Acoustic Separation of Cancer-derived Extracellular Vesicles for Cancer Early Diagnostics
Lead Research Organisation:
Cardiff University
Department Name: Sch of Engineering
Abstract
EPSRC Portfolio Research Areas: The project aligns with RF and microwave devices (Grow), Sensors and Instrumentation (Maintain), Microsystems (Maintain), and Clinical Technologies (Maintain).
Description:
A cancer becomes life-threatening when the primary tumour spreads from the place where it first started to another place in the body. The spreading process is called metastasis, a tumour formed by spreading cancer cells is called a metastatic tumour. Metastasis is the principal cause of death in patients diagnosed with invasive cancer. At present, there is no curative treatment available for patients with metastatic cancer. This clearly demonstrates the urgent need for increased knowledge about the cellular communication mechanisms between tumour cells and normal cells resulting in pathological metabolism in the metastatic site. Extracellular vesicles (EVs) are a distinct population of membranous vesicles of endocytic origin. They range from 30 to 150 nm in diameter and are released by cells upon fusion of intracellular multivesicular bodies with the plasma membrane.
Acoustic waves have been applied in highly sensitive separation of bio-particles such as cells. By increasing the frequency of the acoustic waves, they are capable of separating cancer-derived EVs subpopulation from whole blood. The proposed project, built on the supervisors' available techniques, will fabricate a high frequency acoustic transducer integrating acoustic and microfluidic techniques to identify and separate cancer-derived EV subpopulation from other EVs. The method will be evaluated against conventional EV separation by ultracentrifugation to investigate its sensitivity and specificity. Cancer patient blood will be sourced to test the transducer in a preliminary clinical study. The transducer will be an integrative and sensitive alternative diagnostic technique that targets system-level circulating tumour biomarkers to allow the investigation of the fundamental roles of EVs in cancer progression and treatment response. The candidate will work in a multidisciplinary team including engineers, biologists and clinicians to investigate developing and implementing the novel transducer for new real-time diagnosis and classification of cancer by using EV as a cancer marker.
Description:
A cancer becomes life-threatening when the primary tumour spreads from the place where it first started to another place in the body. The spreading process is called metastasis, a tumour formed by spreading cancer cells is called a metastatic tumour. Metastasis is the principal cause of death in patients diagnosed with invasive cancer. At present, there is no curative treatment available for patients with metastatic cancer. This clearly demonstrates the urgent need for increased knowledge about the cellular communication mechanisms between tumour cells and normal cells resulting in pathological metabolism in the metastatic site. Extracellular vesicles (EVs) are a distinct population of membranous vesicles of endocytic origin. They range from 30 to 150 nm in diameter and are released by cells upon fusion of intracellular multivesicular bodies with the plasma membrane.
Acoustic waves have been applied in highly sensitive separation of bio-particles such as cells. By increasing the frequency of the acoustic waves, they are capable of separating cancer-derived EVs subpopulation from whole blood. The proposed project, built on the supervisors' available techniques, will fabricate a high frequency acoustic transducer integrating acoustic and microfluidic techniques to identify and separate cancer-derived EV subpopulation from other EVs. The method will be evaluated against conventional EV separation by ultracentrifugation to investigate its sensitivity and specificity. Cancer patient blood will be sourced to test the transducer in a preliminary clinical study. The transducer will be an integrative and sensitive alternative diagnostic technique that targets system-level circulating tumour biomarkers to allow the investigation of the fundamental roles of EVs in cancer progression and treatment response. The candidate will work in a multidisciplinary team including engineers, biologists and clinicians to investigate developing and implementing the novel transducer for new real-time diagnosis and classification of cancer by using EV as a cancer marker.
| Description | Acoustophoresis is a technique well-known for actuating and manipulating micro-/nano- particles using acoustic waves and its applications have been demonstrated in a wide-range of biomedical applications such as separating blood cells and platelets, separating circulating tumour cells from whole blood, isolating exosomes, washing and coating of cells. This project in particular focuses on the manipulation of extracellular vesicles (EVs), which range from 30 to 150 nm in diameter. EVs are released by cells and EVs derived from cancer cells can be used as cancer biomarkers for cancer diagnostic application. Commonly, acoustophoretic devices use surface acoustic waves (SAWs) which could also be used to separate EVs from larger cells size population for analysis. SAWs devices consist of two main components; interdigital electrodes (IDEs) on a piezoelectric substrate and a microchannel. Both of these components require clean room faculties to be created, which tend to be expensive and require a significant amount of training with various equipment for any potential user. The patterned IDEs, made using conductive metal, are permanently bonded on top of the substrate. This creates a limitation on experimenting with different orientation and combination of the same IDEs, such as; 4 pair tweezers and tilted angle separation, and if the IDEs or substrate is damaged in any way both components can become obsolete and the user will have to go through the clean room manufacturing again. A novel manufacturing technique was to design the IDEs on a PCB instead of the piezoelectric substrate, this segregation allows the components to be assembled together using a simple mechanical clamp and disassembled and re-located if necessary. This technique was used to create a pair of IDEs on a PCB and was tested on generating a standing SAW (SSAW) to pattern 10 µm particles and non-small lung cancer cells and droplet actuation (10.1039/C9LC01192G). Thus it was demonstrated that the PCB IDEs could perform the same tasks as the clean room made IDEs, which can be manufactured using the standarised PCB industry (widely available to regular consumers). Additionally, If instead of pair of IDEs on a PCB, PCB IDEs chips are created the user can experiment with with single IDEs, tilted IDEs, 2-pairs of IDEs and a 4-pair of IDEs more easily allowing more experimentation and versatility with the same design of IDEs and a single piezoelectric substrate, rather than having to create a single clean room made SAW device for each of those potential configurations. Recently, the student also developed a technique capable of creating microchannel moulds without the use of cleanroom faculties. Typically, a microchannel will be manufactured on a silicon mould that can be used repeatably. Just as the cleanroom IDEs, the silicon mould is again an end product made within the clean room that cannot be modified unless a new mould is created. This limits the potential for experimentation and testing with different microchannel designs since most new user will just stick to the conventional tested model, not wanting to risk creating a mould that will be useless. As an alternative to this, the student created a technique that allows the microchannel mould to be created on top of a plain glass slide and a common fusion deposition modeling 3D printer. This technique permits the creation of a channel minimum width of 500 µm which, even though is much larger than the minimum width achieved by the cleanroom made silicon mould, can still be used by new users for prototyping and testing applications which requires a much less skill that creating a silicon mould. This could allow the expansion of the acoustophoretic application since it will remove a large bottleneck by allowing easy testing and prototyping using the PCB IDEs and 3D printed glass mould. Lastly, microchannels are bonded piezoelectric substrate using plasma treatment, which ensures that the channel is "permanently" bonded to the surface and if removed it will have to be re-treated/ bonded again. As all previous mentioned techniques, this technique as well requires high end facilities or expensive equipment, which may discourage new researchers in the area. Thus a last innovation was to create a mechanical pressing technique for the manufactured microchannel which then would permit the change of channel designs on the fly and a quick repair if leakage occurred. Interestingly enough this technique works well for both silicon mould and 3D printed glass mould made channel, but it has been only tested on lithium niobate which is the substrate the student most often works with. By having all those techniques, the student was capable of more bold experimentation as well as having no need of clean room faculties thus allowing him to have more time to spent testing different device setups. Finally all those technique were implemented to for tilted angled SSAW manipulation of nanoparticles as a pre-step for EVs manipulation and preliminary tests demonstrated that his device is capable of manipulating nanoparticles. But these test remain preliminary and should be handled currently with caution since his tests were cut short due to COVID19 and more thorough confirmation is yet to be performed. Currently, the student is working on re-creating his device which then will be thoroughly characterised using nanoparticles and then tested for EVs separation manipulation. |
| Exploitation Route | Before the EVs testing is performed it is too soon to say in regards of applying the device for diagnostic purposes. But in regards of the PCB IDEs and 3D printed glass moulds, these techniques due to their availability and simplicity can be used a stepping stone by new researchers as preliminary testing for their ideas before thoroughly committing to the field. Additionally, since the PCB IDEs were proven to be capable of manipulating micro sized cells, nothing stops new researchers from using such a device as a clean room made alternative. |
| Sectors | Other |
| Description | CARDIFF SCHOOL OF ENGINEERING: PGR International Experience Fund |
| Amount | £3,000 (GBP) |
| Organisation | Cardiff University |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 07/2019 |
| End | 07/2019 |
| Description | Funding for EPSRC Industrial Strategy Innovation placements/ activities |
| Amount | £10,421 (GBP) |
| Organisation | Cardiff University |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 11/2019 |
| End | 03/2020 |
| Title | Manufacturing of microchannel using a 3D printed mould |
| Description | In acoustofluidics it is most common to use a microchannel with the SAW based devices for microparticle and cell manipulation. Most of the times this channels are manufactured using a silicon mould which is made in clean room facilities. These facilities are both expensive and require a high level of skill/ experience to manufacture a mould both of high quality and consistently. Thus, if a group would like to have multiple testing of channel with different configuration, either a silicon mould with multiple version should be created or by manufacturing new mould after initial trials. This would naturally raise the cost greatly thus it is common to stick with a commonly used and simple version of microchannel that works. Furthermore to bond the channel, plasma treatment is used that again requires expensive equipment and clean room faculties and results in a "permanent" bond, since if the channel is removed from the substrate it has to be re-bonded using this method. In our group we have developed a method that allows to create a microchannel mould using a glass slide and a fusion deposition modeling 3D printer, both which are widely available and cheap in comparison to the clean room facilities. This methods prints the internal geometry of microchannel onto the glass slide which is used as substitute for the silicon mould. Due to the cheapness of this methods and low skill entry, multiple moulds can be manufactured with ease but its dimensional capabilities are inferior to that of the silicon mould, with the minimal channel width achieved using the 3D printed being 500 µm. Lastly, the bonding mechanism used in our group is a simple clamping mechanism which allow the easy relocation of the channel and re-orientation as well as quick and easy assembly and disassembly if a leakage has occurred. |
| Type Of Material | Improvements to research infrastructure |
| Year Produced | 2021 |
| Provided To Others? | No |
| Impact | As previously stated, this method allows the user to avoid the use of clean room facilities and can encourage more testing and prototyping with different channel shapes and configuration since its internal structure is so easy and cheap to manufacture. Additionally, through the use of a mechanical clamp to hold the channel down, the channel can be repositioned in different ways to implement different applications. Thus, this method offers a low-skill entry and a cheap way of manufacturing microchannel which encourages experimentation and testing through trial and error. This methods has been submitted to a journal and our group is awaiting the journal's response. |
| Title | PCB based Interdigital electrodes (IDEs) for SAW devices |
| Description | Typical manufacturing of SAW devices involved the development of the IDEs on top of a piezoelectric substrate. This is achieved with the use of expensive equipment and clean room facilities, which are expensive and require high skill level/ experience personnel. After the manufacturing the fingers are imprinted/ chemically bonded onto the piezoelectric substrate and cannot be removed and if damaged will make both the IDEs and substrate un-operational. This creates a high entry level for using such devices as well as little room for experimentation. In our group we decided to segregate the two components by manufacturing the IDEs on a PCB board, which can be easily performed by the highly developed PCB industry. The bonding between the PCB IDEs and the piezoelectric substrate is achieved via surface contact which is instigated through mechanical clamping. This PCB-SAW device was tested and was proved to be capable of both droplet and particle manipulation as a typical clean room made SAW device. |
| Type Of Material | Improvements to research infrastructure |
| Year Produced | 2020 |
| Provided To Others? | Yes |
| Impact | This methods allows a low entry and low skill requirement to perform SAW based acoustofluidic experiments. By segregating the components ie IDEs and piezoelectric substrate, one components can be salvaged or replaced easily and thus the opportunity of testing and experimenting with the device becomes more widely available. An example would be a cracked lithium niobate, in a normal clean room made device a crack means that both the IDEs and piezoelectric substrate are no longer usable, while with the PCB IDEs the IDEs are still in a good condition and if the lithium niobate has sufficient space it can still be used by simply adjusting the location that the IDEs are pressed on it. Furthermore, this PCB IDEs can be separated onto chips, instead of made as a pair onto one PCB, thus allowing the relocation of each of the chips in different configuration, fine tuning and even the identical operation as a pair of IDEs since their electrical characteristics can easily indicate their alignment quality (submitted paper). |
| Description | Droplet pumping and zinc oxide substrates tests |
| Organisation | Northumbria University |
| Department | Physics and Electrical Engineering |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Provided the PCB IDEs and assembly jig to test for droplet pumping and coupling capabilities with zinc oxide substrates (piezoelectric material). |
| Collaborator Contribution | Provided hydrophobic coating and zinc oxide substrates. |
| Impact | Droplet pumping results used in the following published paper (10.1039/C9LC01192G) Preliminary tests showed that PCB IDEs can couple with the zinc oxide substrate. |
| Start Year | 2019 |
| Description | Investigation of SAW based separation as pre-treatment of cell used in electrotransfection |
| Organisation | Duke University |
| Department | Duke Biomedical Engineering |
| Country | United States |
| Sector | Academic/University |
| PI Contribution | Electrotransfection is a technique commonly used for DNA, RNA and proteins delivery and cancer gene therapy, by applying pulsed electric fields on cells to attract the molecules of interest around the cells and allowing for intracellular transport of materials. Despite its potential advantages and simplicity, this procedure suffers from low transfection efficiency and cell viability, due to the unprotected pDNA degraded by nucleases and the metal ions generated from the metal electrodes. A method to avoid this issue is to use centrifugation to separate the cells from the medium, but the gravitational force might alter morphology of the cells. Another method to reduce cell degradation and improve their viability is to quick remove the cells from the electrotransfection medium, which is also label free and doesn't alter the morphology of the cells. Our group with a collaboration from Duke did exactly that, by having cells go through electrotransfection a group of those cells was then injected into our acoustofludic device and the cells were separated from the electrotranfection medium into PBS using tilted angle based SSAW separation. |
| Collaborator Contribution | The collaborators at Duke performed the electrotranfection procedure and provided our group with the cells for testing at the Duke University. |
| Impact | By performing preliminary tests on groups that after electrotransfection were separated from the electransfection medium, as stated above, and from this preliminary data it was observed that the viability of cells improved after SSAW separation, proving that SSAW could be a methods applied in line with electrotransfection to improved its cell viability. |
| Start Year | 2018 |
| Description | Manufacturing PCB based interdigital electrodes for acoustofludics in-house |
| Organisation | University of Bath |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Provided expertise in manipulation of cells and microparticles using SSAW based application. |
| Collaborator Contribution | Providing clean room facilities, to experiment in manufacturing PCB based interdigital electrodes in-house, and expertise in manufacturing PCB based interdigital electrodes. |
| Impact | Published paper on lab on the chip |
| Start Year | 2018 |
| Description | iRegene, EPSRC Innovation placement |
| Organisation | iRegene |
| Country | China |
| Sector | Private |
| PI Contribution | During the placement, the researcher/ student went to iRegene to develop an acoustofluidic device capable of manipulating exosomes. Initial testing were performed and demonstrated his prototype device's capability in manipulating polystyrene nanoparticles up to 100 nm small. He continued to manipulate nanoparticles to thoroughly characterise his device before proceeding with exosome/ cell manipulation. Unfortunately, due to COVID19 the researcher/ student could not perform any exosome tests during that period due to the national lockdown and then him having to be evacuated from China back to UK. |
| Collaborator Contribution | The collaborator provided space for the user to create a lab space for mechanical/ electrical development of his prototype device as well as cell culturing faculties for cells/ exosome development and exosomes and cells upon request. As stated above, unfortunately the exosomes/ cells were not able to requested due to COVID19 |
| Impact | A development of a prototype acoustofluidic device using a novel manufacturing technique, ie using pre-manufacture PCB with interdigital electrodes, capable of actuating polystyrene nanoparticles. This device is theoretically is capable of manipulating/ separating exosomes within a microfluidic channel, but this statement remains unconfirmed until the biological test are performed. |
| Start Year | 2019 |