Emerging techniques in the isolation and characterization of extracellular vesicles and their roles in cancer diagnostics and prognostics

Sunkara, Vijaya,Woo, Hyun-Kyung,Cho, Yoon-Kyoung The Royal Society of Chemistry 2016 The Analyst Vol.141 No.2

<P>Extracellular vesicles (EVs) are cell-derived nanovesicles, present in almost all types of body fluids, which play an important role in intercellular communication and are involved in the transport of biological signals for regulating diverse cellular functions. Due to the increasing clinical interest in the role of EVs in tumor promotion, various techniques for their isolation, detection, and characterization are being developed. In this review, we present an overview of the current EV isolation and characterization methods in addition to their applications and limitations. Furthermore, EVs as the potential emerging biomarkers in cancer management and their clinical implementation are briefly discussed.</P>

Investigation on the Mechanism of Aminosilane-Mediated Bonding of Thermoplastics and Poly(dimethylsiloxane)

Sunkara, Vijaya,Cho, Yoon-Kyoung American Chemical Society 2012 ACS APPLIED MATERIALS & INTERFACES Vol.4 No.12

<P>A possible mechanism for the aminosilane-mediated room-temperature (RT) bonding of thermoplastics and poly(dimethylsiloxane) (PDMS) is presented. The plasma-activated thermoplastic or PDMS substrates were modified with alkoxy silanes having different organo functional groups, and their bonding characteristics were studied. Manual peeling tests revealed that strong bonding was realized only when the silane had a free amino group and at least two alkoxy groups on the silicon. Silanization was carried out in both aqueous and anhydrous conditions; bonding occurred readily at RT in the former case, but a longer incubation time or a higher temperature was needed for the latter. The presence of the silane on the surface was confirmed by contact-angle measurements and UV spectrophotometric, attenuated total reflectance infrared spectroscopic (ATR-IR) and X-ray photoelectron spectroscopic (XPS) analyses. In the case where the aminosilane was deposited from aqueous solution, the amino functionality of the silane-catalyzed siloxane bond formation between the silanol on the modified thermoplastic surface and the silanol of the plasma-activated PDMS. In the case of anhydrous phase deposition, the aminosilane first catalyzed the hydrolysis of the ethoxy groups on the silicon, and then, catalyzed the condensation between the silanol groups of both materials. Shelf life tests of the modified thermoplastics showed that the aminosilane was stable over 2 weeks, and that bonding occurred at RT when the substrates were soaked in water before bonding.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/aamick/2012/aamick.2012.4.issue-12/am3015923/production/images/medium/am-2012-015923_0007.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/am3015923'>ACS Electronic Supporting Info</A></P>

The Colorectal Cancer Mortality-to-Incidence Ratio as a Potential Cancer Surveillance Measure in Asia

Sunkara, Vasu,Hebert, James R Asian Pacific Journal of Cancer Prevention 2016 Asian Pacific journal of cancer prevention Vol.17 No.9

Background: The cancer mortality-to-incidence ratio (MIR) has been established as an important measure of health disparities in local and global circumstances. Past work has corroborated a linkage between the colorectal cancer MIR and the World Health Organization (WHO) Health System ranking. The literature further documents many Asian countries having incomplete cancer registries and a lack of comprehensive colorectal cancer screening guidelines. Materials and Methods: The colorectal cancer MIR values for 23 Asian countries were calculated from data obtained from the 2012 GLOBOCAN database. The 2000 World Health Organization (WHO) Health System rankings were used as a proxy for health system infrastructure and responsiveness. A regression equation was calculated with the MIR as the dependent variable and the WHO Health System ranking as the independent variable. Predicted MIR values were next calculated based on the regression results. Actual MIR values that exceeded 0.20 from the predicted MIR were removed as 'divergent' points. The regression equation was then re-plotted. Goodness-of-fit for both regressions was assessed by the R-squared test. Results: Asian countries have a relatively wide colorectal cancer MIR range, from a minimum of 0.24 to a maximum of 0.86. For the full dataset, the adjusted R-squared value for this regression was 0.53. The equation was then used to calculate a predicted MIR, whereby two data points were identified as 'divergent' and removed. The adjusted R-squared for the edited dataset increased to 0.66. Conclusions: Asian countries have a marked range in their colorectal cancer MIR values and there is a strong correlationwith the WHO Health System ranking. These results corroborate the contribution of the MIR as a potentially robust tool in monitoring changes in colorectal cancer care for Asian nations.

Simple room temperature bonding of thermoplastics and poly(dimethylsiloxane)

Sunkara, Vijaya,Park, Dong-Kyu,Hwang, Hyundoo,Chantiwas, Rattikan,Soper, Steven A.,Cho, Yoon-Kyoung Royal Society of Chemistry 2011 Lab on a chip Vol.11 No.5

<P>We describe a simple and versatile method for bonding thermoplastics to elastomeric polydimethylsiloxane (PDMS) at room temperature. The bonding of various thermoplastics including polycarbonate (PC), cyclic olefin copolymer (COC), polymethylmethacrylate (PMMA), and polystyrene (PS), to PDMS has been demonstrated at room temperature. An irreversible bonding was formed instantaneously when the thermoplastics, activated by oxygen plasma followed by aminopropyltriethoxysilane modification, were brought into contact with the plasma treated PDMS. The surface modified thermoplastics were characterized by water contact angle measurements and X-ray photoelectron spectroscopy. The tensile strength of the bonded hybrid devices fabricated with PC, COC, PMMA, and PS was found to be 430, 432, 385, and 388 kPa, respectively. The assembled devices showed high burst resistance at a maximum channel pressure achievable by an in-house built syringe pump, 528 kPa. Furthermore, they displayed very high hydrolytic stability; no significant change was observed even after the storage in water at 37 °C over a period of three weeks. In addition, this thermoplastic-to-PDMS bonding technique has been successfully employed to fabricate a relatively large sized device. For example, a lab-on-a-disc with a diameter of 12 cm showed no leakage when it spins for centrifugal fluidic pumping at a very high rotating speed of 6000 rpm.</P> <P>Graphic Abstract</P><P>We report a simple, versatile, instantaneous and irreversible method for bonding thermoplastics to elastomeric polydimethylsiloxane (PDMS) at room temperature. <IMG SRC='http://pubs.rsc.org/services/images/RSCpubs.ePlatform.Service.FreeContent.ImageService.svc/ImageService/image/GA?id=c0lc00272k'> </P>

Electrospun TiO2 nanofiber integrated lab-on-a-disc for ultrasensitive protein detection from whole blood

Lee, W.,Sunkara, V.,Han, J. R.,Park, Y. S.,Cho, Y. K. Royal Society of Chemistry 2015 Lab on a chip Vol.15 No.2

Correction to Lab-on-a-Disc for Fully Integrated Multiplex Immunoassays

Park, Jiwoon,Sunkara, Vijaya,Kim, Tae-Hyeong,Hwang, Hyundoo,Cho, Yoon-Kyoung American Chemical Society 2012 ANALYTICAL CHEMISTRY - Vol.84 No.10

Exodisc for Rapid, Size-Selective, and Efficient Isolation and Analysis of Nanoscale Extracellular Vesicles from Biological Samples

Woo, Hyun-Kyung,Sunkara, Vijaya,Park, Juhee,Kim, Tae-Hyeong,Han, Ja-Ryoung,Kim, Chi-Ju,Choi, Hyun-Il,Kim, Yoon-Keun,Cho, Yoon-Kyoung American Chemical Society 2017 ACS NANO Vol.11 No.2

<P>Extracellular vesicles (EVs) are cell-derived, nano scale vesicles that carry nucleic acids and proteins from their cells of origin and show great potential as biomarkers for many diseases, including cancer. Efficient isolation and detection methods are prerequisites for exploiting their use in clinical settings and understanding their physiological functions. Here, we presented a rapid, label-free, and highly sensitive method for EV isolation and quantification using a lab-on-a-disc integrated with two nanofilters (Exodisc). Starting from raw biological samples, such as cell-culture supernatant (CCS) or cancer patient urine, fully automated enrichment of EVs in the size range of 20-600 nm was achieved within 30 min using a tabletop-sized centrifugal microfluidic system. Quantitative tests using nanoparticle-tracking analysis confirmed that the Exodisc enabled >95% recovery of EVs from CCS. Additionally, analysis of mRNA retrieved from EVs revealed that the Exodisc provided >100-fold higher concentration of mRNA as compared with the gold-standard ultracentrifugation method. Furthermore, on-disc enzyme-linked immunosorbent assay using urinary EVs isolated from bladder cancer patients showed high levels of CD9 and CD81 expression, suggesting that this method may be potentially useful in clinical settings to test urinary EV-based biomarkers for cancer diagnostics.</P>

DNA microarrays on nanoscale-controlled surface

Hong, Bong Jin,Sunkara, Vijaya,Park, Joon Won Oxford University Press 2005 Nucleic acids research Vol.33 No.12

<P>We have developed new surface to ensure a proper spacing between immobilized biomolecules. While DNA microarray on this surface provided each probe DNA with ample space for hybridization with incoming target DNAs, the microarray showed enhanced discrimination efficiency for various types of single nucleotide polymorphism. The high discrimination efficiency holds for all tested cases (100:<1 for internal mismatched cases; 100:<28 for terminal mismatched ones). In addition, by investigating influence of hybridization temperature and washing condition on the fluorescence intensity and the discrimination efficiency with and without controlled mesospacing, it was observed that the nanoscale-controlled surface showed good discrimination efficiency in a wide range of temperature (37–50°C), and hybridization behavior on the surface was in agreement with the solution one. Intriguingly, it was found that washing process after the hybridization was critical for the high discrimination efficiency. For the particular case, washing process was so efficient that only 30 s washing was sufficient to reach the optimal discrimination ratio.</P>

Macromolecular Arabinogalactan Polysaccharide Mediated Synthesis of Silver Nanoparticles, Characterization and Evaluation

Kasva Anuradha,Prakritiranjan Bangal,Sunkara Sakunthala Madhavendra 한국고분자학회 2016 Macromolecular Research Vol.24 No.2

Extremely stable and spherical silver nanoparticles (AgNPs) were synthesized using a simple and ecofriendly approach by using the arabinogalactan mucilage of portulaca, a food grade natural polysaccharide complex. The aqueous solution of mucilage served as both reducing agent and stabilizer in this reaction. The resulting nanoparticles were spherical with a diameter of 20-30 nm. Transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transmission infrared spectroscopy (FTIR), atomic absorption spectroscopy (AAS), dynamic light scattering (DLS) and UV-visible spectroscopy (UV-Vis) techniques were used to characterize and study the reaction mechanism of generated silver nanoparticles. A reaction mechanism was devised for the formation of arabinogalactan stabilized silver nanoparticles. The reaction of resulting AgNPs against bacterial and fungal strains has shown that they are more potent against gram-negative bacterial strains. The reduction reaction of 4-nitrophenol catalyzed by arabinogalactan-stabilized AgNPs showed good catalytic activity towards the formation of 4-amino phenol within 10 min of time.

A lab-on-a-disc with reversible and thermally stable diaphragm valves

Kim, T. H.,Sunkara, V.,Park, J.,Kim, C. J.,Woo, H. K.,Cho, Y. K. Royal Society of Chemistry 2016 Lab on a chip Vol.16 No.19

<P>A lab-on-a-disc is a unique microfluidic platform that utilizes centrifugal force to pump liquids. This offers many benefits for point-of-care devices because it eliminates the need for connections to multiple pumps and complex tubing connections. A wide range of applications including clinical chemistry, immunoassay, cell analysis, and nucleic acid tests could be demonstrated on a spinning disc. To enable the performance of assays in a fully integrated and automated manner, the robust actuation of integrated valves is a prerequisite. However, conventional passive-type valves incur a critical drawback in that their operation is dependent on the rotational frequency, which is easily influenced by the channel geometry and chemistry, in addition to the physical properties of the liquids to be transferred. Even though a few active-type valving techniques permit the individual actuation of valves, independent of the rotational frequency, complex procedures for the fabrication as well as actuation mechanisms have prevented their broader acceptance in general applications. Here, we report on a lab-on-a-disc incorporating individually addressable diaphragm valves (ID valves) that enable the reversible and thermally stable actuation of multiple valves with unprecedented ease and robustness. These ID valves are configured from an elastic epoxy diaphragm embedded on a 3D printed push-and-twist valve, which can be easily actuated by a simple automatic driver unit. As a proof of concept experiment, an enzyme-linked immunosorbent assay (ELISA) and a polymerase chain reaction (PCR) were performed on a disc in a fully automated manner to demonstrate the robust, reversible, leak-free, and thermally stable actuation of the valves.</P>