Status quo of Extracellular Vesicle isolation and detection methods for clinical utility.

Extracellular vesicles (EVs) are nano-sized particles that hold tremendous potential in the clinical space, as their biomolecular profiles hold a key to non-invasive liquid biopsy for cancer diagnosis and prognosis. EVs are present in most bodily fluids, hence are easily obtainable from patients, advantageous to that of traditional, invasive tissue biopsies and imaging techniques. However, there are certain constraints that hinder clinical use of EVs. The translation of EV biomarkers from "bench-to-bedside" is encumbered by the methods of EV isolation and subsequent biomarker detection currently implemented in laboratories. Although current isolation and detection methods are effective, they lack practicality, with their requirement for high bodily fluid volumes, low equipment availability, slow turnaround times and high costs. The high demand for techniques that overcome these limitations has resulted in significant advancements in nanotechnological devices. These devices are designed to integrate EV isolation and biomarker detection into a one-step method of direct EV detection from bodily fluids. This provides promise for the acceleration of EVs into current clinical standards. This review highlights the importance of EVs as cancer biomarkers, the methodological obstacles currently faced in clinical studies and how novel nanodevices could advance clinical translation.

Analysis of secreted small extracellular vesicles from activated human microglial cell lines reveals distinct pro- and anti-inflammatory proteomic profiles.

Microglia are a specialized population of innate immune cells located in the central nervous system. In response to physiological and pathological changes in their microenvironment, microglia can polarize into pro-inflammatory or anti-inflammatory phenotypes. A dysregulation in the pro-/anti-inflammatory balance is associated with many pathophysiological changes in the brain and nervous system. Therefore, the balance between microglia pro-/anti-inflammatory polarization can be a potential biomarker for the various brain pathologies. A non-invasive method of detecting microglia polarization in patients would have promising clinical applications. Here, we perform proteomic analysis of small extracellular vesicles (sEVs) derived from microglia cells to identify sEVs biomarkers indicative of pro-inflammatory and anti-inflammatory phenotypic changes. sEVs were isolated from microglia cell lines under different inflammatory conditions and analyzed by proteomics by liquid chromatography with mass spectrometry. Our findings provide the potential roles of sEVs that could be related to the pathogenesis of various brain diseases.

High Accuracy of Clinical Verification of Electrohydrodynamic-Driven Nanobox-on-Mirror Platform for Molecular Identification of Respiratory Viruses.

The molecular detection of multiple respiratory viruses provides evidence for the rational use of drugs and effective health management. Herein, we developed and tested the clinical performance of an electrohydrodynamic-driven nanobox-on-mirror platform (E-NoM) for the parallel, accurate, and sensitive detection of four respiratory viral antigens. The E-NoM platform uses gold-silver alloy nanoboxes as the core material with the deposition of a silver layer as a shell on the core surfaces to amplify and enable a reproducible Raman signal readout that facilitates accurate detection. Additionally, the E-NoM platform employs gold microelectrode arrays as the mirror with electrohydrodynamics to manipulate the fluid flow and enhance molecular interactions for an improved biosensing response. The presence of viral antigens binds the nanobox-based core-shell nanostructure on the gold microelectrode and creates the nanocavity with extremely strong "hot spots" to benefit sensitive analysis. Significantly, in a large clinical cohort with 227 patients, the designed E-NoM platform demonstrates the capability of screening respiratory infection with achieved clinical specificity, sensitivity, and accuracy of 100.0, 96.48, and 96.91%, respectively. It is anticipated that the E-NoM platform can find a position in clinical usage for respiratory disease diagnosis.

Molecular Stratification and Treatment Monitoring of Lung Cancer Using a Small Extracellular Vesicle-Activated Nanocavity Architecture.

Development of molecular diagnostics for lung cancer stratification and monitoring is crucial for the rational planning and timely adjustment of treatments to improve clinical outcomes. In this regard, we propose a nanocavity architecture to sensitively profile the protein signature on small extracellular vesicles (sEVs) to enable accurate, noninvasive staging and treatment monitoring of lung cancer. The nanocavity architecture is formed by molecular recognition through the binding of sEVs with the nanobox-based core-shell surface-enhanced Raman scattering (SERS) barcodes and mirrorlike, asymmetric gold microelectrodes. By imposing an alternating current on the gold microelectrodes, a nanofluidic shear force was stimulated that supported the binding of sEVs and the efficient assembly of the nanoboxes. The binding of sEVs further induced a nanocavity between the nanobox and the gold microelectrode that significantly amplified the electromagnetic field to enable the simultaneous enhancement of Raman signals from four SERS barcodes and generate patient-specific molecular sEV signatures. Importantly, evaluated on a cohort of clinical samples (n = 76) on the nanocavity architecture, the acquired patient-specific sEV molecular signatures achieved accurate identification, stratification, and treatment monitoring of lung cancer patients, highlighting its potential for transition to clinical utility.

Liquid Biopsy Snapshots of Key Phosphoproteomic Pathways in Lung Cancer Patients for Diagnosis and Therapy Monitoring.

Phosphorylation is a post-translational modification in proteins that changes protein conformation and activity for regulating signal transduction pathways. This mechanism is frequently impaired in lung cancer, resulting in permanently active constitutive phosphorylation to initiate tumor growth and/or reactivate pathways in response to therapy. We developed a multiplexed phosphoprotein analyzer chip (MPAC) that enables rapid (detection time: 5 min) and sensitive (LOD: 2 pg/µL) detection of protein phosphorylation and presents phosphoproteomic profiling of major phosphorylation pathways in lung cancer. We monitored phosphorylated receptors and downstream proteins involved in mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR pathways in lung cancer cell line models and patient-derived extracellular vesicles (EV). Using kinase inhibitor drugs in cell line models, we found that the drug can inhibit the phosphorylation and/or activation of the kinase pathway. We then generated a phosphorylation heatmap by EV phosphoproteomic profiling of plasma samples isolated from 36 lung cancer patients and 8 noncancer individuals. The heatmap showed a clear difference between the noncancer and cancer samples and identify the specific proteins that are activated in the cancer samples. Our data also showed that MPAC could monitor immunotherapy responses by assessment of the phosphorylation states of the proteins, particularly for PD-L1. Finally, with a longitudinal study, we found that the phosphorylation levels of the proteins were indicative of a positive response to therapy. We believe that this study will lead to personalized treatment by providing a better understanding of the active and resistant pathways and will provide a tool for selecting combined and targeted therapies for precision medicine.

SERS Multiplex Profiling of Melanoma Circulating Tumor Cells for Predicting the Response to Immune Checkpoint Blockade Therapy.

Immune checkpoint blockade (ICB) therapy has achieved remarkable success in many cancers including melanoma. However, ICB therapy benefits only a small proportion of patients and produces severe side effects for some patients. Thus, there is an urgent need to identify patients who are more likely to respond to ICB therapy to improve outcomes and minimize side effects. To predict ICB therapy responses, we design a surface-enhanced Raman scattering (SERS) assay for multiplex profiling of circulating tumor cells (CTCs) under basal and interferon-γ (IFN-γ) stimulation. Through simultaneous ensemble and single-cell measurements of CTCs, the SERS assay can reveal tumor heterogeneity and offer a comprehensive CTC phenotype for decision-making. Anisotropic gold-silver alloy nanoboxes are utilized as SERS plasmonic substrates for improved signal readouts of CTC surface biomarkers. By generating a unique CTC signature with four surface biomarkers, the developed assay enables the differentiation of CTCs from three different patient-derived melanoma cell lines. Significantly, in a cohort of 14 melanoma patients who received programmed cell death-1 blockade therapy, the changes of CTC signature induced by IFN-γ stimulation to CTCs show the potential to predict responders. We expect that the SERS assay can help select patients for receiving ICB therapy in other cancers.

Detection of Dengue Virus 2 with Single Infected Mosquito Resolution Using Yeast Affinity Bionanofragments and Plasmonic SERS Nanoboxes.

Dengue disease is an emerging global threat triggered by dengue virus (DENV) transmission, primarily by the mosquito Aedes aegypti. The accurate surveillance and sensitive detection of DENV in mosquito populations are critical for the protection of human populations worldwide that are in the habitat of these mosquito species. There are four DENV serotypes with DENV2 reported to cause the most severe complications. There are limited ultrasensitive methods to early detect DENV2 mosquito infection and prevent human infection. Herein, we report an innovative nanobased immunoassay platform for early, specific, and ultrasensitive detection of DENV2-secreted nonstructural 1 (NS1) protein biomarker in single infected mosquitoes with the limit of detection of 500 fg of recombinant DENV2 NS1. The high sensitivity and DENV2 serotype specificity of the platform are the result of using nanomixing, plasmonic SERS nanoboxes, and yeast affinity bionanofragments displaying single-chain variable fragments (nanoyeast scFvs). Nanoyeast scFvs used for high affinity capture of DENV2 NS1 provided an innovative and cost-efficient alternative to monoclonal antibodies and differentiated DENV2 NS1 from other DENV serotypes and Zika virus NS1. The platform used electrohydrodynamically driven nanomixing to enhance NS1 capture by the nanoyeast scFvs while reducing nonspecific interactions. High sensitivity detection of captured DENV2 NS1 was achieved using NS1-specific surface-enhanced Raman scattering (SERS) nanotags. These nanotechnologies provide a significant innovation for early DENV2 detection in single infected mosquitoes, improving the accurate surveillance of mosquito habitats and preventing infection and severe disease arising from DENV2 transmission.

Dynamic Monitoring of EMT in CTCs as an Indicator of Cancer Metastasis.

Epithelial to mesenchymal transition (EMT) results in the genesis of circulating tumor cells (CTCs) from tumor sites and promotes the metastatic capability of CTCs in circulation. In this study, we develop a multiplex surface-enhanced Raman scattering nanotechnology for comprehensive characterization of EMT-associated phenotypes in CTCs, to monitor cancer metastasis. We observe the downregulation of the CTC marker (EpCAM) and the epithelial marker (E-cadherin), as well as the upregulation of a mesenchymal marker (N-cadherin) and a stem cell marker (ABCB5) during the transforming growth factor-ß-induced EMT process in breast cancer cell line models. Additionally, we also find changes in the heterogeneity levels of these selected markers in cells. With this method, we successfully detect the presence of disease in samples from breast cancer patients and characterize EMT-associated phenotypes in their CTCs. Overall, this approach and findings provide a new means for monitoring the EMT process in cancer, insights into the detailed mechanistic progress of the diseases, and have potential for detecting the early occurrence of cancer metastasis.

Amplification-Free SARS-CoV-2 Detection Using Nanoyeast-scFv and Ultrasensitive Plasmonic Nanobox-Integrated Nanomixing Microassay.

The implementation of accurate and sensitive molecular detection for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is paramount to effectively control the ongoing coronavirus disease 2019 (COVID-19) pandemic. In this regard, we herein propose the specific and highly sensitive SARS-CoV-2 detection based on nanoyeast single-chain-variable fragment (scFv) and ultrasensitive plasmonic nanobox-integrated nanomixing microassay. Importantly, this designed platform showcases the utility of nanoyeast-scFvs as specific capture reagents targeting the receptor-binding domain (RBD) of the virus and as monoclonal antibody alternatives suitable for cost-effective mass production and frequent testing. By capitalizing on single-particle active nanoboxes as plasmonic nanostructures for surface-enhanced Raman scattering (SERS), the microassay utilizes highly sensitive Raman signals to indicate virus infection. The developed microassay further integrated nanomixing for accelerating molecular collisions. Through the synergistic working of nanoyeast-scFv, plasmonic nanoboxes, and nanomixing, the highly specific and sensitive SARS-CoV-2 detection is achieved as low as 17 virus/µL without any molecular amplification. We successfully demonstrate SARS-CoV-2 detection in saliva samples of simulated patients at clinically relevant viral loads, suggesting the possibility of this platform for accurate and noninvasive patient screening.

On the Behavior of Nanoparticles beyond the Nanopore Interface.

Recent advances in solid-state and biological nanopore sensors have produced a deluge of analytical techniques for in situ characterization of bio-nano colloidal dispersions; however, the transport forces governing particle movement into and out of the nanopore are not yet fully understood. Herein, we study the motion of particles outside the smaller opening of an elastomeric size-tunable nanopore and relate this motion to existing transport forces known to act on particles within the pore. Subsequently, we develop a combined optoelectronic approach which allows the comparison of both resistive pulse sensing and single particle tracking-based techniques for particle size characterization and, intriguingly, measurements of the ensemble particle motion induced by a combination of particle electrophoresis as well as pressure-driven and electroosmotic flows through the sensor nanopore. We find evidence suggesting that although bulk fluid flow from the pore tends to drive particle motion, in certain circumstances, electrophoretically driven motion can dominate bulk fluid flow-driven motion even at large distances from the pore opening. By permitting direct observation of the behavior of fluids at the nanopore interface, this approach enables a greater understanding of the transport forces acting on particles as they migrate toward and move through nanopore sensors-with implications for future particle characterization systems and for nanopore methods in general.

Tracking Drug-Induced Epithelial-Mesenchymal Transition in Breast Cancer by a Microfluidic Surface-Enhanced Raman Spectroscopy Immunoassay.

Epithelial-mesenchymal transition (EMT) is a primary mechanism for cancer metastasis. Detecting the activation of EMT can potentially convey signs of metastasis to guide treatment management and improve patient survival. One of the classic signatures of EMT is characterized by dynamic changes in cellular expression levels of E-cadherin and N-cadherin, whose soluble active fragments have recently been reported to be biomarkers for cancer diagnosis and prognosis. Herein, a microfluidic immunoassay (termed "SERS immunoassay") based on sensitive and simultaneous detection of soluble E-cadherin (sE-cadherin) and soluble N-cadherin (sN-cadherin) for EMT monitoring in patients' plasma is presented. The SERS immunoassay integrates in situ nanomixing and surface-enhanced Raman scattering readout to enable accurate detection of sE-cadherin and sN-cadherin from as low as 10 cells mL-1 . This assay enables tracking of a concurrent decrease in sE-cadherin and increase in sN-cadherin in breast cancer cells undergoing drug-induced mesenchymal transformation. The clinical potential of the SERS immunoassay is further demonstrated by successful detection of sE-cadherin and sN-cadherin in metastatic stage IV breast cancer patient plasma samples. The SERS immunoassay can potentially sense the activation of EMT to provide early indications of cancer invasions or metastasis.

Toward Personalized Cancer Treatment: From Diagnostics to Therapy Monitoring in Miniaturized Electrohydrodynamic Systems.

Historically, cancer was seen and treated as a single disease. Over the years, this image has shifted, and it is now generally accepted that cancer is a complex and dynamic disease that engages multiple progression pathways in each patient. The shift from treating cancer as single disease to tailoring the therapy based on the individual's characteristic cancer profile promises to improve the clinical outcome and has also given rise to the field of personalized cancer treatment. To advise a suitable therapy plan and adjust personalized treatment, a reliable and fast diagnostic strategy is required. The advances in nanotechnology, microfluidics, and biomarker research have spurred the development of powerful miniaturized diagnostic systems that show high potential for use in personalized cancer treatment. These devices require only minute sample volumes and have the capability to create instant cancer snapshots that could be used as tool for cancer risk indication, early detection, tumor classification, and recurrence. Miniaturized systems can combine a whole sample-to-answer workflow including sample handling, preparation, analysis, and detection. As such, this concept is also often referred to as "lab-on-a-chip". An inherit challenge of monitoring personalized cancer treatment using miniaturized systems is that cancer biomarkers are often only detectable at trace concentrations present in a complex biological sample rich in interfering molecules, necessitating highly specific and sensitive biosensing strategies. To address the need for trace level detection, highly sensitive fluorescence, absorbance, surface-enhanced Raman spectroscopy (SERS), electrochemical, mass spectrometric, and chemiluminescence approaches were developed. To reduce sample matrix interferences, ingenious device modifications including coatings and nanoscopic fluid flow manipulation have been developed. Of the latter, our group has exploited the use of alternating current electrohydrodynamic (ac-EHD) fluid flows as an efficient strategy to reduce nonspecific nontarget biosensor binding and speed-up assay times. ac-EHD provides fluid motion induced by an electric field with the ability to generate surface shear forces in nanometer distance to the biosensing surface (known as nanoshearing phenomenon). This is ideally suited to increase the collision frequency of cancer biomarkers with the biosensing surface and shear off nontarget molecules thereby minimizing nonspecific binding. In this Account, we review recent advancements in miniaturized diagnostic system development with potential use in personalized cancer treatment and monitoring. We focus on integrated microfluidic structures for controlled sample flow manipulation followed by on-device biomarker interrogation. We further highlight the progress in our group, emphasis fundamentals and applications of ac-EHD-enhanced miniaturized systems, and outline promising detection concepts for comprehensive cancer biomarker profiling. The advances are discussed based on the type of cancer biomarkers and cover circulating tumor cells, proteins, extracellular vesicles, and nucleic acids. The potential of miniaturized diagnostic systems for personalized cancer treatment and monitoring is underlined with representative examples including device illustrations. In the final section, we critically discuss the future of personalized diagnostics and what challenges should be addressed to make these devices clinically translatable.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2016-2018).

One of the most cited limitations of capillary and microchip electrophoresis is the poor sensitivity. This review continues to update this series of biannual reviews, first published in Electrophoresis in 2007, on developments in the field of online/in-line concentration methods in capillaries and microchips, covering the period July 2016-June 2018. It includes developments in the field of stacking, covering all methods from field-amplified sample stacking and large-volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to online or in-line extraction methods that have been used for electrophoresis.

Single droplet detection of immune checkpoints on a multiplexed electrohydrodynamic biosensor.

Monitoring soluble immune checkpoints in circulating fluids has the potential for minimally-invasive diagnostics and personalised therapy in precision medicine. Yet, the sensitive detection of multiple immune checkpoints from small volumes of liquid biopsy samples is challenging. In this study, we develop a multiplexed immune checkpoint biosensor (MICB) for parallel detection of soluble immune checkpoints PD-1, PD-L1, and LAG-3. MICB integrates a microfluidic sandwich immunoassay using engineered single chain variable fragments and alternating current electrohydrodynamic in situ nanofluidic mixing for promoting biosensor-target interaction and reducing non-specific non-target binding. MICB provides advantages of simultaneous analysis of up to 28 samples in <2 h, requires as little as a single sample drop (i.e., 20 µL) per target immune checkpoint, and applies high-affinity yeast cell-derived single chain variable fragments as a cost-effective alternative to monoclonal antibodies. We investigate the assay performance of MICB and demonstrate its capability for accurate immune checkpoint detection in simulated patient serum samples at clinically-relevant levels. MICB provides a dynamic range of 5 to 200 pg mL-1 for PD-1 and PD-L1, and 50 to 1000 pg mL-1 for LAG-3 with a coefficient of variation <13.8%. Sensitive immune checkpoint detection was achieved with limits of detection values of 5 pg mL-1 for PD-1, 5 pg mL-1 for PD-L1, and 50 pg mL-1 for LAG-3. The multiplexing capability, sensitivity, and relative assay simplicity of MICB make it capable of serving as a bioanalytical tool for immune checkpoint therapy monitoring.

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.

Derivatisation for separation and detection in capillary electrophoresis (2015-2017).

Derivatisation is an integrated part of many analytical workflows to enable separation and detection of the analytes. In CE, derivatisation is adapted in the four modes of pre-capillary, in-line, in-capillary, and post-capillary derivatisation. In this review, we discuss the progress in derivatisation from February 2015 to May 2017 from multiple points of view including sections about the derivatisation modes, derivatisation to improve the analyte separation and analyte detection. The advancements in derivatisation procedures, novel reagents, and applications are covered. A table summarising the 46 reviewed articles with information about analyte, sample, derivatisation route, CE method and method sensitivity is provided.

Recent advances in enhancing the sensitivity of electrophoresis and electrochromatography in capillaries and microchips (2014-2016).

One of the most cited limitations of capillary (and microchip) electrophoresis is the poor sensitivity. This review continues to update this series of biennial reviews, first published in Electrophoresis in 2007, on developments in the field of on-line/in-line concentration methods in capillaries and microchips, covering the period July 2014-June 2016. It includes developments in the field of stacking, covering all methods from field amplified sample stacking and large volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to on-line or in-line extraction methods that have been used for electrophoresis.

Sensitivity enhancing injection from a sample reservoir and channel interface in microchip electrophoresis.

The stacking of a cationic analyte (i.e., rhodamine B) at the interface between a sample reservoir and channel in a microchip electrophoresis device is described for the first time. Stacking at negative polarity was by micelle to solvent stacking where the dye was prepared in a micellar solution (5 mM sodium dodecyl sulfate in 25 mM phosphoric acid, pH 2.5) and the channel was filled with high methanol content background solution (70% methanol in 50 mM phosphoric acid, pH 2.5). The injection of the stacked dye into the channel was by simple reversal of the voltage polarity with the sample solution and background solution at the anodic and cathodic reservoirs of the straight channel, respectively. The enrichment of rhodamine B at the interface and injection of the stacked dye into the channel was clearly visualized using an inverted fluorescence microscope. A notable sensitivity enhancement factor of up to 150 was achieved after 2 min at 1 kV of micelle to solvent stacking. The proposed technique will be useful as a concentration step for analyte mixtures in simple and classical cross-channel microchip electrophoresis devices or for the controlled delivery of enriched reagents or analytes as narrow plugs in advanced microchip electrophoresis devices.

Derivatisation for separation and detection in capillary electrophoresis (2012-2015).

Derivatisation is a well-established and mature form of sample preparation for CE. The modification of the analyte can cause superior analysis characteristics such as better sensitivity and selectivity, however, derivatisation of the analyte introduces an additional step into the analytical workflow. This review covers articles from January 2012 to January 2015 on derivatisation in CE. The main sections are on the derivatisation modes (i.e. pre-capillary, in-line, in-capillary and post-capillary), separation and detection modes (i.e. LIF and others). LIF is discussed in more detail since this detection mode was most prevalent. A table of the common labelling agents and wavelengths for excitation and emission and the common derivatisation reactions are included. In addition, a comprehensive table which summarises all research articles is provided. This review is suitable for analytical chemists as a guide for 'how to get started' with derivatisation for separation and detection in CE.