Ramanathan Vaidyanathan

Analysis of exosome purification methods using a model liposome system and tunable-resistive pulse sensing

Exosomes are vesicles which have garnered interest due to their diagnostic and therapeutic potential. Isolation of pure yields of exosomes from complex biological fluids whilst preserving their physical characteristics is critical for downstream applications. In this study, we use 100 nm-liposomes from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as a model system as a model system to assess the effect of exosome isolation protocols on vesicle recovery and size distribution using a single-particle analysis method. We demonstrate that liposome size distribution and ζ-potential are comparable to extracted exosomes, making them an ideal model for comparison studies. Four different purification protocols were evaluated, with liposomes robustly isolated by three of them. Recovered yields varied and liposome size distribution was unaltered during processing, suggesting that these protocols do not induce particle aggregation. This leads us to conclude that the size distribution profile and characteristics of vesicles are stably maintained during processing and purification, suggesting that reports detailing how exosomes derived from tumour cells differ in size to those from normal cells are reporting a real phenomenon. However, we hypothesize that larger particles present in most purified exosome samples represent co-purified contaminating non-exosome debris. These isolation techniques are therefore likely nonspecific and may co-isolate non-exosome material of similar physical properties.

Detecting Exosomes Specifically: A Multiplexed Device Based on Alternating Current Electrohydrodynamic Induced Nanoshearing

Exosomes show promise as noninvasive biomarkers for cancer, but their effective capture and specific detection is a significant challenge. Herein, we report a multiplexed microfluidic device for highly specific capture and detection of multiple exosome targets using a tunable alternating current electrohydrodynamic (ac-EHD) methodology, referred to as nanoshearing. In our system, electrical body forces generated by ac-EHD act within nanometers of an electrode surface (i.e., within the electrical layer) to generate nanoscaled fluid flow that enhances the specificity of capture and also reduce nonspecific adsorption of weakly bound molecules from the electrode surface. This approach demonstrates the analysis of exosomes derived from cells expressing human epidermal growth factor receptor 2 (HER2) and prostate specific antigen (PSA), and is also capable of specifically isolating exosomes from breast cancer patient samples. The device also exhibited a 3-fold enhancement in detection sensitivity in comparison to hydrodynamic flow based assays (LOD 2760 exosomes/μL for ac-EHD vs LOD 8300 exosomes/μL for hydrodynamic flow; (n = 3)). We propose this approach can potentially have relevance as a simple and rapid quantification tool to analyze exosome targets in biological applications.

Enabling Rapid and Specific Surface-Enhanced Raman Scattering Immunoassay Using Nanoscaled Surface Shear Forces

A rapid and simple approach is presented to address two critical issues of surface-enhanced Raman scattering (SERS)-based immunoassay such as removal/avoiding nonspecific adsorption and reducing assay time. The approach demonstrated involves rationally designed fluorophore-integrated gold/silver nanoshells as SERS nanotags and utilizes alternative current electrohydrodynamic (ac-EHD)-induced nanoscaled surface shear forces to enhance the capture kinetics. The assay performance was validated in comparison with hydrodynamic flow and conventional immunoassay-based devices. These nanoscaled physical forces acting within nanometer distances from the electrode surface enabled rapid (40 min), sensitive (10 fg/mL), and highly specific detection of human epidermal growth factor receptor 2 in breast cancer patient samples. We believe this approach presents potential for the development of rapid and sensitive SERS immunoassays for routine clinical diagnosis.

Real time and label free profiling of clinically relevant exosomes

Tumor-derived exosomes possess significant clinical relevance due to their unique composition of genetic and protein material that is representative of the parent tumor. Specific isolation as well as identification of proportions of these clinically relevant exosomes (CREs) from biological samples could help to better understand their clinical significance as cancer biomarkers. Herein, we present a simple approach for quantification of the proportion of CREs within the bulk exosome population isolated from patient serum. This proportion of CREs can potentially inform on the disease stage and enable non-invasive monitoring of inter-individual variations in tumor-receptor expression levels. Our approach utilises a Surface Plasmon Resonance (SPR) platform to quantify the proportion of CREs in a two-step strategy that involves (i) initial isolation of bulk exosome population using tetraspanin biomarkers (i.e., CD9, CD63), and (ii) subsequent detection of CREs within the captured bulk exosomes using tumor-specific markers (e.g., human epidermal growth factor receptor 2 (HER2)). We demonstrate the isolation of bulk exosome population and detection of as low as 10% HER2(+) exosomes from samples containing designated proportions of HER2(+) BT474 and HER2(−) MDA-MB-231 cell derived exosomes. We also demonstrate the successful isolation of exosomes from a small cohort of breast cancer patient samples and identified that approximately 14–35% of their bulk population express HER2.

Molecular nanoshearing: an innovative approach to shear off molecules with AC-induced nanoscopic fluid flow.

Early diagnosis of disease requires highly specific measurement of molecular biomarkers from femto to pico-molar concentrations in complex biological (e.g., serum, blood, etc.) samples to provide clinically useful information. While reaching this detection limit is challenging in itself, these samples contain numerous other non-target molecules, most of which have a tendency to adhere to solid surfaces via nonspecific interactions. Herein, we present an entirely new methodology to physically displace nonspecifically bound molecules from solid surfaces by utilizing a newly discovered “tuneable force”, induced by an applied alternating electric field, which occurs within few nanometers of an electrode surface. This methodology thus offers a unique ability to shear-off loosely bound molecules from the solid/liquid interface. Via this approach, we achieved a 5-fold reduction in nonspecific adsorption of non-target protein molecules and a 1000-fold enhancement for the specific capture of HER2 protein in human serum.

Electrohydrodynamic-induced SERS immunoassay for extensive multiplexed biomarker sensing

Cancer diagnosis and patient monitoring require sensitive and simultaneous measurement of multiple cancer biomarkers considering that single biomarker analysis present inadequate information on the underlying biological transformations. Thus, development of sensitive and selective assays for multiple biomarker detection might improve clinical diagnosis and expedite the treatment process. Herein, a microfluidic platform for the rapid, sensitive, and parallel detection of multiple cancer-specific protein biomarkers from complex biological samples is presented. This approach utilizes alternating current electrohydrodynamic-induced surface shear forces that provide exquisite control over fluid flow thereby enhancing target-sensor interactions and minimizing non-specific binding. Further, the use of surface-enhanced Raman scattering-based spectral encoding with individual barcodes for different targets enables specific and simultaneous detection of captured protein biomarkers. Using this approach, the specific and sensitive detection of clinically relevant biomarkers including human epidermal growth factor receptor 2 (HER2); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor; and Mucin 16, cell surface associated (MUC16) at concentrations as low as 10 fg mL-1 in patient serum is demonstrated. Successful target detection from patient samples further demonstrates the potential of this current approach for the clinical diagnosis, which envisages a clinical translation for a rapid and sensitive appraisal of clinical samples in cancer diagnostics.

Tunable "nano-shearing": a physical mechanism to displace nonspecific cell adhesion during rare cell detection.

We report a tunable alternating current electro-hydrodynamic (ac-EHD) force which drives lateral fluid motion within a few nanometers of an electrode surface. Because the magnitude of this fluid shear force can be tuned externally (e.g., via the application of an ac electric field), it provides a new capability to physically displace weakly (nonspecifically) bound cellular analytes. To demonstrate the utility of the tunable nanoshearing phenomenon, we present data on purpose-built microfluidic devices that employ ac-EHD force to remove nonspecific adsorption of molecular and cellular species. Here, we show that an ac-EHD device containing asymmetric planar and microtip electrode pairs resulted in a 4-fold reduction in nonspecific adsorption of blood cells and also captured breast cancer cells in blood, with high efficiency (approximately 87%) and specificity. We therefore feel that this new capability of externally tuning and manipulating fluid flow could have wide applications as an innovative approach to enhance the specific capture of rare cells such as cancer cells in blood.

Alternating current electrohydrodynamics in microsystems: Pushing biomolecules and cells around on surfaces

Electrohydrodynamics (EHD) deals with the fluid motion induced by an electric field. This phenomenon originally developed in physical science, and engineering is currently experiencing a renaissance in microfluidics. Investigations by Taylor on Gilberts theory proposed in 1600 have evolved to include multiple contributions including the promising effects arising from electric field interactions with cells and particles to influence their behaviour on electrode surfaces. Theoretical modelling of electric fields in microsystems and the ability to determine shear forces have certainly reached an advanced state. The ability to deftly manipulate microscopic fluid flow in bulk fluid and at solid/liquid interfaces has enabled the controlled assembly, coagulation, or removal of microstructures, nanostructures, cells, and molecules on surfaces. Furthermore, the ability of electrohydrodynamics to generate fluid flow using surface shear forces generated within nanometers from the surface and their application in bioassays has led to recent advancements in biomolecule, vesicle and cellular detection across different length scales. With the integration of Alternating Current Electrohydrodynamics (AC-EHD) in cellular and molecular assays proving to be highly fruitful, challenges still remain with respect to understanding the discrepancies between each of the associated ac-induced fluid flow phenomena, extending their utility towards clinical diagnostic development, and utilising them in tandem as a standard tool for disease monitoring. In this regard, this article will review the history of electrohydrodynamics, followed by some of the recent developments in the field including a new dimension of electrohydrodynamics that deals with the utilization of surface shear forces for the manipulation of biological cells or molecules on electrode surfaces. Recent advances and challenges in the use of electrohydrodynamic forces such as dielectrophoresis and ac electrosmosis for the detection of biological analytes are also reviewed. Additionally, the fundamental mechanisms of fluid flow using electrohydrodynamics forces, which are still evolving, are reviewed. Challenges and future directions are discussed from the perspective of both fundamental understanding and potential applications of these nanoscaled shear forces in diagnostics.

A Multiplexed Device Based on Tunable Nanoshearing for Specific Detection of Multiple Protein Biomarkers in Serum

Microfluidic flow based multiplexed devices have gained significant promise in detecting biomarkers in complex biological samples. However, to fully exploit their use in bioanalysis, issues such as (i) low sensitivity and (ii) high levels of nonspecific adsorption of non-target species have to be overcome. Herein, we describe a new multiplexed device for the sensitive detection of multiple protein biomarkers in serum by using an alternating current (ac) electrohydrodynamics (ac-EHD) induced surface shear forces based phenomenon referred to as nanoshearing. The tunable nature (via manipulation of ac field) of these nanoshearing forces can alter the capture performance of the device (e.g., improved fluid transport enhances number of sensor-target collisions). This can also selectively displace weakly (nonspecifically) bound molecules from the electrode surface (i.e., fluid shear forces can be tuned to shear away nonspecific species present in biological samples). Using this approach, we achieved sensitive (100 fg mL−1) naked eye detection of multiple protein targets spiked in human serum and a 1000-fold enhancement in comparison to hydrodynamic flow based devices for biomarker detection. We believe that this approach could potentially represent a clinical diagnostic tool that can be integrated into resource-limited settings for sensitive detection of target biomarkers using naked eye.

Cell Interactions at the Nanoscale: Piezoelectric Stimulation

Nanometric movements of the substrate on which endothelial cells are growing, driven by periodic sinusoidal vibration from 1 Hz to 50 Hz applied by piezo actuators, upregulate endothelin-1 and Kruppel-like factor 2 expression, and increase cell adhesion. These movements are in the z (vertical) axis and ranges from 5 to 50 nm and are similar in vertical extent to protrusions from the cells themselves already reported in the literature. White noise vibrations do not to produce these effects. Vibrational sweeps, if suitably confined within a narrow frequency range, produce similar stimulatory effects but not at wider sweeps. These effects suggest that coherent vibration is crucial for driving these cellular responses. In addition to this, the applied stimulations are observed to be close to or below the random seismic noise of the surroundings, which may suggest stochastic resonance is being employed. The stimulations also interact with the effects of nanometric patterning of the substrates on cell adhesion and Kruppel-like factor 2 and endothelin-1 expression thus linking cell reactions to nanotopographically patterned surfaces with those to mechanical stimulation.