A Transparent Semiconducting Surface for Capturing and Releasing Single Cells from a Complex Cell Mixture.

Selective isolation of individual target cells from a heterogeneous population is technically challenging; however, the ability to retrieve single cells can have high significance in various aspects of biological research. Here, we present a new photoelectrochemical surface based on a transparent electrode that is compatible with high-resolution fluorescence microscopy for isolating individual rare cells from complex biological samples. This is underpinned by two important factors: (i) careful design of the electrode by patterning discrete Au disks of micron dimension on amorphous silicon-indium tin oxide films and (ii) orthogonal surface chemistry, which modifies the patterned electrode with self-assembly layers of different functionalities, to selectively capture target cells on the Au disks and resist cell binding to the amorphous silicon surface. The co-stimulation of the surface using light from a microscope and an electric potential triggers the reductive desorption of the alkanethiol monolayer from the Au disks to release the single cells of interest from the illuminated regions only. Using circulating tumor cells as a model, we demonstrate the capture of cancer cells on an antibody-coated surface and selective release of single cancer cells with low expression of epithelial cell adhesion molecules.

Altered Cerebrospinal Fluid Exosomal microRNA Levels in Young-Onset Alzheimer's Disease and Frontotemporal Dementia.

BACKGROUND: micro-RNAs (miRNAs) are stable, small, non-coding RNAs enriched in exosomes. Their variation in levels according to different disease etiologies have made them a promising diagnostic biomarker for neurodegenerative diseases such as Alzheimer's disease (AD). Altered expression of miR-320a, miR-328-3p, and miR-204-5p have been reported in AD and frontotemporal dementia (FTD). OBJECTIVE: To determine their reliability, we aimed to examine the expression of three exosomal miRNAs isolated from cerebrospinal fluid (CSF) of patients with young-onset AD and FTD (< 65 years), correlating with core AD biomarkers and cognitive scores. METHODS: Exosomes were first isolated from CSF samples of 48 subjects (8 controls, 28 AD, and 12 FTD), followed by RNA extraction and quantitative PCR to measure the expression of miR-320a, miR-328-3p, and miR-204-5p. RESULTS: Expression of all three markers (miR-320a (p = 0.005), miR-328-3p (p = 0.049), and miR-204-5p (p = 0.036)) were significantly lower in AD versus controls. miR-320a was reduced in FTD versus controls (p = 0.049) and miR-328-3p was lower in AD versus FTD (p = 0.054). Notably, lower miR-328-3p levels could differentiate AD from FTD and controls with an AUC of 0.702, 95% CI: 0.534- 0.870, and showed significant correlation with lower CSF Aß42 levels (r = 0.359, p = 0.029). Pathway enrichment analysis identified potential targets of miR-328-3p implicated in the AMPK signaling pathway linked to amyloid-ß and tau metabolism in AD. CONCLUSION: Overall, we demonstrated miR-320a and miR-204-5p as reliable biomarkers for AD and FTD and report miR-328-3p as a novel AD biomarker.

Methylation dependent gold adsorption behaviour identifies cancer derived extracellular vesicular DNA.

Extracellular vesicles (EV) play a major role in intercellular communication by transmitting cellular materials (e.g. protein, RNA) among distant cells. Recent evidence suggests that they could also contribute to carrying DNA which could inform on the mutational status of the parent tumour DNA. Thus, the fundamental analysis of evDNA could open a better understanding of tumour metastasis and provide new pathways for noninvasive detection and monitoring of cancer. To explore the potential of evDNA for diagnostics, the isolation of pure evDNA from body fluids free of cfDNA contamination is crucial. Herein, we use a liposome based model system to develop an improved evDNA isolation protocol free from cfDNA contamination and evaluate the methylation dependent physicochemical properties of evDNA to develop a simple test for detecting cancer evDNA. Using a highly sensitive multiplex microelectrode device, we demonstrate that serum-evDNA derived from cancer patients show different solution and surface based properties than normal evDNA due to their different methylation landscape (i.e. methylscape). This microdevice allows simultaneous analysis of multiple samples in a single platform from as low as 500 pg µL-1 of evDNA.

Retooling phage display with electrohydrodynamic nanomixing and nanopore sequencing.

Phage display methodologies offer a versatile platform for the isolation of single-chain Fv (scFv) molecules which may be rebuilt into monoclonal antibodies. Herein, we report on a complete workflow termed PhageXpress, for rapid selection of single-chain Fv sequences by leveraging electrohydrodynamic-manipulation of a solution containing phage library particles to enhance target binding whilst minimizing non-specific interactions. Our PhageXpress technique is combined with Oxford Nanopore Technologies' MinION sequencer and custom bioinformatics to achieve high-throughput screening of phage libraries. We performed 4 rounds of biopanning against Dengue virus (DENV) non-structural protein 1 (NS1) using traditional methods (4 week turnaround), which resulted in the isolation of 19 unique scFv clones. We validated the feasibility and efficiency of the PhageXpress method utilizing the same phage library and antigen target. Notably, we successfully mapped 14 of the 19 anti-NS1 scFv sequences (∼74%) with our new method, despite using ∼30-fold less particles during screening and conducting only a single round of biopanning. We believe this approach supersedes traditional methods for the discovery of bio-recognition molecules such as antibodies by speeding up the process for the development of therapeutic and diagnostic biologics.

Label-free detection of exosomes using a surface plasmon resonance biosensor.

The development of a sensitive and specific detection platform for exosomes is highly desirable as they are believed to transmit vital tumour-specific information (mRNAs, microRNAs, and proteins) to remote cells for secondary metastasis. Herein, we report a simple method for the real-time and label-free detection of clinically relevant exosomes using a surface plasmon resonance (SPR) biosensor. Our method shows high specificity in detecting BT474 breast cancer cell-derived exosomes particularly from complex biological samples (e.g. exosome spiked in serum). This approach exhibits high sensitivity by detecting as low as 8280 exosomes/µL which may potentially be suitable for clinical analysis. We believe that this label-free and real-time method along with the high specificity and sensitivity may potentially be useful for clinical settings.

Nanomechanical Microfluidic Mixing and Rapid Labeling of Silica Nanoparticles using Allenamide-Thiol Covalent Linkage for Bioimaging.

Rapid surface functionalization of nanomaterials using covalent linkage following "green chemistry" remains challenging, and the quest for developing simple protocols is persisting. We report a nanomechanical microfluidic approach for the coupling of allenamide functionalized organic derivatives on the surface of thiol-modified silica nanoparticles using allenamide-thiol chemistry. The coupling principle involves the use of a microfluidic surface acoustic wave device that generates acoustic streaming-based chaotic fluid micromixing that enables mixing of laterally flowing fluids containing active components. This approach was used to demonstrate the direct surface labeling of thiol-modified silica nanoparticles using a selected group of modified fluorescent tags containing allenamide handles and achieved a total labeling efficiency of 83-90%. This green approach enabled a highly efficient surface functionalization under aqueous conditions, with tunable control over the conjugation process via the applied field. The dye-labeled silica particles were characterized using various analytical techniques and found to be biocompatible with potential in live cell bioimaging. It is envisaged that this bioconjugation strategy will find numerous applications in the field of bioimaging and drug delivery.

Cancer diagnosis: from tumor to liquid biopsy and beyond.

Technological advancements in research on circulating biomarkers from patient derived blood have enabled a less invasive means of diagnosing non-hematologic cancers. Considered a more practical way of real-time patient monitoring than traditional tumor biopsy, liquid biopsy markers including circulating tumor cells (CTCs), circulating tumor DNA (ctDNA) and extracellular vesicles (EVs) and exosomes certainly have the potential to change the dynamics of cancer management and treatment. Liquid biopsy essentially presents a snapshot of the disease from the primary and/or distant tumor locations and can be utilized for repeated sampling of tumor markers to adjust therapy according to the patient's response to treatment, also known as personalized or precision treatment. In this review, we discuss the research progress in this field with respect to each of the liquid biopsy markers ranging from CTCs, EVs to ctDNA. First, we highlight key CTC technologies that have been commercialized and extensively employed for patient sample analysis. Next, we present some recent developments with regards to exosome and ctDNA research. We then conclude with some future perspectives on the areas of research for these biomarkers. Taken together, we believe these non-invasive capabilities and their potential for diagnostic development can influence treatment selections and aid precision cancer therapies.

Microfluidics for cell sorting and single cell analysis from whole blood.

The complexity and dynamic evolution of cancer often result in tumor subpopulations containing distinctly heterogeneous cells. During metastasis, these also give rise to heterogeneous circulating tumor cells (CTCs) which are considered to be a hematogenous dissemination from the primary tumor. CTCs represent a viable less-invasive sampling opportunity, also known as liquid biopsy. However, current technological platforms that analyze entire CTC population are not effective due to cell-to-cell variability within the same population and this can manifest differences in genomic expression, cell cycle stages and eventually cellular responses to drug treatments. Here, we present a novel microfluidic approach that involves combination of two microfluidic chips operating under inertial fluid forces and hydrodynamic focusing to rapidly isolate and selectively retrieve bulk as well as single CTCs from whole blood for downstream single cell analysis. It is envisioned that this combinational approach to retrieve single CTCs can cater to several applications including more accurate disease diagnosis as well as formulation of personalized therapeutic strategies.

Circulating tumor microemboli: Progress in molecular understanding and enrichment technologies.

Circulating tumor cells (CTCs) and their clusters, also known as circulating tumor microemboli (CTM), have emerged as valuable tool that can provide mechanistic insights into the tumor heterogeneity, clonal evolution, and stochastic events within the metastatic cascade. However, recent investigations have hinted that CTM may not be mere aggregates of tumor cells but cells comprising CTM exhibit distinct phenotypic and molecular characteristics in comparison to single CTCs. Moreover, in many cases CTM demonstrated higher metastatic potential and resistance to apoptosis as compared to their single cell counterparts. Thus, their evaluation and enumeration may provide a new dimension to our understanding of cancer biology and metastatic cancer spread as well as offer novel theranostic biomarkers. Most of the existing technologies for isolation of hematogenous tumor cells largely favor single CTCs, hence there is a need to devise new approaches, or re-configure the existing ones, for specific and efficient CTM isolation. Here we review existing knowledge and insights on CTM biology. Furthermore, a critical commentary on current and emerging trends in CTM enrichment and characterization along with recently developed ex-vivo CTC expansion methodologies is presented with the aim to facilitate researchers to identify further avenues of research and development.

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.

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.

Capture and On-chip analysis of Melanoma Cells Using Tunable Surface Shear forces.

With new systemic therapies becoming available for metastatic melanoma such as BRAF and PD-1 inhibitors, there is an increasing demand for methods to assist with treatment selection and response monitoring. Quantification and characterisation of circulating melanoma cells (CMCs) has been regarded as an excellent non-invasive candidate but a sensitive and efficient tool to do these is lacking. Herein we demonstrate a microfluidic approach for melanoma cell capture and subsequent on-chip evaluation of BRAF mutation status. Our approach utilizes a recently discovered alternating current electrohydrodynamic (AC-EHD)-induced surface shear forces, referred to as nanoshearing. A key feature of nanoshearing is the ability to agitate fluid to encourage contact with surface-bound antibody for the cell capture whilst removing nonspecific cells from the surface. By adjusting the AC-EHD force to match the binding affinity of antibodies against the melanoma-associated chondroitin sulphate proteoglycan (MCSP), a commonly expressed melanoma antigen, this platform achieved an average recovery of 84.7% from biological samples. Subsequent staining with anti-BRAF(V600E) specific antibody enabled on-chip evaluation of BRAF(V600E) mutation status in melanoma cells. We believe that the ability of nanoshearing-based capture to enumerate melanoma cells and subsequent on-chip characterisation has the potential as a rapid screening tool while making treatment decisions.

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 Gilbert's 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.

Enhancing Protein Capture Using a Combination of Nanoyeast Single-Chain Fragment Affinity Reagents and Alternating Current Electrohydrodynamic Forces.

New high-performance detection technologies and more robust protein capture agents can be combined to both rapidly and specifically capture and detect protein biomarkers associated with disease in complex biological samples. Here we demonstrate the use of recently developed recombinant affinity reagents, namely nanoyeast-scFv, in combination with alternating current electrohydrodynamic (ac-EHD)-induced shear forces, to enhance capture performance during protein biomarker analysis. The use of ac-EHD significantly improves fluid transport across the capture domain, resulting in enhanced sensor-target interaction and simultaneous displacement of nonspecific molecules from the electrode surface. We demonstrate this simple proof-of-concept approach for the capture and detection of Entamoeba histolytica antigens from disinfected stool, within a span of 5 min using an ac-EHD microfluidic device. Under an ac-EHD field, antigens were captured on a nanoyeast-scFv immobilized device and subsequently detected using a quantum dot conjugated antibody. This immunosensor specifically detected antigen in disinfected stool with low background noise at concentrations down to 58.8 fM with an interassay reproducibility (%RSD of n = 3) < 17.2%, and in buffer down to 5.88 fM with an interassay reproducibility (% RSD, n = 3) of 8.4%. Furthermore, antigen detection using this immunosensor was 10 times more sensitive than previously obtained with the same nanoyeast-scFv reagents in a microfluidic device employing surface-enhanced Raman scattering (SERS) detection in buffer and at least 200 times more sensitive than methods using screen printed gold electrodes in disinfected stool. We predict this rapid and sensitive approach using these stable affinity reagents may offer a new methodology to detect protein disease biomarkers from biological matrices.

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.

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.

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.

Tuneable surface shear forces to physically displace nonspecific molecules in protein biomarker detection.

We report a simple method to remove nonspecifically adsorbed species from sensor surface and also improve the detection sensitivity of the sensor using tuneable alternating current (ac) electrohydrodynamics (ac-EHD) forces. These forces generated within few nanometers of an electrode surface (i.e., double layer) engender fluid flow within a serpentine channel containing a long array of the asymmetric electrode pairs, and can easily be tuned externally by changing the frequency and amplitude of the ac-EHD field. Under the optimized experimental conditions, we achieved a 3.5-fold reduction in nonspecific adsorption of non-target proteins with a 1000-fold enhancement in detection sensitivity of the device for the analysis of human epidermal growth factor receptor 2 (HER2) protein spiked in serum. This approach can be applicable in diverse fields including biosensors, cellular and molecular separation systems and biomedical applications to remove/reduce nonspecific adsorption of molecular and cellular species.

Alternating current electrohydrodynamics induced nanoshearing and fluid micromixing for specific capture of cancer cells.

We report a new tuneable alternating current (ac) electrohydrodynamics (ac-EHD) force referred to as "nanoshearing" which involves fluid flow generated within a few nanometers of an electrode surface. This force can be externally tuned via manipulating the applied ac-EHD field strength. The ability to manipulate ac-EHD induced forces and concomitant fluid micromixing can enhance fluid transport within the capture domain of the channel (e.g., transport of analytes and hence increase target­sensor interactions). This also provides a new capability to preferentially select strongly bound analytes over nonspecifically bound cells and molecules. To demonstrate the utility and versatility of nanoshearing phenomenon to specifically capture cancer cells, we present proof-of-concept data in lysed blood using two microfluidic devices containing a long array of asymmetric planar electrode pairs. Under the optimal experimental conditions, we achieved high capture efficiency (e.g., approximately 90%; %RSD=2, n=3) with a 10-fold reduction in nonspecific adsorption of non-target cells for the detection of whole cells expressing Human Epidermal Growth Factor Receptor 2 (HER2). We believe that our ac-EHD devices and the use of tuneable nanoshearing phenomenon may find relevance in a wide variety of biological and medical applications.