| 1. |
- Afrasiabi, Roodabeh, et al.
(författare)
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Integration of a droplet-based microfluidic system and silicon nanoribbon FET sensor
- 2016
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Ingår i: Micromachines. - : MDPI AG. - 2072-666X. ; 7:8
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Tidskriftsartikel (refereegranskat)abstract
- We present a novel microfluidic system that integrates droplet microfluidics with a silicon nanoribbon field-effect transistor (SiNR FET), and utilize this integrated system to sense differences in pH. The device allows for selective droplet transfer to a continuous water phase, actuated by dielectrophoresis, and subsequent detection of the pH level in the retrieved droplets by SiNR FETs on an electrical sensor chip. The integrated microfluidic system demonstrates a label-free detection method for droplet microfluidics, presenting an alternative to optical fluorescence detection. In this work, we were able to differentiate between droplet trains of one pH-unit difference. The pH-based detection method in our integrated system has the potential to be utilized in the detection of biochemical reactions that induce a pH-shift in the droplets.
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| 2. |
- Cavallaro, Sara, et al.
(författare)
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Comparison and optimization of nanoscale extracellular vesicle imaging by scanning electron microscopy for accurate size-based profiling and morphological analysis
- 2021
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Ingår i: Nanoscale Advances. - : Royal Society of Chemistry. - 2516-0230. ; 3:11, s. 3053-3063
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Tidskriftsartikel (refereegranskat)abstract
- Nanosized extracellular vesicles (EVs) have been found to play a key role in intercellular communication, offering opportunities for both disease diagnostics and therapeutics. However, lying below the diffraction limit and also being highly heterogeneous in their size, morphology and abundance, these vesicles pose significant challenges for physical characterization. Here, we present a direct visual approach for their accurate morphological and size-based profiling by using scanning electron microscopy (SEM). To achieve that, we methodically examined various process steps and developed a protocol to improve the throughput, conformity and image quality while preserving the shape of EVs. The study was performed with small EVs (sEVs) isolated from a non-small-cell lung cancer (NSCLC) cell line as well as from human serum, and the results were compared with those obtained from nanoparticle tracking analysis (NTA). While the comparison of the sEV size distributions showed good agreement between the two methods for large sEVs (diameter > 70 nm), the microscopy based approach showed a better capacity for analyses of smaller vesicles, with higher sEV counts compared to NTA. In addition, we demonstrated the possibility of identifying non-EV particles based on size and morphological features. The study also showed process steps that can generate artifacts bearing resemblance with sEVs. The results therefore present a simple way to use a widely available microscopy tool for accurate and high throughput physical characterization of EVs.
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| 3. |
- Cavallaro, Sara, 1992-
(författare)
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Development of Techniques for Characterization, Detection and Protein Profiling of Extracellular Vesicles
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Nanosized extracellular vesicles (EVs, ∼30-2000 nm) have emerged as important mediators of intercellular communication, offering opportunities for both diagnostics and therapeutics. In particular, small EVs generated from the endolysosomal pathway (∼30-150 nm), referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics and treatment monitoring based on minimally invasive liquid biopsies. This is because exosomes carry valuable biological information (proteins, lipids, genetic material, etc.) reflecting their cells of origin. Using EVs as biomarkers or drug delivery agents in clinical applications requires a full understanding of their cellular origin, functions, and biological relevance. However, due to their small size and very high heterogeneity in molecular and physical features, the analysis of these vesicles is challenged by the limited detection ranges and/or accuracy of the currently available techniques. To overcome some of these challenges, this thesis focuses on developing different techniques for characterization, detection and protein profiling of EVs at both bulk and single particle levels. Specifically, the three methods investigated are scanning electron microscopy, electrokinetic sensing, and combined fluorescence - atomic force microscopy. First, a protocol for scanning electron microscopy imaging of EVs was optimized to improve the throughput and image quality of the method while preserving the shape of the vesicles. Application of the developed protocol for analysis of EVs from human serum showed the possibility to use scanning electron microscopy for morphological analysis and high-resolution size-based profiling of EVs over their entire size range. Comparison with nanoparticle tracking analysis, a commonly used technique for EV size estimation, showed a superior sensitivity of scanning electron microscopy for particles smaller than 70-80 nm. Moreover, the study showed process steps that can generate artifacts resembling sEVs and ways to minimize them. Secondly, a novel label-free electrokinetic sensor based on streaming current was developed, optimized and multiplexed for EV protein analysis at a bulk level. Using multiple microcapillary sensors functionalized with antibodies, the method showed the capacity for multiplexed detection of different surface markers on small EVs from non-small-cell lung cancer cells. The device performance in the multichannel configuration remained similar to the single-channel one in terms of noise, detection sensitivity, and reproducibility. The application of the technique for analysis of EVs isolated from lung cancer patients with different genomic alterations and after different applied treatments demonstrated the prospect of using EVs from liquid biopsies as a source of biomarker for cancer monitoring. Moreover, the results held promise for the application of the developed method in clinical settings. Finally, to increase the understanding of EV subpopulations and heterogeneity, a platform combining fluorescence and atomic force microscopy was developed for multiparametric analysis of EVs at a single particle level. The use of a precise spot identification approach and an efficient vesicle capture protocol allowed to study and correlate for the first time the membrane protein composition, size and mechanical properties (Young modulus) on individual small EVs. The application of the technique to vesicles isolated from different cell lines identified both common and cell line-specific EV subpopulations bearing distinct distributions of the analyzed parameters. For example, a sEV population co-expressing all the three analyzed proteins in relatively high abundance, yet having average diameters of <100 nm and relatively low Young moduli was found in all cell lines. The obtained results highlighted the possibility of using the developed platform to help decipher unsolved questions regarding EV biology.
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| 4. |
- Cavallaro, Sara, et al.
(författare)
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Label-Free Surface Protein Profiling of Extracellular Vesicles by an Electrokinetic Sensor
- 2019
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Ingår i: ACS Sensors. - : AMER CHEMICAL SOC. - 2379-3694. ; 4:5, s. 1399-1408
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Tidskriftsartikel (refereegranskat)abstract
- Small extracellular vesicles (sEVs) generated from the endolysosomal system, often referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics, as they carry valuable biological information and reflect their cells of origin. Herein, we propose a simple and inexpensive electrical method for label-free detection and profiling of sEVs in the size range of exosomes. The detection method is based on the electrokinetic principle, where the change in the streaming current is monitored as the surface markers of the sEVs interact with the affinity reagents immobilized on the inner surface of a silica microcapillary. As a proof-of-concept, we detected sEVs derived from the non-small-cell lung cancer (NSCLC) cell line H1975 for a set of representative surface markers, such as epidermal growth factor receptor (EGFR), CD9, and CD63. The detection sensitivity was estimated to be similar to 175000 sEVs, which represents a sensor surface coverage of only 0.04%. We further validated the ability of the sensor to measure the expression level of a membrane protein by using sEVs displaying artificially altered expressions of EGFR and CD63, which were derived from NSCLC and human embryonic kidney (HEK) 293T cells, respectively. The analysis revealed that the changes in EGFR and CD63 expressions in sEVs can be detected with a sensitivity in the order of 10% and 3%, respectively, of their parental cell expressions. The method can be easily parallelized and combined with existing microfluidic-based EV isolation technologies, allowing for rapid detection and monitoring of sEVs for cancer diagnosis.
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| 5. |
- Cavallaro, Sara, et al.
(författare)
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Multiparametric Profiling of Single Nanoscale Extracellular Vesicles by Combined Atomic Force and Fluorescence Microscopy : Correlation and Heterogeneity in Their Molecular and Biophysical Features
- 2021
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Ingår i: Small. - : Wiley. - 1613-6810 .- 1613-6829. ; 17:14
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Tidskriftsartikel (refereegranskat)abstract
- Being a key player in intercellular communications, nanoscale extracellular vesicles (EVs) offer unique opportunities for both diagnostics and therapeutics. However, their cellular origin and functional identity remain elusive due to the high heterogeneity in their molecular and physical features. Here, for the first time, multiple EV parameters involving membrane protein composition, size and mechanical properties on single small EVs (sEVs) are simultaneously studied by combined fluorescence and atomic force microscopy. Furthermore, their correlation and heterogeneity in different cellular sources are investigated. The study, performed on sEVs derived from human embryonic kidney 293, cord blood mesenchymal stromal and human acute monocytic leukemia cell lines, identifies both common and cell line-specific sEV subpopulations bearing distinct distributions of the common tetraspanins (CD9, CD63, and CD81) and biophysical properties. Although the tetraspanin abundances of individual sEVs are independent of their sizes, the expression levels of CD9 and CD63 are strongly correlated. A sEV population co-expressing all the three tetraspanins in relatively high abundance, however, having average diameters of <100 nm and relatively low Young moduli, is also found in all cell lines. Such a multiparametric approach is expected to provide new insights regarding EV biology and functions, potentially deciphering unsolved questions in this field.
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| 6. |
- Cavallaro, Sara, et al.
(författare)
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Multiplexed electrokinetic sensor for detection and therapy monitoring of extracellular vesicles from liquid biopsies of non-small-cell lung cancer patients
- 2021
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Ingår i: Biosensors & bioelectronics. - : Elsevier. - 0956-5663 .- 1873-4235. ; 193
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Tidskriftsartikel (refereegranskat)abstract
- Liquid biopsies based on extracellular vesicles (EVs) represent a promising tool for treatment monitoring of tumors, including non-small-cell lung cancers (NSCLC). In this study, we report on a multiplexed electrokinetic sensor for surface protein profiling of EVs from clinical samples. The method detects the difference in the streaming current generated by EV binding to the surface of a functionalized microcapillary, thereby estimating the expression level of a marker. Using multiple microchannels functionalized with different antibodies in a parallel fluidic connection, we first demonstrate the capacity for simultaneous detection of multiple surface markers in small EVs (sEVs) from NSCLC cells. To investigate the prospects of liquid biopsies based on EVs, we then apply the method to profile sEVs isolated from the pleural effusion (PE) fluids of five NSCLC patients with different genomic alterations (ALK, KRAS or EGFR) and applied treatments (chemotherapy, EGFR- or ALKtyrosine kinase inhibitors). The vesicles were targeted against CD9, as well as EGFR and PD-L1, two treatment targets in NSCLC. The electrokinetic signals show detection of these markers on sEVs, highlighting distinct interpatient differences, e.g., increased EGFR levels in sEVs from a patient with EGFR mutation as compared to an ALK-fusion one. The sensors also detect differences in PD-L1 expressions. The analysis of sEVs from a patient prior and post ALK-TKI crizotinib treatment reveals significant increases in the expressions of some markers (EGFR and PD-L1). These results hold promise for the application of the method for tumor treatment monitoring based on sEVs from patient liquid biopsies.
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| 7. |
- Chulapakorn, Thawatchart, 1988-, et al.
(författare)
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Influence of Swift Heavy Ion Irradiation on the Photoluminescence of Si-nanoparticles and Defects in SiO2
- 2017
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Ingår i: Nanotechnology. - : IOP PUBLISHING LTD. - 0957-4484 .- 1361-6528. ; 28:37
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Tidskriftsartikel (refereegranskat)abstract
- The influence of swift heavy ion (SHI) irradiation on the photoluminescence (PL) of silicon nanoparticles (SiNPs) and defects in SiO2-film is investigated. SiNPs were formed by implantation of 70 keV Si+ and subsequent thermal annealing to produce optically active SiNPs and to remove implantation-induced defects. Seven different ion species with energy between 3-36 MeV and fluence from 10(11)-10(14) cm(-2) were employed for irradiation of the implanted samples prior to the thermal annealing. Induced changes in defect and SiNP PL were characterized and correlated with the specific energy loss of the employed SHIs. We find that SHI irradiation, performed before the thermal annealing process, affects both defect and SiNP PL. The change of defect and SiNP PL due to SHI irradiation is found to show a threshold-like behaviour with respect to the electronic stopping power, where a decrease in defect PL and an anticorrelated increase in SiNP PL after the subsequent thermal annealing are observed for electronic stopping exceeding 3-5 keV nm(-1). PL intensities are also compared as a function of total energy deposition and nuclear energy loss. The observed effects can be explained by ion track formation as well as a different type of annealing mechanisms active for SHI irradiation compared to the thermal annealing.
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| 8. |
- Chulapakorn, Thawatchart, 1988-, et al.
(författare)
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Luminescence of silicon nanoparticles from oxygen implanted silicon
- 2018
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Ingår i: Materials Science in Semiconductor Processing. - : Elsevier. - 1369-8001 .- 1873-4081. ; 86, s. 18-22
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Tidskriftsartikel (refereegranskat)abstract
- Oxygen with a kinetic energy of 20 keV is implanted in a silicon wafer (100) at different fluences, followed by post-implantation thermal annealing (PIA) performed at temperatures ranging from 1000 to 1200 degrees C, in order to form luminescent silicon nanoparticles (SiNPs) and also to reduce the damage induced by the implantation. As a result of this procedure, a surface SiOx layer (with 0 < x < 2) with embedded crystalline Si nanoparticles has been created. The samples yield similar luminescence in terms of peak wavelength, lifetime, and absorption as recorded from SiNPs obtained by the more conventional method of implanting silicon into silicon dioxide. The oxygen implantation profile is characterized by elastic recoil detection (ERD) technique to obtain the excess concentration of Si in a presumed SiO2 environment. The physical structure of the implanted Si wafer is examined by grazing incidence X-ray diffraction (GIXRD). Photoluminescence (PL) techniques, including PL spectroscopy, time-resolved PL (TRPL), and photoluminescence excitation (PLE) spectroscopy are carried out in order to identify the PL origin. The results show that luminescent SiNPs are formed in a Si sample implanted by oxygen with a fluence of 2 x 10(17) atoms cm(-2) and PIA at 1000 degrees C. These SiNPs have a broad size range of 6-24 nm, as evaluated from the GIXRD result. Samples implanted at a lower fluence and/or annealed at higher temperature show only weak defect-related PL. With further optimization of the SiNP luminescence, the method may offer a simple route for integration of luminescent Si in mainstream semiconductor fabrication.
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| 9. |
- Chung, Nguyen Xuan, et al.
(författare)
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Optimized electrochemical breakdown etching using temporal voltage variation for formation of nanopores in a silicon membrane
- 2021
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Ingår i: Sensors and actuators. B, Chemical. - : Elsevier BV. - 0925-4005 .- 1873-3077. ; 331
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Tidskriftsartikel (refereegranskat)abstract
- Dielectric breakdown etching is a well-known method of making nanopores on thin (similar to 50 nm) dielectric membranes. However, voltage driven translocation of biomolecules through such nanopores becomes extremely fast. For improved detection, for instance by the current blockage, a high-aspect-ratio nanopore could be beneficial for slowing down the translocation. High-aspect-ratio nanopore on silicon fabrication requires a well-controlled process and is dependent on specific crystal orientation, dopant type and resistivity of substrate. Therefore, an optimized method of processing high-aspect-ratio nanopores is necessary considering the advantage of a silicon membrane being able to be integrated with standard CMOS processing. Here, we present an optimized fabrication method for mass-producing a single and an array of nanopores on a thick (2 mu m) silicon device layer based on a silicon-on-insulator (SOI) wafer. A method of temporal voltage variation is exploited to optimize the etching parameters for the nanopore formation during electrochemical breakdown etching, diameters of nanopores around 12 nm have been achieved. Besides, the correlation between the parameters of etching and nanopore diameter is deduced. The processed high-aspect-ratio nanopore enables applications in single-molecule sensing such as DNA, exosomes, viruses, and protein markers. The developed process is inexpensive, fast and can be batch fabricated.
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| 10. |
- Ciobanu, V., et al.
(författare)
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Large-Sized Nanocrystalline Ultrathin β-Ga2 O3 Membranes Fabricated by Surface Charge Lithography
- 2022
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Ingår i: Nanomaterials. - : MDPI AG. - 2079-4991. ; 12:4
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Tidskriftsartikel (refereegranskat)abstract
- Large-sized 2D semiconductor materials have gained significant attention for their fascinat-ing properties in various applications. In this work, we demonstrate the fabrication of nanoperforated ultrathin β-Ga2 O3 membranes of a nanoscale thickness. The technological route includes the fabrication of GaN membranes using the Surface Charge Lithography (SCL) approach and subsequent thermal treatment in air at 900◦ C in order to obtain β-Ga2 O3 membranes. The as-grown GaN membranes were discovered to be completely transformed into β-Ga2 O3, with the morphology evolving from a smooth topography to a nanoperforated surface consisting of nanograin structures. The oxidation mechanism of the membrane was investigated under different annealing conditions followed by XPS, AFM, Raman and TEM analyses.
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