vibration induced microstreaming for navigating microscale objects along arbitrary trajectories and controllably rotating them in microfluidics, which however has not yet been achieved till now.

In particular, for the first time, we demonstrate the submicron particle patterning and focusing with bulk vibration-based manipulation using ultra-low frequency (800 Hz) within viscoelastic fluid. The non-Newtonian viscoelastic fluids have already been extensively applicated into microfluidics microparticles/cells sorting and submicron scale target such as exosomes and DNA molecules. Compared with other polymer (polyvinylpyrrolidone (PVP) and polyacrylamide (PAA)) which normally is utilized to produce the viscoelastic fluids with stable properties and great biocompatibility, polyethylene oxide (PEO) is chosen here not only due to the aforementioned advantages, more importantly, its intermediate molecular weight exhibiting strong viscoelastic with a weak shear-thinning effect. Here, a micropillar array placed on the piezoelectric vibration stage will generate simultaneous and uniform axis- symmetric acoustic microstreaming around each pillar with rectilinear vibration. Compared with traditional IDT, which are typically limited by localized impact region due to their initial design, bulk vibration-induced boundary microstreaming around micropillar array can achieve batches of simultaneous and uniform particle manipulation. Also, the vibration stage allows the freely exchanging of microchips and flexible frequency tuning, thus greatly save the potential cost in real application. Numerically and experimentally methods are adopted here to explore the particle manipulation affected by the cooperation between bulk vibration- induced microstreaming and viscoelastic fluid introduced elastic lift force. The developed submicron particle manipulation method exhibits both simplicity and uniformity with low- cost nature, significantly broaden the particle control way and potential applications in various biomedical fields. Fig. 12(a) shows a microfluidic device 5 according to an embodiment of the invention. Here, the microfluidic device comprises a first substrate 10. An inlet 15, an outlet 20, and a channel or chamber 25 are defined in the first substrate 10. The inlet 15, chamber 25 and outlet 20 are in fluid communication such that a fluid suspension 45 will flow through the device 5 in the direction A so by the arrow, i.e. from the inlet 15 through to the chamber 25, and finally out of the device 5 via the outlet 20. Disposed in the chamber 25 are micro-structures 30. The first substrate 10 is disposed on the surface of a second substrate 35. The first substrate 10 and the second substrate 35 are bonded together by any suitable means to form the microfluidic device 5. At least one transducer40 (2 shown in Fig. 12a) is disposed adjacent the second substrate 35, said transducer 40 is adapted to generate and deliver ultrasonic waves along an X-axis and/or Y-axis into the chamber 25 so as to manipulate a particle (a microparticle) present in a fluid suspension 45 that flows through the device 5.

In an alternative embodiment shown in Fig. 1, and as will be described in further detail below, the bonded first substrate 10 and second substrate 35 is disposed or mounted on a vibration stage 50 instead of the transducers 40. The vibration stage, with the relevant built-in electronics, is equivalent to the two transducers 40 shown in Figs. 7 and 12.

As shown in the figure, the second substrate 35 is a block having a substantially rectangular shape. The cuboidal block has top and bottom surfaces and four sides. In an embodiment, a transducer 40 is disposed on each of two adjacent sides of the second substrate 35 (as shown in Fig. 12a) to generate and deliver ultrasonic waves along an X-axis and/or Y-axis into the chamber. The two transducers 40 may be disposed on the adjacent sides of the second substrate 35 that forms right angles with each other, so that if one transducer is on, it generates waves in a particular direction. If both transducers are on, then the waves meet at right angles. In various embodiments, the transducer 40 may be a piezoelectric patched- transducer. The transducer 40 is adapted to generate and deliver ultrasonic waves having a frequency less than or equal to 1000 kHz. Preferably, the transducer 40 is adapted to generate and deliver ultrasonic waves having a frequency is 50 kHz.

In the embodiment shown in Fig. 12(a), the micro-structures 30 are cylindrical in shape. Fig. 12(b) shows the device 5 in an alternative embodiment where the micro-structures 30 are rotors having a hub and propellers. In this application, terms such as pillars, cylinders etc. may be used to refer to the micro-structures 30.

The figure also shows that there are a plurality of micro-structures 30 disposed in the chamber 25 such that the plurality of the micro-structures 30 are equally spaced apart to form a grid array. Here, in Fig. 12a, it is a 7 x 4 grid. The distance between each micro-structure in the array may be about between 10-500 pm. The size of the grid may be 7 mm x 1 mm. The diameter and height of each micro-structure 30 may be about between 10-300 pm.

In Fig. 12(b), the propeller blade of each rotor is curved in a clockwise or counter-clockwise direction and tapers at an end opposite the rotor hub. Such a shape configuration resembles that of a ninja's shuriken. Alternatively, the rotor can have any number of propeller blades and shape configurations of the blades. Figs. 5a and 6 show the various numbers of propeller blades that may be included in the shuriken. The rotor is adapted to rotate in a counter clock wise direction when a frequency signal input of the transducer is in an X direction, and the rotor is adapted to rotate in a clock-wise direction when the frequency signal input of the transducer is in a Y direction.

In various embodiments, the first 10 and second 35 substrates are made of a material suitable for conducting an acoustic wave. The material is any one selected from the group comprising: PDMS, glass, polymethylmethacrylate (PMMA), and polycarbonate (PC).

In various embodiments, the second substrate 35 is 1mm thick.

Fig. 7 shows a perspective photo image of the device 5 shown in the schematic Figs. 12(a) and 12(b).

The invention also provides a method for manipulating a particle in a fluid suspension using the device 5. The method includes (a) providing a chamber 25 defined in a first substrate 10, at least one micro-structure 30 disposed in the chamber 25; (b) providing an inlet 15 in fluid communication with the chamber 25 disposed at one end of the chamber 25, the inlet 15 for receiving the fluid suspension; (c) providing an outlet 20 in fluid communication with the chamber 25 disposed at an end opposite the inlet 15, the outlet 20 for discharging the fluid suspension; and (d) providing a second substrate 35 having a surface, the first substrate 10 is disposed on the surface of the second substrate 35; and (e) manipulating the particle in the chamber 25 by introducing the fluid suspension to the chamber 25 via the inlet 15 and by providing at least one transducer 40 disposed adjacent the second substrate 35 to generate and deliver ultrasonic waves along an X-axis and/or Y-axis into the chamber 25.

In various embodiments, the second substrate 35 has a substantially rectangular shape having top and bottom surfaces and four sides, and a transducer 40 is disposed on each of two adjacent sides of the second substrate 35 to generate and deliver ultrasonic waves along an X-axis and/or Y-axis into the chamber 25.

Advantageously, the particle is suspended in a suspension of a non-Newtonian viscoelastic fluid. In an embodiment, the fluid is polyethylene oxide. The concentration of the polyethylene oxide may be between 0.01 to 5 wt%.

As with the description above, the transducer 40 is adapted to generate and deliver ultrasonic waves having a frequency less than or equal to 1000 kHz. A plurality of micro-structures 30 are disposed in the chamber 25, the plurality of the micro-structures 30 are equally spaced apart to form a grid array.

Each of the micro-structure 30 is substantially cylindrical in shape. The distance between each micro-structure 30 in the array is about between 10-500 pm. The diameter and height of each micro-structure 30 may be about between 10-300 pm.

The micro-structure 30 is a rotor and each rotor comprises a hub and at least one propeller blade. The propeller blade of each rotor is curved in a clockwise or counter-clockwise direction and tapers at an end opposite the rotor hub. The rotor is adapted to rotate in a counter clock-wise direction when a frequency signal input of the transducer 40 is in an X direction, and the rotor is adapted to rotate in a clock-wise direction when the frequency signal input of the transducer 40 is in a Y direction. The transducer 40 may be a piezoelectric patched-transducer. Going further to describe in detail the invention, Fig. 1 shows the schematic design of bulk vibration-based submicron particle manipulation setup, in which a pillar array 35 microfluidic device 5 is affixed onto a piezoelectric vibration stage 50 that can be oscillated in both X and Y directions. The microfluidic chip 5 was fabricated using a standard PDMS soft-lithography process, in which the master mold for PDMS casting was fabricated with SU-8 (SU-8 2025, MicroChem, Newton, MA, USA) on a silicon wafer. Later the prefabricated microchip layer together with the glass substrate were treated under air plasma (Harrick Plasma PDC-32G, Ithaca, NY, USA), then were brought into contact to form the permanent bonding and closed channel. The microfluidic chip 5 is affixed onto a piezoelectric vibration stage 50 that introduces oscillations along the X and Y axes with arbitrary waveforms through a function generator A sinusoidal wave input signal was amplified through a piezoelectric ceramic controller before applying to the piezoelectric vibration stage. The micropillar array 30 possess the diameter of 40 pm (c^ in Fig. 2a) for each pillar and a height of 40 pm, with pillar center point distance of 120 pm (d₂ in Fig. 2a) in both horizontal and vertical directions. The vibration-induced acoustic streaming flow fields around the pillar array and the first normal stress difference (jV- were simulated using a finite element method (FEM)-based numerical model (COMSOL Multiphysics 5.3, USA), and the laminar flow module with a steady-state study was adopted. The model had the same geometric dimensions shown in Fig. 2a. N₁ is derived via Oldroyd-B model as N₁ = 2h_rlg², where h_r and l is the polymeric contribution to the viscosity of a diluted PEO solution and the relaxation time, respectively y represents the fluid shear rate over channel cross-section, which is defined as (2D: D)z. Here D refers to the deformation rate tensor and is expressed as D ⁼ U means the

streaming flow velocity³⁵.

After the setup of microfluidic device 5, the particle suspension sample 45 was then infused slowly into the microfluidic cavity or chamber 25 from the inlet 15 (see Fig. 1). Fluorescent polystyrene spheres (3 pm, 1 pm, 800 nm and 500 nm, in diameter) were purchased without any further modification (Magsphere, USA). The particles were diluted with Dulbecco's phosphate-buffered saline (DPBS, Thermo Fisher Scientific, USA) containing 0.6% Pluronic F127 (Sigma-Aldrich, USA) to avoid particle agglomeration and adhesion onto the channel / chamber wall. Each individual experiment was conducted with a new microfluidic device 5 to avoid cross-contamination and residual particles or bubbles adhered in used devices. The PEO solutions were made by dissolving PEO (M_w = 600 kDa, Sigma-Aldrich, USA) powder into DPBS with concentrations of 0.05 wt% (500 ppm), 0.1 wt% (1000 ppm) and 0.15 wt% (1500 ppm). After adding PEO powder into DPBS, the solution was gently stirred overnight to keep uniform solution property. The bulk vibration-based submicron particle manipulation was recorded using a CCD camera on an inverted microscope (Olympus, CKX53, Japan) and a high-speed camera (Photron Inc., San Diego, CA, USA) to capture the particle behavior.

In these results, we demonstrated the submicron particle patterning and focusing with bulk vibration-based manipulation using ultra-low frequency within viscoelastic fluid. In order to understand the mechanism, we first conducted the numerical simulation of streaming fluid flow and N₁ gradient map generated around pillar array with various PEO concentrations fluids. Only four pillars among the pillar array were randomly presented in Fig. 2 to clearly show the flow streaming and N₁ details. With horizontal direction vibration of the piezoelectric stage, four axis-symmetric microstreaming group will occur around the pillar (Fig. 2a~2d). Among which two counter rotating microstreaming appear on each side of the pillar, pointing from the pillar center to outside along with the rectilinear vibration direction. Fig. 2a~2d also reveals the microstreaming flow velocity changing with the increase of viscoelastic fluid concentrations. From 0 wt% to 0.15 wt% PEO, a quantitative comparison shows the maximum flow velocity has decreased around ~1.3, ~1.5 and 2 times with every 0.05 wt% PEO concentrations increasing. Thus, the streaming flow velocity presents an inverse ratio to the PEO concentration, implying that the addition of small amount large molecules polymer can tune the microstreaming flow. Due to the absorption of vibration energy by the fluid medium and inevitable attenuation, the vibration-induced streaming flow around the pillar will form when the affixed stage vibrated with rectilinear motion. The streaming flow will also induce a drag force on the suspended particle, F_D = 3pmάn, where m is the dynamic viscosity, d refers to the particle diameter and v is the particle velocity relative to the flow. Generally, the streaming flow velocity is proportional to the power of the input source. The input peak-to-peak voltage was fixed at the maximum value 10V with offset of 5V and amplified by piezoelectric ceramic controller (output voltage: 0~120V) at the maximal capacity for all experiments here. Fig. 2e~2h presents the

gradient difference among various PEO concentrations fluids. In non-Newtonian viscoelastic fluids, an additional elastic lift force ( F_E ) generated on particles will affect the equilibrium focusing positions. The F_E exerted on the particle pointing to the smaller shear rate region can be expressed as F_E = C_£d³V/V₁~d³(V t_cc — VT₂₂), where C_E refers to the non-dimensional elastic lift coefficient. N₁ = t_cc — t₂₂ presents the tension along main flow direction, where t_cc and t₂₂ represents the normal stress in flow and velocity gradient direction. According to previous listed Oldroyd-B model N₁ = 2h_rlg² , polymeric viscosity h_r for Newtonian fluid with 0 wt% PEO is zero, i.e., no N₁ gradient. The relaxation time l of the PEO solution increase with c⁰·⁶⁵ (c refers to PEO concentration), h_r increase with c and g can be viewed as an average constant in PEO solutions at low concentrations (0.05~0.15 wt% in current study), thus increasing c can enhance the F_E and lead to tuning of the particle manipulation. From 0.05 wt%~0.15 wt% PEO, the N₁ gradually throw its weight and the maximum N₁ in 0.1 wt% PEO is around three times larger than the valve in 0.05 wt% PEO, implying the F_E exhibit rare effect on particle trajectory within 0.05 wt% PEO fluid. Noted that the F_E is directly related to the previous described tensor D, which is contributed by streaming velocity U. Based on the previous analysis, we can achieve the particle focusing manipulation by tuning the cooperation of both streaming flow induced drag force (F_d ) and elastic lift force (F_E) at various PEO concentrations. With the horizontal rectilinear vibration, particles initially flow along with the microstreaming trajectory will be affected by F_E and then apply focusing near the region where N₁ pointed, i.e., the two horizontal region where possess the minimum shear rate at two sides of the pillar. For 0.15 wt% PEO with larger N₁ affected region, the even slower streaming flow velocity with higher fluid viscosity have to be taken into consideration, as the extremely low flow velocity will lead to the sluggish moving of the particle.

The previous numerical simulation results and analysis were continued verified with following experiments. Fig. 3a~3d revealed the different 800 nm particle behavior within various PEO concentrations at horizontal vibration frequency of 800 Hz. From Fig. 3a~3d, it was observed that within 0 wt% PEO, axis-symmetric streaming flow appeared as the same form presented in the simulation results. Along with the concentration increasing, particle flow velocity exhibited relative slowness. In 0.1 wt% PEO (Fig. 3c), the 800 nm fluorescent particles were patterned and focused nicely besides the pillar, presenting exactly the same form as we predicted before. From Fig. 3d, it was observed that the particle could hardly move with high fluid viscosity in 0.15 wt% PEO at frequency of 800 Hz, while the particle initially stayed around the pillar still could be pushed into the target region. Thus, the focusing efficiency in 0.15 wt% PEO was far from the perfect form appeared within 0.1 wt% PEO. By tuning the PEO concentration, we found the optimized cooperation of streaming flow induced F_D and F_E on submicron particle manipulation. Fig. 3g presented the maximum flow velocity comparison of simulated and experimented bulk vibration-based microstreaming, which exhibited monotonically increasing along with the input frequency rising. Fig. 4a~4d showed the differently sized particle focusing and patterning performance within 0.1 wt% PEO at 800 Hz. 3 pm, 1 pm and 800 nm all performed excellently focusing ability. The 3 pm particle focusing dot besides pillar was relative dim compared with 1 pm and 800 nm due to the initial particle fluorescent intensity and lower stock concentration density.

In this section, we established the optimized cooperation of bulk vibration-based streaming flow induced F_D and F_E, and even demonstrated the submicron particle patterning and focusing using ultra-low frequency (800 Hz) within viscoelastic fluid. This novel phenomenon and method enable a versatile easy and low-cost multiple submicron particle manipulation. The remote, simultaneous batches submicron particle manipulation with uniformity on a single piezoelectric vibration stage greatly simplifies the microchip device fabrication procedure and enhances flexibility, significantly broaden the promising applications in various biomedical fields.

In this invention, we combine the acoustically vibration-induced actuation and two-photon polymerization fabrication to achieve controllable simultaneously and uniformly manipulation of multiple micro-components. In alternative embodiments, the microfluidic device of the present invention may be fabricated using a nano-scale 3D printing method.

Taken the micro-rotor as example, Fig. 5 shows the schematic design of acoustically vibration- induced actuation achieving uniform multiple micro-rotors manipulation. The paired asymmetric acoustic streaming generated by series of curved sharp tips on micro-rotor will form the net torque to propel rotor rotation. The micro-rotor rotation speed can be easily tuned with input frequency and rotor arm numbers (as shown in Fig. 6), also the input signal direction can control the rotor rotation orientation. In this section, the invention presents the remote simultaneously and uniformly control of multiple micro-rotors with piezoelectric vibration stage with ultra-low frequency (800 Hz), which is needless for involving of transducer, greatly simplify the micro-rotor device fabrication procedure and enhance flexibility. Meanwhile, no specific requirement of aqueous solution in the micro-rotor device together with versatile fabrication and low cost will provide great potential application in practical biomedical field.

Precise manipulation of microscale objects (e.g. biological cells, polymer beads, and multi-cell cellular organisms) becomes an essential technique in biomedical analysis, which benefits a variety of fields including cell characterization, drug discovery, and tumor heterogeneity. Microfluidics with the focus on studying fluid control in microscopic length scale has led to the creation of powerful tools for biomedical analysis, as well as enabled a variety of microparticle manipulation methods, including magnetophoresis, optical tweezing, dielectrophoresis, and acoustophoresis. In magnetophoresis method, manipulation efficiency is limited by the complexity of cell sample pretreatment, where cells commonly need to be conjugated or embedded with magnetic particles. Optical tweezers are able to trap and transport individual cells via a high-power laser, which however may cause irreversible damages to biological cells and compromise their viability and functionality. Dielectrophoresis method requires the use of strong electric fields that might also cause damages to biological cells by Joule heating. Acoustophoresis makes use of the interactions between acoustic waves and fluids or particles for precise microscale manipulation. Benefiting from its low power consumption and excellent biocompatibility with cell manipulation, it recently has emerged as a promising technique for non-contact particle manipulation in microfluidics.

On-demand controllable movement of selected microparticles, either navigating to specific positions via a pre-defined trajectory or rotating about a pre-defined axis, has become a useful and important technique in microfluidic manipulation. In this section, we presented a microfluidic device 5 that enables controllable transport and rotation of individual microparticles 45 and cells via ultrasonic streaming flow effect produced by 50 kHz oscillation of embedded microstructures 30. The ultrasonic (i.e. >20 kHz) streaming flow field is dependent on the shape and dimensions of microstructures, as well as the ultrasonic vibration mode. Numerical modeling has been conducted to study the effects of these factors on the produced ultrasonic microstreaming in the microfluidic device. In particular, we have demonstrated the ability to transport individual microparticles along pre defined trajectories by actuating ultrasonic transducers 40 affixed to the microfluidic device 5. In-situ capture and rotation of individual cells has also been demonstrated via a similar ultrasonic streaming effect in the microfluidic device 5. The work presents a novel method for precise manipulation of particles and cells 45 in microscale, which could enable new capabilities for single-cell analysis.

Fig. 7 depicts the ultrasonic streaming flow produced in a microfluidic device 5 via two different vibrational modes. As shown in Fig. 7a, a polydimethylsiloxane (PDMS) made microfluidic chamber 25 is bonded on a glass substrate, where two Pb based Lanthanum doped Zirconate Titanates (PZT) transducers 40 are affixed onto the boarders of the glass substrate along with X-axis and Y-axis. When the PZT transducers are actuated with alternating current (AC) electric signals, they can generate ultrasonic waves propagating along X-axis and Y-axis directions of the glass substrate, respectively. The ultrasonic waves then propagate into the PDMS microstructures via their firm bonding with the glass substrate, driving the microstructures oscillation along with the wave propagation. As the fluid- microstructure interface oscillates, the ultrasonic energy dissipates into the boundary layer of surrounding fluid in proximity to the structure boundary. As a result, a steep velocity gradient along the direction perpendicular to the interface builds up, and a strong streaming flow is produced inside the boundary layer, called the inner boundary layer streaming. Streaming in the bulk of the fluid, known as the outer boundary layer, is then induced by the inner boundary layer streaming. The inner and outer boundary layer streaming are also known as Schlichting streaming and Rayleigh streaming, named by the two scientists who firstly mathematically modelled the two effects, respectively. When the X-axis and Y-axis propagating ultrasonic wave has a half-p phase difference, they couple as a circular movement of the microstructure, termed circular vibration mode. As shown in the Fig. 7b, this circular vibration mode generates an ultrasonic streaming flow as a circular profile along the counter of the microstructure. When only one PZT transducer is actuated, the ultrasonic wave works as a rectilinear vibration mode to oscillate the microstructure, then a four-vortices streaming flow is generated aside the microstructure. Due to the fluid drag force on the microparticles, they flow along the ultrasonic streaming flow, i.e. circular and four-vortices movements in the circular and rectilinear vibration modes, respectively.

Materials and Methods

Ultrasonic microfluidic device

The ultrasonic microfluidic device consists of a microfluidic channel for loading aqueous samples and two PZT transducers for producing ultrasonic waves. Microstructures were fabricated inside a 40 pm thick PDMS microfluidic chamber via a widely used soft-lithography technique with SU-8 photoresist (SU-8 25, MicroChem Corp., Newton, MA) on a silicon wafer. The PDMS microfluidic channel was then bonded onto a 1-mm thick glass substrate after an air plasma surface treatment. To generate the ultrasonic wave propagating along the glass substrate, two PZT transducers (Farnell elementl4, Leeds, UK) were firmly affixed onto the two perpendicular boarders of the glass substrate of the microfluidic channel, as shown in Figure 12a. The thin film electrodes of the PZT transducers were soldered to a signal generator (Tektronix, Beaverton, OR), from which 50 kHz AC signals were applied on the transducer for wave generation. Samples of cell suspension or polystyrene microsphere suspension was injected to the PDMS microfluidic chamber before the actuation of PZT transducers, and then the inlet and outlet of the microfluidic device were sealed by tape to ensure a stationary flow environment. The experiments were conducted under observation of an inverted fluorescence microscope with a CCD camera (Leica, Germany).

Numerical simulation Finite element method (FEM) based simulations were conducted using COMSOL Multiphysics 5.3 to study the ultrasonic streaming flow field. To reduce the computational cost, a two- dimensional model (X-Y plane as shown in Figure 12a) were used to perform the FEM simulation. The actual dimensions of the microfabricated structure along the X-Y plane were applied in the FEM model.

The simulation was implemented based on a numerical method developed and validated in Muller eto/.'s work. Generally, the numerical simulation consists of two steps, which compute the inner and outer boundary layer streaming in series. The first step employed Thermoviscous Acoustics module in frequency domain to solve the linearized compressional Navier-Stokes equations, the continuity equation, and the thermodynamic heat transfer equation. The PDMS microstructures, with the same geometry as experimental setup, are defined with periodic velocity boundary conditions for generating the vibration. This step was mainly to resolve the thermoviscous effect for calculating the inner boundary layer streaming in proximity to the oscillating microstructure. In the second step, the results calculated from the first step were used as the source terms to solve the Navier-Stokes equation in Laminar Flow module. The numerical solution of the second step was the ultrasonic outer boundary layer streaming flow field, which was typically observed under the microscope.

Sample preparation

Microparticles used in the manipulation demonstration were 5 pm red fluorescent polystyrene microspheres purchased from Magsphere Inc., Pasadena, CA, USA. These particles were suspended in deionized water, containing 0.5% (m/m) Pluronic F127 (Sigma- Aldrich) to avoid particle agglomeration and adhesion onto the channel wall.

A breast cancer cell line (MCF-7) was used in the demonstration of controllable cell rotation experiments. The cell line purchased from the American Type Culture Collection (ATCC Cat. No. HB-72) was cultured in Dulbecco's modification of Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin). The cells were collected from petri dish and suspended in Phosphate-Buffered Saline (PBS) solution containing 0.5% (m/m) Pluronic F127 (Sigma-Aldrich). Results and Discussion

Ultrasonic microstreaming around a cylinder array

A simplex micro-structure array was at first used as the microstructure to generate the ultrasonic microstreaming. In an embodiment, the micro-structure are cylindrical in shape so as to form a cylinder array. The cylinder array was constructed by 50 pm diameter cylinders arrayed in a triangular pattern, in which every three adjacent cylinders were allocated at the vertexes of an equilateral triangle with a central distance between each adjacent cylinder of 150 pm. The whole array area is 7 mm x 1 mm. As shown in Fig. 8a, the circular vibration mode produces an ultrasonic streaming flow field around the cylinder. A 5 pm fluorescent microparticle was dragged by the streaming flow to circulate around the cylinder in a circular vibration mode (Fig. 8b).

It should be noted that the frequency of the ultrasonic wave applied on the vibrating boundary is 50 kHz in the simulation, which is the same frequency as the AC signal applied on the PZTs. However, the amplitude of the boundary vibration in the simulation is difficult to be accurately measured in practical experiments, because the vibration amplitude is typically on the order of magnitude of ~10 nm that is far below the observation limit of optical microscope. Thus, the boundary vibration amplitude was calibrated by matching the maximum streaming flow velocity in the simulation to the experimental measurement of particle velocity in the ultrasonic streaming. In the experimental measurement, the average instantaneous velocity of the circulating fluorescent microsphere (an example shown in Fig. 8b) was derived based on more than 2500 frames of images captured at a frame rate of 100 fps. Then a boundary vibration amplitude swept from 100 to 600 nm with a step of 10 nm to match the simulated streaming flow with experimental measurements. It was found that the boundary vibration amplitude of 280 nm best fit the instantaneous velocities from experimental results. Fig. 8c shows a quantitative agreement of the streaming flow velocity as a function of the distance from the cylindrical center between numerical simulation with 280 nm vibration amplitude and experimental measurement. The ultrasonic streaming flow field produced by the cylinder array in a rectilinear vibration mode is shown in Fig. 9. The numerical simulation (Fig. 9a) shows that the rectilinear vibration mode produces four symmetric vortices with respect to the cylindrical center, which agrees with the experimental observation. In particular, Fig. 9b shows the circulating motion of a single 5 pm microsphere within one of the four vortices about the cylinder, demonstrating the potential of this ultrasonic streaming effect to trap and rotate microparticles.

Cell trapping and rotation by ultrasonic streaming

Having understood the ultrasonic streaming around microcylinders, we then studied the microstreaming around an array of double-slit microstructures which have been demonstrated for high-throughput hydrodynamic cell trapping at the single-cell level. By "double-slit", it is meant to refer to that configuration and arrangement of the micro structures as shown in Fig. 10 (a to c). In these figures, the micro-structures are arranged in a "camber" configuration where the micro-structures are angled or tilted to form a curved outline while maintaining a distance between each other. Similar to the previous microcylinders array, the circular and rectilinear vibration modes also produce different forms of the ultrasonic streaming flow fields around the double-slit microstructures. Fig. 10a shows the simulated streaming flow field around a single double-slit microstructure unit in a circular vibration mode, which is still a closed-loop circulating flow with varying velocity magnitude along its circumference. Although the streaming flow velocity is weakened in the space between adjacent sub-structures, this closed-loop circulating flow can still circulate neighboring particles around the double-slit microstructure unit, as demonstrated in Fig. 10b.

In contrast, when the single double-slit microstructure unit was actuated in a rectilinear vibration mode, the streaming effect formed two circulating vortices aside the inner part of the double-slit microstructures, as shown by the simulation in Fig. 10c. Neighboring small particles (5 pm microsphere) were thus trapped and circulated inside the vortex, as shown in Fig. 10c. When a larger ~15 pm MCF-7 cell was aside the vortex, because the cell size was comparable to the overall size of this streaming vortex, the single cell rotated about itself instead of a circulating motion, as shown in Fig. lOe. Versatile particle transport by ultrasonic streaming

In addition to the capability of cell capture and rotation, versatile transport of microparticles along an arbitrary pre-defined trajectory is also one of the essential manipulation capabilities highly demanded in single-cell level studies. To demonstrate the capability of versatile particle transport in a 2D microscale space using ultrasonic streaming, we next showcased the ability to move individual particles along trajectories defined by four alphabet letters, S, U, T and D, as shown in Fig. 11. These alphabet letters were constructed by a set of smaller rectangles and trapezoids with close spacings, which were fabricated by a standard soft-lithography technique. These "rectangles and trapezoids" form the micro-structures according to this invention. The 4 alphabet letters may be considered as arbitrary complex trajectories formed by the micro-structures. Fig. lla-d show that the circular vibration mode can produce a closed-loop circulating flow around the overall letter structure, which is generally similar to Fig. 10a. The 5 pm red fluorescent microspheres were then introduced into the microfluidic chamber with these letter structures. Fig. lle-h show the superimposed trajectories of microspheres in close proximity to these letter structures in a circular vibration mode. It was found that these neighboring small particles circulated around the outer borderline of these letter structures. As a result, their superimposed trajectories represented the outlining of the four alphabet letters, forming SUTD, as shown in Fig. lle-h. This demonstration reveals the capability of transporting microparticles along any arbitrary trajectory using the ultrasonic streaming effect.

Conclusion

In this invention, a versatile ultrasonic microstreaming-based manipulation method that enables active and precise control of transport and rotation of individual microscale particles and biological cells in a microfluidic device is presented. We have further showcased the ability to transport individual microparticles along the outlines of complex alphabet letters, demonstrating its versatility and simplicity of single-particle level manipulation with a bulk vibration. The developed ultrasonic streaming based precise particle manipulation could provide new capabilities for single-cell level analysis. Different with the prior art, we have extended the application not only limited to certain cell, but also applied for submicron particles manipulation and various micro-components. For the first time, we demonstrate the submicron particle patterning and focusing with bulk vibration-based manipulation using ultra-low frequency (800 Hz). This submicron particle manipulation with ultra-low frequency (< lk Hz) can also be extended to microscale. This novel phenomenon and method enable easy and low-cost particle manipulation with simplicity and uniformity, significantly broaden the promising applications in various biological and biomedical fields.

We also present a versatile patched-transducer (50 kHz) based manipulation method that enables active and precise control of transport and rotation of single microscale particles and biological cells in a microfluidic device. We have further showcased the ability to transport individual microparticles along the outlines of complex alphabet letters, demonstrating its versatility and simplicity of single-particle level manipulation with a bulk vibration. The developed ultrasonic streaming based precise particle manipulation could provide new capabilities for single-cell level analysis.

Among the acoustically actuation micro-rotor, this invention was also different from an earlier work with the micro-rotor fabricated through UV polymerization, where the rotor was capable to rotate within ethanol medium to avoid adhering to stator axle or substrate. Our micro-structures, including the rotor propellers, possess distinct geometry and is fabricated with two-photon polymerization, which could freely rotate within the Dl water/PBS or any other aqueous solution, meanwhile embraced the ability of three-dimensional modification and relatively high rotation speed, therefore greatly extended the application in biomedical and other related fields.

The key advantage/improvement over existing state-of-the-art methods is that here for the first time, we demonstrate the submicron particle patterning and focusing with bulk vibration-based manipulation using ultra-low frequency (800 Hz). This submicron particle manipulation with ultra-low frequency ( < lk Hz) can also be extended to microscale. Compared with existing high energy consumable techniques, which are typically limited by localized impact region due to their initial design, bulk vibration-induced method can achieve batches of simultaneous and uniform particle manipulation. Also, the vibration stage allows the freely exchanging of microchips and flexible frequency tuning, thus greatly save the potential cost in real application. This means that microchip substrate containing the micro- structures can be detached and removed from the glass substrate it is attached to such that the glass substrate and the transducers may be re-used. We achieved not only the submicron particle and micro-rotor manipulation with ultra-low frequency (< lk Hz), but also precisely manipulated the single microscale target movement, even formed the complex alphabet letters. This novel phenomenon and method enable a versatile easy and low-cost single/multiple particle manipulation. The remote, simultaneous batches particle/micro components manipulation with uniformity on a single bulk vibration-based stage greatly simplifies the microchip device fabrication procedure and enhances flexibility, significantly broaden the promising applications in various biomedical fields. Whilst there has been described in the foregoing description preferred embodiments of the present invention, it will be understood by those skilled in the technology concerned that many variations or modifications in details of design or construction may be made without departing from the present invention.

Claims

1. A microfluidic device for manipulating a particle in a fluid suspension, the device comprising:

(a) a chamber defined in a first substrate, at least one micro-structure disposed in the chamber;

(b) an inlet in fluid communication with the chamber disposed at one end of the chamber, the inlet for receiving the fluid suspension;

(c) an outlet in fluid communication with the chamber disposed at an end opposite the inlet, the outlet for discharging the fluid suspension; and

(d) a second substrate having a surface, the first substrate is disposed on the surface of the second substrate; wherein at least one ultrasonic wave generator is disposed adjacent the second substrate to generate and deliver ultrasonic waves to the chamber.

2. The device according to claim 1, wherein the second substrate having a substantially rectangular shape having top and bottom surfaces and four sides, the ultrasonic wave generator is disposed on the bottom surface opposite the first substrate to generate and deliver ultrasonic waves along an X-axis and/or Y-axis to the chamber.

3. The device according to claim 1, wherein the second substrate having a substantially rectangular shape having top and bottom surfaces and four sides, and an ultrasonic wave generator is disposed on each of two adjacent sides of the second substrate to generate and deliver ultrasonic waves along an X-axis and/or Y-axis into the chamber.

4. The device according to any one of the preceding claims, wherein a plurality of micro structures are disposed in the chamber, the plurality of the micro-structures are equally spaced apart to form a grid array.

5. The device according to any one of the preceding claims, wherein each micro structure is substantially cylindrical in shape.

6. The device according to any one of claims 2 to 5, wherein the distance between each micro-structure in the array is about between 10-500 miti.

7. The device according to claim 6, wherein the grid array is 7 mm x 1 mm.

8. The device according to any one of the preceding claims, wherein the diameter and height of each micro-structure is about between 10-300 pm.

9. The device according to any one of the preceding claims, wherein the micro-structure is a rotor and each rotor comprises a hub and at least one rotatable propeller blade attached to the hub.

10. The device according to claim 9, wherein the propeller blade of each rotor is curved in a clockwise or counter-clockwise direction and tapers at an end opposite the rotor hub.

11. The device according to any one of claims 9 or 10, wherein the rotor is adapted to rotate in a counter clock-wise direction when a frequency signal input of the ultrasonic wave generator is in an X direction, and the rotor is adapted to rotate in a clock-wise direction when the frequency signal input of the ultrasonic wave generator is in a Y direction.

12. The device according to any one of the preceding claims, wherein the ultrasonic wave generator is a vibration stage or a piezoelectric patched-transducer.

13. The device according to claim 12, wherein the transducer is adapted to generate and deliver ultrasonic waves having a frequency less than or equal to 1000 kHz.

14. The device according to claim 12, wherein the transducer is adapted to generate and deliver ultrasonic waves having a frequency is 50 kHz.

15. The device according to any one of the preceding claims, wherein the first and second substrates are made of a material suitable for conducting an acoustic wave.

16. The device according to claim 14, wherein the material is any one selected from the group comprising: PDMS, glass, polymethylmethacrylate (PMMA), and polycarbonate (PC).

17. The device according to any one of the preceding claims, wherein the second substrate is about 1mm thick.

18. A method for manipulating a particle in a fluid suspension, the method comprising:

(a) providing a chamber defined in a first substrate, at least one micro-structure disposed in the chamber;

(b) providing an inlet in fluid communication with the chamber disposed at one end of the chamber, the inlet for receiving the fluid suspension;

(c) providing an outlet in fluid communication with the chamber disposed at an end opposite the inlet, the outlet for discharging the fluid suspension; and

(d) providing a second substrate having a surface, the first substrate is disposed on the surface of the second substrate; and

(e) manipulating the particle in the chamber by introducing the fluid suspension to the chamber via the inlet and by providing at least one ultrasonic wave generator disposed adjacent the second substrate to generate and deliver ultrasonic waves to the chamber.

19. The method according to claim 18, wherein the particle is suspended in a suspension of a non-Newtonian viscoelastic fluid.

20. The method according to claim 19, wherein the fluid contains polyethylene oxide.

21. The method according to claim 20, wherein the concentration of the polyethylene oxide is about between 0.01 to 5 wt%.

22. The method according to any one of claims 18 to 21, wherein the ultrasonic wave generator is adapted to generate and deliver ultrasonic waves having a frequency less than or equal to 1000 kHz.

23. The method according to any one of claims 18 to 22, wherein a plurality of micro structures are disposed in the chamber, the plurality of the micro-structures are equally spaced apart to form a grid array.

24. The method according to claim 23, wherein each of the micro-structure is substantially cylindrical in shape.

25. The method according to claim 23, wherein the distance between each micro structure in the array is about between 10-500 pm.

26. The method according to any one of claims 18 to 25, wherein the diameter and height of each micro-structure is about between 10-300 pm.

27. The method according to any one of claims 18 to 26, wherein the micro-structure is a rotor and each rotor comprises a hub and at least one rotatable propeller blade attached to the hub.

28. The method according to claim 27, wherein the propeller blade of each rotor is curved in a clockwise or counter-clockwise direction and tapers at an end opposite the rotor hub.

29. The method according to any one of claims 27 or 28, wherein the rotor is adapted to rotate in a counter clock-wise direction when a frequency signal input of the ultrasonic wave generator is in an X direction, and the rotor is adapted to rotate in a clock-wise direction when the frequency signal input of the ultrasonic wave generator is in a Y direction.

30. The method according to any one of claims 18 to 29, wherein the ultrasonic wave generator is a vibration stage or a piezoelectric patched-transducer.

31. A device for manipulating a particle in a fluid suspension substantially as herein described with reference to any one of the examples or to any one of the accompanying drawings.