DEGREE PROJECT IN MEDICAL ENGINEERING, SECOND CYCLE, 30 CREDITS
STOCKHOLM, SWEDEN 2017
Microfluidic Chip development for acoustophoresis assisted selective cell sorting
Utveckling av mikrofluidiskt chip för akustoforesassisterad selektiv cellsortering
MOHD ADNAN FAQUI SHAHZAD
KTH ROYAL INSTITUTE OF TECHNOLOGY SCHOOL OF ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH
KTH ROYAL INSTITUTE OF TECHNOLOGY
SCHOOL OF TECHNOLOGY AND HEALTH
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www.kth.se
i ABSTRACT
Analysis of blood samples is one of the major steps in diagnosing pathological conditions like cancer.
The upstream sample preparation for the pathological cell analysis from complex biological fluid like blood, involves selective cell sorting. It can be achieved using fluorescently activated or magnetically activated cell sorters. Another way is to sort them using acoustophoresis which is cheaper, gives better spatial control and is also rapid apart from the fact that, it does not affect the cellular viability.6,9 In acoustophoresis, particles depending upon their density and compressibility relative to the suspended medium migrate to either pressure anti-nodes or nodes, when subjected to acoustic field. Poly vinyl alcohol-based microbubbles have a strong negative acoustic contrast factor and hence migrate to the anti-nodes in a standing ultrasonic wave. Previously, this property was utilized for cell separation by conjugating the bubbles to cells and subjecting them to ultrasonic waves in a silicon glass based microfluidic channel.55 A protocol for coating the microbubbles with avidin, so that these can readily attach to the cells has been developed in this work. However, microfluidic channel is obtained from a master mold which is developed in a clean room facility using photolithography. A cost-effective way has been developed for the production of a mold using a Computerized Numerical Control system (where the positive master for the microfluidic channel is drilled onto a PMMA sheet) for continuous separation of cancer cells. Alternate methods like a cutting plotter (which uses a double sided adhesive tape as a positive master) and a 3-D printer have been investigated, in order to be used as a mold for the microfluidic channel. As a proof, microbubbles-cell complex was focused in a PDMS based microfluidic channel, by utilizing standing Bulk acoustic waves. At flow rate of 10µl/min, efficiency greater than 80% has been achieved. This technique is low cost and can be implemented in places without a clean room facility for size independent cell sorting.
ii ACKNOWLEDGEMENTS
I am immensely thankful to Allah, The Almighty for every aspect of this work and I ask forgiveness for my shortcomings.
My supervisor Aman Russom, a big Thank You, for all the patience in supervising and mentoring my thesis all the way. All the inputs from the Monday group meetings from you have been very vital in the shaping up of this thesis. I can never thank you enough for giving me this opportunity to work under you.
Special thanks to Dmitry Grishenkov for the Microbubbles provided by you even when you were busy. They were very crucial for the beginning of the project.
All my colleagues in the Scilife lab especially Tharagan, Sharath, Harisha, Indra, Niklas, Zenib for being patient with me when I had so many questions in the lab and about the chemicals. Thank you for making me feel comfortable in the lab when I was new. It has been fun to work with you. All the time spent with you people made these months very enjoyable.
I would also like to thank Noa, Harsha, Kryzstof, Amin, Jorge, Martin, Gustav for being so kind and helpful in the Scilife lab.
Thanks to all my colleagues at STH, Flemingsberg for the great reviews during the meetings and especially Rodrigo Moreno for the dedication in reviewing our theses all the way from the start to the end.
Special thanks to Asim Faridi for all the guidance and help with the project. You are someone I look up to and have learnt many things from you in the time that we spent. You have been like an elder brother and I could not have asked for a better friend than you. My colleagues from the subcontinent, Ghufran and Sharif, we had a lot of fun together with Asim during your stay in Sweden. Those memories will always be cherished and stay with me forever.
Lastly my Parents, Grandmother and Sisters, to whom I dedicate this thesis to. You are always my strength. You encouraged me and supported me in every possible way. Without your prayers, the Masters studies was not possible.
iii List of abbreviations
CAD Computer aided drawing CAM Computer aided modelling CNC Computerized numerical control
EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide EpCAM Epithelial cell adhesion molecule
FITC Fluorescein isothiocyanate HCT Hematocrit
IDT Inter digital transducers
MES 2-(N-Morpholino)ethanesulfonic acid PBS Phosphate buffered saline
PDMS Poly dimethyl siloxane PLA Poly lactic acid
PMMA Poly methyl methacrylate PVA Poly vinyl alcohol RPM Rotations per minute 3D Three dimensional
iv Contents
ABSTRACT ... i
ACKNOWLEDGEMENTS ...ii
List of abbreviations ... iii
1. Introduction ... 1
2. Aim and Objectives ... 3
3. MATERIALS AND METHODS ... 4
3.1 Manufacturing of Microbubbles ... 4
3.2 Avidin coating on microbubbles ... 4
3.2.1 Oxidation of PVA Microbubbles ... 4
3.2.2 Hydrazide-Biotin coupling to Microbubbles ... 4
3.2.3 Binding Avidin to Biotinylated PVA Microbubbles ... 5
3.3 Conjugating Cells/Spheres and Avidin coated Microbubbles ... 5
3.4 Microfluidic Channel Design ... 6
3.4.1 3D printer ... 6
3.4.2 Cutting Plotter ... 7
3.4.3 Milling using a CNC system ... 7
3.5 Experimental Setup ... 8
4. Results ... 10
4.1 Avidin coating on the microbubble’s surface ... 10
4.2 Conjugation of Biotin coated spheres with Microbubbles ... 11
4.3 Mold from 3-D printer ... 12
4.4 Mold from Cutting Plotter ... 13
4.5 Mold from CNC milling ... 13
4.6 Bubble-cell complex under acoustophoresis and separation ... 14
5. Discussion ... 17
6. Conclusion and Future work... 18
APPENDIX ... 20
1 1. Introduction
Acoustophoresis is a phenomenon in which sound waves are utilized for the movement of particles in a medium. It is one among many other principles used for the manipulation of cells or particles in a microfluidic channel. The others being electrophoresis and magnetophoresis which uses electric forces and magnetic forces, respectively, for the motion of particles and thereby separation from the original mixture.6 The effects of Ultrasound on red blood cells were reported as early as 1971 by Dyson et al.61 In acoustophoresis the cells can be manipulated by the formation of ultrasonic standing waves which can be Bulk acoustic waves or Standing surface acoustic waves.62 Figure 1 illustrates the particle behavior when subjected to an ultrasonic standing wave. Johansson et al62 used Bulk acoustic waves for the first time, when a fluorescently labeled cell is detected by a camera to trigger an ultrasonic transducer. This causes the cell to change its path in the streamline towards a node and hence separating it through a different outlet.
Figure 1. Particle behaviour when a) no Ultrasonic wave is present, b) movement of particles due to ultrasonic standing wave to node(towards the center, represented by blue particles having a positive acoustic contrast factor) and antinodes (away from the center, represented by yellow particles having a negative acoustic contrast factor), c) particles in equilibrium after being exposed to ultrasonic standing wave, d) under flow conditions, the particles are separated in the microfluidic channels through the bifurcating channel. Image Source: Acoustophoresis60
Faridi et al55 used a glass silicone microfluidic chip with a single-outlet, single-inlet to acoustically manipulate a sample containing microbubble-cell conjugate, towards the antinodes, for separation from the original mixture. Here antibody functionalized, Poly(vinyl alcohol)-PVA, based microbubbles, which are gas filled and have a strong negative acoustic contrast factor54 were used. The microbubbles used were having a surface coating of Avidin, due to which they readily attach to biotinylated anti-EpCAM (epithelial Cell Adhesion Molecule). Here EpCAM was used as a cell surface marker to isolate circulating tumor cells like, HCT 116 colon cancer cells. The development of a robust protocol for the functionalization of the PVA based microbubbles is needed.
The use of a silicon-glass chip with microfluidic channels require expensive fabrication in a clean- room facility. An alternative, soft lithography, provides a non-expensive way of producing
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microfluidic channel device65, where an elastomeric material like Poly(dimethyl siloxane), PDMS63, is cast against a master, then peeled off and bonded to a glass slide64. However, the positive master’s manufacturing is done by photolithography. Instead, low cost methods like a 3D-printer or a double sided adhesive tape or milling using a computerized numerical control system66 can be investigated for the production of a positive master, on which PDMS can be poured and peeled off to obtain a microfluidic channel. Earlier works involving the usage of PDMS used Standing surface acoustic waves for the acoustic manipulation of the cells67. It needed the fabrication of metallic interdigital transducers on the surface of the piezoelectric substrate. This is again an expensive process. Another approach is to generate Bulk acoustic standing waves in a PDMS channel sandwiched between glass slides and then, use acoustophoresis to focus the microbubbles. If it can be done without the use of a master from photolithography, the cost of having a clean-room facility can be avoided and thereby, a decrease in the time taken to produce a mold in which PDMS can be cast. But, the thickness of the PDMS layer has to be controlled, as PDMS has a high attenuation coefficient for the ultrasonic waves.72,73
3 2. Aim and Objectives
The final aim of the project is to separate HCT116 cancer cells by attaching them to the avidin coated microbubbles. To achieve this a microfluidic channel is also needed. The objectives are divided as under:
a) Develop a protocol for the deposition of avidin on the surface of the microbubbles, in order to conjugate them to biotinylated anti-EpCAM antibodies.
b) These Microbubbles will then be tested for their ability to specifically bind to the target cells.
c) Developing a low-cost way of producing a microfluidic chip in order to continuously separate the microbubble-cell conjugate.
4 3. MATERIALS AND METHODS
3.1 Manufacturing of Microbubbles
The microbubbles were manufactured at the School of Technology and Health, KTH, based on the method developed by Cavalieri et al.70 Vigorous stirring of an aqueous solution of telechelic PVA, at acidic pH, resulted in interface crosslinking, and the resulting foam acted as colloidal stabilizer and coating on the air bubbles. The microbubbles had an average diameter of 3.5 microns and the thickness of the shell was 400nm.
3.2 Avidin coating on microbubbles
Avidin can be coupled to Biotin on the surface of a microbubble. But, first there has to be enough reactive carbonyl groups for the biotin to be deposited on the surface of the microbubbles. Otherwise, first the surface has to be oxidized before proceeding. Also, it is important to check for the floating ability of the bubbles. Overtime, the bubbles may lose their properties and sediment to the bottom. The best way is to centrifuge the bubbles at 300 rotations per minute (rpm) for about 5 minutes and check if the bubbles are still floating. Discard the sample if they are not floating and go for other batch of microbubbles. Below is the protocol for the avidin coating on the microbubbles starting from the oxidation of the surface to achieve enough reactive carbonyl groups.
3.2.1 Oxidation of PVA Microbubbles
a) Sodium Acetate buffer (Oxidation buffer) of 100mM concentration at a pH of 5.5 was prepared.
b) 1ml of Microbubbles (approximately 108) were washed in about 0.3ml of Oxidation buffer, at 300 rpm for 3 minutes and the bubbles from the top were removed using a sharp tip or needle.
c) 1ml of 20mM cold Sodium periodate (Sigma Aldrich) solution was prepared in the oxidation buffer and added to the above sample of microbubbles. The mixture was incubated for about 30 minutes on ice. The reaction vessel should be protected from light in all the steps.
d) The sample was washed using 1X phosphate buffered saline solution (PBS), (0.5ml) at 300rpm for 3 min. and the buffer was removed while the Microbubbles floated.
e) The bubbles are suspended in 0.5ml of MES buffer [2-(N-Morpholino)ethanesulfonic acid], (Sigma aldrich) having a pH of 5.
3.2.2 Hydrazide-Biotin coupling to Microbubbles
a) 50mM hydrazide-Biotin(Sigma Aldrich) reagent in dimethyl sulfoxide is prepared.
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b) Freshly prepared 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-EDC71(a thermo fisher product) is useful for activating the carbonyl groups on the microbubbles and also it reacts with amines on the Biotin Hydrazide to form amide bond. 10mg of EDC in 100µl of MES buffer is dissolved.
c) 75µl of Biotin hydrazide was added to 250µl of the microbubbles sample from the previous step.
d) 6.25µl of the EDC solution was added to the above sample and incubated with gentle mixing overnight.
e) The sample was washed by adding 0.1ml of 1X PBS at 300rpm for 3 min. The floating bubbles were removed using a sharp tip or needle. The resulting sample was biotin coated.
3.2.3 Binding Avidin to Biotinylated PVA Microbubbles
a) From the above sample, about 20µl of bubbles are taken in an amber vial and to this 20µl of Avidin-FITC (Thermo-fisher product) was added. The mixture was incubated at room temperature for 20 to 30 minutes with gentle mixing.
b) The sample is now washed in 20µl of 1X PBS buffer at 300rpm for 3 min and analysed under an inverted light microscope.
3.3 Conjugating Cells/Spheres and Avidin coated Microbubbles
In order to mimic the attachment of the microbubbles to the biotinylated anti-EpCAM antibodies, the microbubbles were first checked for conjugation with biotin coated nile red fluorescent spheres with an average diameter of 7µm( a product of Spherotech, Inc).
a) 20µl of avidin coated microbubbles from the previous step were taken in an amber vial and added to 5 µl of the biotin coated spheres. The sample was incubated at room temperature with gentle mixing for 30 min.
b) To the above sample 10 µl of 1X PBS is added and it is washed at 300rpm for 3 min.
c) The sample was analysed under a bright field microscope.
Subsequently, the microbubbles were conjugated to HCT 116 colon cancer cells by adding biotinylated anti-EpCAM antibodies and incubating for 30 minutes. The cells were stained using Calcein red-orange AM (a product of Thermo Fisher Scientific).
6 3.4 Microfluidic Channel Design
The Microfluidic channel was drawn in the computer aided design software, Autodesk AutoCAD 2018 software, as shown in figure 2. Of the three inlets, the outer two are used for the flow of Phosphate buffer solution and the inner inlet is for the injection of the sample. Of the two outlets, the first is used to collect the focused sample after the standing ultrasonic waves align the Microbubbles along the antinode. The second or the outer outlet is used for the remaining sample and buffer solution.
Figure 2. 3inlet-2outlet Microfluidic channel
3.4.1 3D printer
3-D printing has been considered as an alternative to soft lithography.68 The CAD file is initially converted to a .stl (standard triangulation file) and then it is sliced to individual layers digitally. This is realized sequentially to build an object in 3D.
The ultimaker2+ (3d printer) was used to create a mould. It extrudes Poly(Lactic Acid), a biodegradable, thermoplastic polyester, through a high temperature nozzle to build 3D structures. The idea is to use the printed channels as a positive master and pour PDMS liquid onto it and cure it in an oven at 650C and peel it off to bond the PDMS layer to glass using O2 plasma treatment.
Inlets for buffer and MB-cell complex
0.535mm wide channel Outlets
7 3.4.2 Cutting Plotter
While laser cutting is a tool with good precision and non-contact cutting, for microfluidic channels, it requires significant capital investment. Moreover, it also suffers from left over burnt residue after cutting and a vacuum pump for clearing out fumes and debris69.
A Cutting plotter (Graphtec CE6000, Graphtec Corporation., USA) using a double sided adhesive tape (ARseal™ 90880, Adhesives Research, Inc) has been used to carve out the microfluidic channel. The double-sided tape is protected by a release liner on both sides and the polypropylene film is in the middle. The thickness of the tape is about 200µm which would form the height of the microfluidic channel. The drawing in the drawing Exchange File (DXF) format was designed in the CAD software.
First a simple channel with single inlet and single outlet was cut in order to optimize the settings in the cutting plotter. After the optimization of the settings in the cutting plotter the complex, 3-inlets 2- outlets channel was fed. The cutting usually took about less than 30 seconds for the entire channel. It was then glued onto a glass slide, in order to serve as a mold to pour PDMS. After pouring the liquid PDMS, curing is done in an oven at 650C for about one to two hours. The PDMS is then removed and then O2 plasma treated to bond onto a glass plate. This mold can be used until the adhesive comes off from the glass slide.
3.4.3 Milling using a CNC system
A milling method using computerised numerical control system (Roland, Benchtop CNC mill MDX- 40A) was used to directly mill on a Poly methyl methacrylate (PMMA) sheet of dimensions 150mm*300mm. A computer aided machining software, Autodesk ArtCAM, was used to feed the drawing to the CNC milling machine.
a) The surface of the PMMA sheet had to be checked for uniform thickness, as slight variations in the thickness of the surface resulted in the mold having different heights at the inlets and outlets or the whole channel altogether. Therefore, a surface area clearance with a thickness of 0.3mm using a tool with diameter of 4mm (Φ = 4) was done to even out the surface. Another set of surface area clearance with a tool of diameter 2.5mm was carried out for a depth of 0.1mm. Now on top of this, the final structure was carved as shown in figure 3, along with a cover having holes with a height of about 1.5mm, exactly on top of the PDMS inlets and outlets. These holes can be used later to insert the inlet/outlet tubings.
b) The final height of 100µm, of the milled channels is verified using a digital caliper.
c) The PMMA master is removed from its sheet and cleaned for any residue left due to the milling tool. It is then washed with detergents and dried.
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d) It is then placed in a petri dish and liquid PDMS mixture along with curing agent (10:1 w/w) is poured on it.
e) The cover with holes for the inlets and outlets, which is milled at a depth of 250µm is placed on top of the master having the channels to squeezed out any excess PDMS. Hence, a PDMS layer of about 150µm (250 µm depth of the cover minus 150 µm height of the channels) remains above the channels. This control is necessary to have Standing Bulk Acoustic waves forming in the channel for acoustophoresis.
f) Any air bubbles formed are removed by placing the channel with cover in a degassing container for about 3-5 hours.
g) The dish is placed in an oven at 65oC for curing of the PDMS for about 2 hours.
h) The dish is then taken out and the master is taken to a Laminar Air flow hood. The cover is carefully taken out, in order not to tear off the PDMS layer from the master. Most of the times half the channel sticks to either the cover or the master and if it is torn while removing, then the entire procedure from step iv has to be repeated until a clean PDMS layer is obtained.
i) The PDMS layer is O2 plasma treated at 5 Bar pressure for 30 seconds and bonded onto a glass slide, thus forming a microfluidic channel.
Figure. 3 Milled channel of height 100µm on a PMMA block
3.5 Experimental Setup
The experimental setup has a PDMS channel with 100µm height and 535µm width as dimensions, bonded between two glass slides and a PZT (lead zirconate titanium) transducer with the fundamental frequency of 2.75Mhz, glued onto one of the glass slides by a water soluble adhesive (Tensive adhesive gel, Perker labs Inc.,) as shown in figure 4. The transducer is driven by a continuous sinusoidal wave generator (Tektronix Inc., AFG 3022). The microbubbles-cell complex was injected
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in the channel using a syringe pump (Harvard PHD 2000, USA) at a flow rate of 10µl/min and observed under a light microscope.
Figure.4 PDMS channel between the glass slide with transducer
The avidin coated microbubbles were examined in a microscopic glass slide under a light microscope and checked for fluorescence. Similarly, the microbubbles were also analyzed for conjugation with Biotin coated Nile red fluorescent spheres under a light microscope as a control step, before conjugating with HCT 116 colon cancer cells.
10 4. Results
4.1 Avidin coating on the microbubble’s surface
10µl of the avidin coated microbubble sample was taken and analysed under a light microscope. The use of FITC-conjugated avidin to coat the surface helps to identify if the bubble surface has been functionalized or not. There are no definite ways of quantifying the amount of avidin coated on the surface of the microbubble. As the FITC fluorophore has an emission and excitation spectrum of 519/495nm respectively, it gives off green colour when analysed under a light microscope as can be seen in figure 5. The corresponding bright field image is on the left in figure 5. The other faint dots represent microbubbles in the other plane. To visualize these the focus of the lens has to be changed.
Figure 5. Bright field and Fluorescent field image at 40X magnification
In-order to verify that the microbubbles which have been surface modified are not behaving as water filled capsules, control experiments were performed for the avidin coated microbubbles in the presence of microbeads having a diameter of 10µm. Under the influence of ultrasound, the gas filled microbubbles should focus to the antinodes and the microbeads to the nodes, which is illustrated in figure 6a, 6b. As the microbubbles raise above the beads due to buoyancy, they are in a different plane and hence the microscope objective has to be refocused inorder to visualize them.
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Figure: 6a,b showing the microbubbles and beads focussed at antinodes and nodes after the surface modification of Microbubbles at 20X magnification.
4.2 Conjugation of Biotin coated spheres with Microbubbles
Avidin has a strong affinity towards biotin. 10µl of the avidin coated microbubble sample which was mixed with Biotin coated Nile red Spheres was used to analyse under the microscope. The Nile red dye emitted a red colour when analysed under fluorescent microscope. It is shown in the fluorescent image of figure 7. The Microbubbles are clearly visible in the corresponding bright field image in figure 7, and those that had avidin coating on the surface readily attached to the Biotinylated spheres.
Here, most of the bubbles attach over the two spheres which are partially hidden due to the bubbles on top of them. In the corresponding bright field image, clearly the spheres being of a larger diameter (approximately 7 µm) are out of focus, in another plane. The microbubbles had formed clusters and are on top of the spheres. This shows the proof that the microbubbles have been functonalized and can conjugate to biotinylated anti-EpCAM antibodies. These can be further conjugated to HCT 116 colon cancer cells for separation from the non-conjugated cells.
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Figure. 7 Bright field and Fluorescent field image of the microbubbles coupled to the Biotinylated Nile red dye spheres.
4.3 Mold from 3-D printer
In figure 8 the relatively rough surface finish on the top of the printing is a disadvantage while casting PDMS over the mold. Also, the small inlets and outlets are clear but the channels connecting them are quite weak to hold onto the PDMS liquid when it is poured over them. Although, the channel’s design as a whole is printed, due to the limitations of accuracy, the fine structures like the outlet’s bifurcation is lost and cannot be recognized.
Figure. 8 Top view of the 3D printed channel. Channel ‘a’ has a thickness of 0.2mm, b is 0.5mm, c is 1mm and d is 2mm thick
13 4.4 Mold from Cutting Plotter
The complexities arose in the peeling off of the release liner in the 3-inlet, 2-outlet channels as the semicircles in the inlet/outlet had to be firmly pointed in order to not lose them while removing the outside area. This would sometimes expose off the adhesive below the top release liner, due to the pressure applied on the top surface to hold down the inlet/outlet’s semicircles. The PDMS when poured onto this would stick to the film and hence it would be difficult to peel off the PDMS from the master after the curing procedure.
Fig.9 A 3-Inlet,2-outlet(left) and 1-inlet,1-outlet(right) adhesive tape cut-out from the cutting plotter
4.5 Mold from CNC milling
The surface of the acrylic sheet had roughness (in the form of ridges and grooves due to the milling tool’s stepover) to it once the milling was done. This was overcome by optimizing the settings for amount of stepover (it is the distance the tool should move after milling in a particular direction) for the milling tool. The best results were obtained with as small a stepover as possible, but then it would increase the time taken to finish the milling job. A step over of one-third of the diameter of the milling tool was chosen (for example a milling tool with a diameter of 0.6mm with step over size of 0.2mm), as it gave a smooth surface over a stepover size of half the diameter of the milling tool.
The channel’s outlet has to have as low width as possible, in order to collect only the non-conjugated stream of cells flowing through the node which in this case would be at the center of the 535µm wide channel. And the rest of the bubble-cell conjugates flow through the anti-nodes and from the bifurcation leading to the side outlet where they would be collected. Initially, the width of the outlet was chosen to be 0.1mm as shown in the figure below. But this channel when tested for flow through, had no fluid coming out from the outlet. Upon close examination under the microscope, as shown in the figure, the channel’s width was not good enough for the fluid to pass through. This was tested with multiple molds done on the CNC machine at different times.
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Fig 10. The Blockage in the outlet.
Although the channels of about 100µm were easy to mill, the flow was blocked in the PDMS channel as can be seen in the figure 10, where a water droplet is blocked at the entrance of the outlet. Hence, the width of the outlet channel was increased to 200 µm and flow through it was checked. After optimising the width of the outlet channel in the positive master, acoustophoresis on the final PDMS channel was tested.
4.6 Bubble-cell complex under acoustophoresis and separation
The microbubbles have a strong negative acoustic contrast factor(ACF).54 ACF depends on the compressibility and the density of the particle relative to the medium it is suspended in. The direction of the radiation forces depend on the ACF of a particle. Particles with a negative ACF, focus at the anti-nodes (like PVA microbubbles) and those with a positive ACF, focus at the nodes (like cells).16,57 The microbubbles here were conjugated with the cells and injected in the microfluidic channel. Images were acquired under no flow conditions and without activating the transducer as shown in figure 11a.
After the transducer was triggered, the voltage was increased slowly from 0 to 10V (𝑉𝑝𝑝= 10𝑣𝑜𝑙𝑡𝑠).
The cell-bubble complex moved near the edge of the channel and the unconjugated cells remain at the center as shown in figure 11b.
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Figure 11 a: bubbles and cells (in different plane, out of focus) when the transducer is off. b: Migration of bubbles-cell complex (near the top edge of the wall) toward the antinode and cell towards the center of the channel.
In flow-through conditions, flow rates from 0.5 to 10µl/min at a voltage of 5V were used. The bubble- cell complex moved in the antinodes near the edge the of the channel and were collected from the bifurcating side outlet. Almost all the individual microbubbles which were not attached to the cells, also traversed the same the path as the cell-bubble complex. The cells which were unconjugated moved along the center of the channel (position of the nodes) and were collected at the other outlet as shown in figure 11b. Figure 12a represents the bubble-cell conjugate near the edge of the channel as a bright field image. The red and green spots in figure 12b represent the bubble-cell complex taken as a fluorescent image. The Calcein red-orange dye gives a red fluorescence from the cell and FITC in avidin FITC on the microbubbles gives a green fluorescence. The streak in figure 12c shows the path of the cell-bubble complex. A sorting efficiency of more than 80% was achieved at rates upto 10µl/min as shown in table 1.
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Figure 12. a) Bright field image of cell-bubble complex at the antinodes. b)Green and red Fluorescent dots representing the bubble-cell complex near the wall of the channel. c) The motion of the cell-bubble complex near the edge of the channel in green streaks.
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Table 1 Different sorting efficiencies at different rates
5. Discussion
The relatively less number of microbubbles in figure 5, at the end of the surface coating with avidin, can be attributed to the many number of washing steps in the protocol. The washing steps are necessary to visualize the microbubble surface’s fluorescence at the end of avidin-FITC coating, as it washes away any unattached avidin-FITC. Otherwise, it may give rise to background noise in the form of fluorescence and hinders the visualization of avidin deposition on the surface of the microbubbles while analyzing them under a light microscope. This washing step can be avoided partially after these trials, which are aimed at establishing a robust protocol for avidin coating on microbubbles. For this, avidin not conjugated with any fluorophore can be used to reduce the loss of microbubbles to some extent. Still the relatively high loss of microbubbles cannot be avoided as the washing steps after treatment with oxidation buffers is also necessary, inorder to remove the sodium acetate buffer. Its presence prevents the cross linking of Biotin with the reactive groups on the surface of the microbubble. Microbubbles with reactive surfaces can be one of the solutions to avoid the initial oxidation steps and directly start with biotin conjugation, which in turn would reduce the loss of microbubbles.
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
100.00%
0.5µl/min 1µl/min 10µl/min
Sorting Efficiency in %
Anti-node cell count Node cell count
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The molds from the cutting plotter were the fastest to fabricate, typically taking a few seconds to cut out the channels. But they were very difficult to be worked upon due to the overall dimensions of the channels, the inlets and the outlets. A 3d printer with a better resolution might have yielded a better mold, but it has to be seen if a problem still remains while peeling off the PDMS channel after baking it in the oven at 65ͦ C. So far, the mold from the CNC milling has given the best results to cast PDMS on it. The channels can be changed according to the requirement of the application into a single or double or more inlets and outlets which is facilitated by the use of a CNC machine. And hence it is a robust way to fabricate molds for further applications in microfluidics.
A spin coater can be utilized instead of a top cover (which squeezes out the excess PDMS) to have a very thin layer of PDMS on the mold. This may help reduce the operating voltage while focusing the cells in the microfluidic chip. Higher voltages would detach the transducer from the glass slide due to heating effects as was experienced during the experiments.
Buoyant property of the microbubble has been utilized for the separation of tumor cells previously.76 Here, the acoustic properties of microbubbles have been relied upon to focus the cells, after conjugating them with anti-EpCAM antibody. However, conjugating the microbubbles with blood sample containing tumor cells can be investigated due to the better spatial control and cell viability provided by acoustic manipulation.
6. Conclusion and Future work
Protocol for the surface functionalization of the microbubbles has been developed with repeatable results in coating avidin on them. Although, the final samples after avidin coating have lesser number of microbubbles, they readily conjugate to HCT 116 colon cancer cell line.
A microfluidic master chip has been developed by using CNC milling, which has a resolution in the order of 50µm. The PDMS microfluidic channel has been thickness controlled (between 550 to 750µm) using a cover on top of the master mold and hence, standing bulk acoustic waves have been generated which focus the microbubbles to antinode. Alternatively, Off stoichiometric thiol-ene polymer (OSTE), can be tried, instead of a PDMS based channel, as the mechanical properties of OSTE can be tuned for acoustofluidic applications.74,75 It can be used for a faster prototyping in soft lithography of microfluidic applications.
The focusing of the bubble-cell complex can be changed so that they flow through the center of the channel rather than near its sides. It can be achieved by further optimizing the driving frequency and the channel width accordingly. The inlets 1 and 2 in the channel can have PBS solution as sheath flow
19
and the inlet 3 can have only the sample containing the bubble-cell conjugate. A better efficiency can be expected using this method.
In future, the surface roughness of the 3D printer’s mold can be worked upon. Although, the cutting plotter yields a master mold in the matter of seconds using a double sided adhesive tape, ways to glue it on the glass slides without the exposure of adhesive layer towards the PDMS can be investigated thus providing a microfluidic channel with the height of about 50µm.
20 APPENDIX
State of the art
21 1. INTRODUCTION
One of the most crucial steps in the field of diagnostics is sample preparation and this involves isolation, separation, filtering and sorting of cells from complex bio-fluids like urine, blood, sputum etc. Sorting is also used to concentrate the selection of cells for further diagnostic downstream analysis or research investigations. Due to the developments in the cell sorting techniques, even the rare target cells like circulating fetal cells or CTCs (circulating tumor cells) are being enriched. The already existing problem of rapid sample preparation and the need to take diagnostics to remote regions for fast and inexpensive solution to a problem has driven the need to sort the cells in a faster, efficient and reliable way.1 Moreover the standards for the next stage evolvement of the sorting devices are set high as there is a need to have sorting ability to differentiate diverse cells, higher throughput, fully automated systems coupled with ease of operation, taking the use of aerosols out of the equation for reduced biological hazards and size reduction for user convenience and transportation. This is where microfluidic instruments play an important role in cell sorting as they give more control and that too on a very small platform.2 The microfluidic cell sorting techniques has been investigated but their translation and application into the real world is rare.
2. BACKGROUND
The use of fluorescence for sorting the cells, FACS (Flourescence activated cell sorting), was the first major step towards developing a cell sorter, and sorting has come a long way since then3, and can now sort around 50000 per second.3 More rapid sorting is achieved by magnetic activated cell sorting instruments4 which isolate magnetic labeled cells from other cells. The processes have now evolved so much that it is easier to differentiate them on the basis of either the way a cell is recognized or by the physical principles that are employed. The way of recognizing a cell can be Fluorescence based or Bead based or Label free sorting. Depending on the physical principles involved it can be divided into either Active sorting or Passive sorting. Active sorting techniques like electrophoresis5, acoustophoresis6 or magnetophoresis7 , use external energy to take advantage of the forces imparted on the cells to sort them. Passive techniques use inertial microfluidics8, geometry of the microfluidic channel to sort the cells. Laurell and Lenshof used ultrasound coupled to a microfluidic channel to manipulate particles.6,9 This acoustophoretic technique is useful for many purposes like washing the samples inside the microfluidic channel, concentration and separation. A disadvantage is its inability to differentiate between particles of different nature but having the same size, as acoustic radiation force is the driving factor. However, a recent method developed is size independent in separation of particles10 taking advantage of the altering of the cell’s medium in order to use acoustophoretic contrast factor (ACF) which can be either positive or negative or neutral for different particles. But all the media are not suitable for all the human cells and hence biologically activated negative acoustic
22
contrast particles can be used an alternative.11 The study of behavior of PVA microbubbles was studied under the ultrasound standing waves and it was concluded that they move to antinodes due to the high negative ACF(Satyapalli et al., 2016). This was furthered by Faridi et al(2017) by using an antibody funtionalized microbubble to separate cells in a microfluidic channel. The protocol used here needs to be made more robust for the functionalization of the microbubbles and for use in a multiple inlet, multiple outlet channel for continuous separation of cells from blood.
3. CELL SORTING TECHNIQUES
As described cell sorting can be divided depending on the physical principles deployed, into active or passive sorting. Passive sorting relies upon the differences in the physical characteristics of the cells like density, size and compressibility.
3.1 PASSIVE METHODS
3.1.1 INERTIAL SEPARATION IN CURVED CHANNELS
Particles migration is possible in a Newtonian fluid as the inertial force causes the particles to experience lift forces, taking them to the boundary of the microfluidic channel.17 It has been used for separation, focusing and sorting. High through put separation was achieved in this technique using micro channels besides each other.18 This is one of the ways of Passive separation. Another way in inertial microfluidics is to separate particles using a curved channel. The forces act in a way that the size of the particles is taken advantage of.17 The larger particles tend to migrate close to the inner wall as has been shown in figure 1B, C and an advantage of this method is high throughput. Cell isolation was also achieved by using deterministic lateral displacement and using inertial focusing having a spiral channel along with a magnetic label. Just centrifugal forces have also been used to sort and focus cells.8 A trapezoidal section was shown to improve the performance of the microfluidic device to sort the cells.20
23
3.1.2 STRAIGHT CHANNELS
In straight channels, pinched flow fractionation uses a pinched segment, which pushes the smaller particles closer to the wall and the larger ones to the center of the wall. As the segment ends and the large area is traversed the particles are separated based on size due to the laminar flow.21 In this case a particle free fluid is also simultaneously injected which pushes the particle containing fluid in the pinched section. The use of multiple outlets and the change in spatial orientation, to take advantage of the gravitational force, further enhances the cell separation.22 Inertial forces were used to separate bacteria from the blood at an efficiency of 80% when a radial array of 40 channels is used with a single inlet and double outlet. The channels have a short focusing and slow expanding regions culminating in the collecting region. The expanding regions impart a change of equilibrium positions to the particles so that they go towards the walls, hence giving good efficiency.23
Figure 1. Separation of particles in A. a serpentine path. B. a spiral channel where the bigger (red paticles) are separated along the IW(inner wall) and the smaller (green particles) along the OW(outer wall) and C.
the forces acting on the particles Fl are the lift forces and the dean forces FD. Copyright 2009 Royal Society of Chemistry.
24 3.1.3 DLD
DLD (Deterministic lateral displacement) is an approach where the particles encounter fixed points of hindrances to change their path depending on their size. The control over separation is dictated by the critical diameter of the arrays (serving as obstacles here) where the particles lesser than the critical diameter follow convective flow and the ones having larger than critical diameter change their course according to the placement of the hindrances.24 The fractionation of blood as well as cancer cell isolation was also achieved using this process.25
3.1.4 FILTRATION
Pores arranged either at the ceiling or at the bottom of a channel are used to trap larger cells in the process of filtration. One can also have slanted arrays which create a pressure gradient in the channel and hence align the cells for separation.26 The problem can sometimesbe choking of the pores or the clogging which arises due to unwanted waste or bigger cells. A solution was devised by having a larger pore size and a magnetic component to trap magnetically labeled cells at the pore edges27, as the other cells just pass away into the filters. In hydrodynamic filtration multiple outlets (of different sizes) which drain the fluid are used to separate the particles. The smaller particles are close to the border of the channels and the outlets increase in size gradually, separating smaller cells through the smaller outlets.28
3.2 ACTIVE TECHNIQUES
Active separation utilizes different mechanisms like Dielectrophoresis, Magnetophoresis, Optics and Acoustophoresis combined with fluorescence or beads or without the use of any label for sorting particles.
3.2.1 ELECTROKINETIC
Electrokinetic is an effect where movement of particles is initiated by applying an electric field.29 It has electrophoresis, dielectrophoresis and electroosmotic flow as principles. Initiation of motion of particles towards an opposite charge using direct current constitutes electrophoresis. The cells are slightly negatively charged due to the chemical groups present on their surface. Hence, in an electric field, they tend to migrate to the positive electrode. The force exerted depends on the charge on the cell.56 Fluorescently labeled cells can also be sorted electrostatically downstream using a detector which detects the fluorescence in upstream57 which is a non-destructive way of sorting cells continuously. The forces exerted on the particles also depend on their size and hence can be used to
25
sort larger particles from the smaller ones.29 Electrophoresis uses direct current, which can produce Hydrogen peroxide and can also cause a change in pH of the solution due to electrolysis of water. This is a drawback in this method of sorting. Another way is Dielectrophoresis that uses alternating current and polarizes the particles in order to transport them. Cells depending on their comparative permeability to the medium migrate to the stronger field or weaker field.30 Cells having a higher permeability than the medium, migrate towards the stronger electric field (positive Dielectrophoresis) and those having a lesser permeability than the medium, migrate to the weaker electric field (negative Dielectrophoresis) as shown in figure 2.
The forces acting on the particles are manipulated by binding them to beads and hence separating.31 Insulating Arrays can also be used to create a non-uniform electric field and hence exert force on the passing by cells.32 One more way is to induce the motion of the cells or the particles by actually moving the solvated ions electrically. As the ions move, so does the fluid medium and thereby moving the cells in it. This is the phenomena behind electro osmotic flow. In order to avoid the generation of Hydrogen Peroxide or bubbles and also the pH variations in the solution, alternating current electro- osmosis using negative Dielectrophoresis is used to sort cells.33
3.2.2 SEPARATION BY OPTICS
The refractive index of the cells is different from the surrounding medium that they are suspended in.
When a beam is focused onto them, it gives rise to scattering and gradient forces. The gradient forces pull the particles towards the point of maximum intensity of the beam and the scattering forces push them away from it. When the scattering forces are overcome by the gradient forces the particles are
Figure 2 showing positive DEP and negative DEP.
26
trapped, creating optical tweezers. This method can be used to trap particles from several nanometers to several micrometers in size. Due to the development of miniaturized optics based devices, fluorescently labeled cells at the rate of 20,000/sec are being sorted.34 It also does not change the cell function and hence is very advantageous for particle manipulation.
3.2.3 MAGNETOPHORESIS
Cells can either be magnetically responsive or labeled magnetically. And by using electromagnetic coils, forces can be exerted on them for isolation or separation. This also depends on the size of the cells. Depending on the magnetic moments, magnetic beads could be sorted along 25 channels.35 Cells can also be labeled with larger magnetic beads to separate them from cells labeled with smaller beads and finally separating these from the unlabeled cells.36 The labeled cells can also be trapped in parallel chambers which are perpendicular to the inlet-outlet channel37 as can be seen in the figure below. This method has shown to have very good efficiency to capture circulating tumor cells.37
Figure 3. Trapping cells in parallel chambers.
Unlike labeling, erythrocytes have a natural iron content and hence can be isolated from other cells using magnetophoresis.38 Different sized cells like macrophages experience different amount of magnetic force compared to monocytes. Under externally applied magnetic field this can be taken advantage of and used for sorting them.39
27 3.2.4 ACOUSTIC SORTING
An ultrasonic wave is used to separate the particles by aligning them along points of maximum amplitude or minimum amplitude. The phenomenon does not alter the viability of the cells and gives good spatial control. A standing wave is created when two waves of the same frequency and magnitude traverse in opposite directions. The points of maximum amplitude are called anti nodes while points of minimum amplitude are called nodes in a standing wave. These points can be a source of separating particles from one another depending on their size, density and compressibility.58,59 The different ways acoustic waves are used are in the form of Bulk standing acoustic waves, Standing surface acoustic waves and travelling waves. The first two approaches are more commonly used to manipulate particles. Bulk standing acoustic waves are formed when the system is in resonance i.e., the microfluidic channel is excited by matching the wavelength of the wave and the width of the channel. The particles are deflected depending on their compressibility and density to either node or antinode. A camera initiates a transducer when a fluorescent particle is detected by it and thereby deflecting it from the rest of the particles.40
Standing surface acoustic waves are formed by using inter digital Transducers, where the standing waves are formed along the floor of the microfluidic channel. Bead based manipulation is also possible as they show a negative Acoustophoretic Contrast Factor (ACF) and in a standing wave are focused towards antinodes. This has been used to biofunctionalize the beads and coat them with streptavidin to conjugate them with cells having biotinylated antibodies.41, 42 Lenshof et al. also used this to label cells with positive ACF microbeads and separate them from non target cells.43 The larger cells experience a higher force compared to the smaller cells and this concept has been used to separate WBCs from prostate cancer cells.44
4. THEORY
Acoustically induced motion of particles is possible using waves in the frequency range of 1Mhz – 10MHz. This motion is due to the acoustic radiation force12 which was experimentally demonstrated by Kundt and Lehman in 1874 in a standing wave. Two waves having the same frequency, wavelength and amplitude but traveling in the opposite directions produce a standing wave. It is represented by the following equations in terms of longitudinal position y and time variable t as:
𝑓(𝑦, 𝑡) = 𝑎𝑠𝑖𝑛 (𝟐𝝅𝒚𝝀 − 𝝎𝒕) Eq. 1
𝑔(𝑦, 𝑡) = 𝑎𝑠𝑖𝑛 (𝟐𝝅𝒚
𝝀 + 𝝎𝒕) Eq. 2
The resultant wave X is given by adding Eq. 1 and Eq. 2
28 𝑋 = 2𝑎𝑐𝑜𝑠(𝝎𝒕)𝑠𝑖𝑛(𝟐𝝅𝒚
𝝀 ) Eq. 3
Where a is the amplitude of the wave, 𝜔 is the angular frequency and 𝜆 is the wavelength. Although most acoustic manipulations are performed using the Ultrasonic standing waves (USW), traveling waves are also used in some cases.13-15 In an USW at positions where, y = 𝒌𝝀
𝟐, (k= …., -2, -1, 0, 1, 2….) the amplitude is zero and are called as nodes and where, y = 𝒌𝝀
𝟒, (k= ….,-5, -3, -1, 1, 3, 5….) the amplitude is the maximum and are called as antinodes.
4.1 FORCES ON PARTICLES
As described earlier, acoustic radiation forces are responsible for the motion of particles in travelling and standing waves. They are of two types, primary radiation force and secondary interaction force(also known as Bjerknes force).16 The magnitude of primary radiation force depends on the size of the particle and the direction of the force depends on the density and compressibility of the particle relative to the medium the particle is suspended in. There is a condition which is to be considered here in the microfluidic channel and that is that the diameter of the spherical particles is far less than the wavelength of the sound wave imposed ( d << 𝝀 ). The equation for the primary radiation force57 acting on the particle is given by
𝐹𝑝𝑟𝑖 = 4
3𝜋𝑘 sin(2𝑘𝑠) 𝜑(𝜅, 𝜌)𝐸𝑎𝑐𝑟3 Eq. 4
where r, Eac, k, s, φ(κ, ρ) are the radius of the particle, energy density (acoustic), wave number, distance from node, acoustophoretic contrast factor (ACF) of the partice respectively. The ACF φ (κ, ρ) of the particle is given by
𝜙(𝜅, 𝜌) = 1
3𝑓1+1
2𝑓2 Eq. 5 Where 𝑓1= 1 − 𝜅𝑝/𝜅0 and 𝑓2=2(𝜌𝑝− 𝜌0)
(2𝜌𝑝+ 𝜌0)
⁄
Here κp, κ0, ρp, ρ0 are the compressibilities and densities of the particle and the medium respectively.
The ACF is negative if the compressibility ratio κp/κ0 is bigger than the density ratio ρp/ ρ0. The particle therefore experiences a force towards the node or the antinode depending on the sign of the ACF. A positive ACF would mean the particle tends to be pushed towards the node and the opposite would mean particle migrating towards the antinode.
29 4.2 SECONDARY INTERACTION FORCE
In an acoustic field where many particles are exposed to an acoustic wave, there will be scattering of the waves from one particle and these interact with the neighbouring particles giving rise to secondary interaction forces or Bjerknes forces.16 The particles repel each other if the Bjerknes force is positive and they attract each other when it is negative.
5. MICROBUBBLES
One of the most important contrast agents used in Ultrasound imaging is Microbubbles, that are used to enhance the visualization of different organs. The gas present inside the microbubbles is responsible for ultrasound backscatter mechanism for imaging. The microbubbles resonate under the influence of an ultrasonic wave and also reflect the waves better than the human tissues which enables enhancement of the grey scale images. Currently, the microbubbles are not only used in diagnostic applications but therapeutic applications as well. They can be used as drug or gene carrying agents or to conjugate them with cells for sorting.
When microbubbles are subjected to ultrasound, there are many events taking place, ranging from the very delicate action of gradient forces on them to the bigger cavitational effect. Therefore a wide range of applications are possible with microbubbles in between these two extreme phenomena.
Due to the surface tension experienced, while being suspended in a liquid, the microbubbles are unstable and hence require a robust shell.45 The thickness of the shell is used to define a microbubble as thick or thin. If the ratio of the thickness of the shell to the radius of the whole microbubble is less than 5%, then it is classified as a thin-shelled microbubble, otherwise it is known as a thick-shelled microbubble.53 This depends on the material the microbubble’s shell is made up of. Therefore the microbubbles can be further classified according to the composition of their shell. The different types are explained below.
5.1 PROTEIN BASED
Human serum albumin, when heated in the presence of air, undergoes sonication and forms albumin coated microbubbles.47 AlbunexTM (GE Healthcare), a type of albumin coated microbubbles were the first to be approved by the USFDA for use in healthcare applications. It is found to be stable for long periods if refrigerated. The thickness of these microbubbles range is about 15 nm and the radius around 0.5 to 7.5 µm.46 The initial use of these microbubbles was largely as Ultrasound contrast
30
agents. Other than this a mixture having albumin and avidin was also used to make microbubbles couple to biotinylated targets as avidin has affinity to biotin.
5.2 LIPID BASED
These are widely used in drug delivery and imaging. Lipid based microbubbles have a hydrophilic outer end at the shell-water boundary and a hydrophobic inner end towards the shell-gas boundary.
The low surface tension for the lipid layer is what makes the microbubble stable and also the fact that the lipid monolayers have van der waals attractions, make them cohese. As these forces are weak, it is also taken advantage during ultrasonic expansion and compression of the gas inside. These also resonate with minimum damping48 and can also be conjugated with different substances to act as drug delivery agents.49
5.3 POLYMER BASED MICROBUBBLES
As the term suggests, these microbubbles have a shell comprising of cross linked polymers which is thicker than protein based and lipid based microbubbles. Due to the same fact that the thickness of polymer shells is higher, the echogenicity as well as the compressibility is lower than the above mentioned microbubbles. Also comparatively the effects of subjecting a polymeric microbubble to pressures is different, for example, it does not oscillate and a small defect in its shell takes place at higher pressures and releases the gas bubble while the shell remains largely intact and repelled from the gas. This effect can be utilised in drug delivery.50 A sodium alginate solution is used for ionotropic gelation, causing microencapsulation of air and then hardened by calcium ion containing solution. The size of the microbubbles produced this way was too large for medical applications.51 Bjerknes et al., produced microbubbles by freeze drying a biodegradable polyester dissolved in hydrophobic methylene groups i.e., oil in water emulsions. The resulting microbubbles had an elongated shape like microcapsules with a diameter of 1-20 micrometer.52
PVA, Poly (vinyl alcohol) is a synthetic biocompatible polymer used by Cavalieri et al.to make microbubbles in the diameter of around 6 micron and a shell thickness of about 900nm to 700nm ( at 40 C). It is made by high stirring of a low pH solution of telechelic PVA. They showed good shelf life with the surface having hydrophilic groups like OH and aldehyde. And the inner boundary of gas- polymer shell has a hydrophobic part.
5.4 APPLICATIONS OF MB’s
Microbubbles serve as good ultrasound contrast agent due to the co incidence that the thin shelled microbubbles resonate at the frequency of 1-10 Mhz. At the resonance frequency the backscattered signals have a high strength and hence can be used to visualize organs under ultrasound. Microbubbles
31
can also be used as gene and drug delivery agents, which can either carry or bind drugs to specific targets in the body. Ultrasound can then be used to fragment or direct the microbubbles to accomplish its task.
Figure 4. The layer by layer depiction of microbubble used for drug delivery.
Nucleic acids can be loaded onto a protein based microbubble by either incorporating it in the shell while manufacturing or by making the surface adsorb the nucleic acid. Both methods do not significantly alter the acoustic properties of the microbubbles. However the microbubble cannot cross the endothelial layer which is a potential barrier in terms of its usage as drug delivery agent. In the field of microfluidics using a PZT transducer with a resonance frequency of 2.8 MHz to produce Ultrasonic standing waves in a microfluidic channel, the motion of the PVA based microbubbles has been studied.54 The fact that ACF of the microbubbles is negative, has been taken advantage of in order to achieve a 75% efficiency to sort the cancer cells by first conjugating them to the surface of the microbubble.55 Hence there is potential in developing the microbubble in a cell sorting environment to capture, for example cancer cells, for diagnostic applications.
NOTE: All images have been reproduced after due permission from copyright owners.
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