LowTempGAL: a highly responsive low temperature-inducible GAL system in Saccharomyces cerevisiae.

Temperature is an important control factor for biologics biomanufacturing in precision fermentation. Here, we explored a highly responsive low temperature-inducible genetic system (LowTempGAL) in the model yeast Saccharomyces cerevisiae. Two temperature biosensors, a heat-inducible degron and a heat-inducible protein aggregation domain, were used to regulate the GAL activator Gal4p, rendering the leaky LowTempGAL systems. Boolean-type induction was achieved by implementing a second-layer control through low-temperature-mediated repression on GAL repressor gene GAL80, but suffered delayed response to low-temperature triggers and a weak response at 30°C. Application potentials were validated for protein and small molecule production. Proteomics analysis suggested that residual Gal80p and Gal4p insufficiency caused suboptimal induction. 'Turbo' mechanisms were engineered through incorporating a basal Gal4p expression and a galactose-independent Gal80p-supressing Gal3p mutant (Gal3Cp). Varying Gal3Cp configurations, we deployed the LowTempGAL systems capable for a rapid stringent high-level induction upon the shift from a high temperature (37-33°C) to a low temperature (≤30°C). Overall, we present a synthetic biology procedure that leverages 'leaky' biosensors to deploy highly responsive Boolean-type genetic circuits. The key lies in optimisation of the intricate layout of the multi-factor system. The LowTempGAL systems may be applicable in non-conventional yeast platforms for precision biomanufacturing.

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.

Defining the relationship between cellular and extracellular vesicle (EV) content in breast cancer via an integrative multi-omic analysis.

Much recent research has been dedicated to exploring the utility of extracellular vesicles (EVs) as circulating disease biomarkers. Underpinning this work is the assumption that the molecular cargo of EVs directly reflects the originating cell. Few attempts have been made, however, to empirically validate this on the -omic level. To this end, we have performed an integrative multi-omic analysis of a panel of breast cancer cell lines and corresponding EVs. Whole transcriptome analysis validated that the cellular transcriptome remained stable when cultured cells are transitioned to low serum or serum-free medium for EV collection. Transcriptomic profiling of the isolated EVs indicated a positive correlation between transcript levels in cells and EVs, including disease-associated transcripts. Analysis of the EV proteome verified that HER2 protein is present in EVs, however neither the estrogen (ER) nor progesterone (PR) receptor proteins are detected regardless of cellular expression. Using multivariate analysis, we derived an EV protein signature to infer cellular patterns of ER and HER2 expression, though the ER protein could not be directly detected. Integrative analyses affirmed that the EV proteome and transcriptome captured key phenotypic hallmarks of the originating cells, supporting the potential of EVs for non-invasive monitoring of breast cancers.

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.

Tetraplex Immunophenotyping of Cell Surface Proteomes via Synthesized Plasmonic Nanotags and Portable Raman Spectroscopy.

Multiplex immunophenotyping of cell surface proteomes is useful for cell characterization as well as providing valuable information on a patient's physiological or pathological state. Current methods for multiplex immunophenotyping of cell surface proteomes still have associated technical pitfalls in terms of limited multiplexing capability, challenging result interpretation, and large equipment footprint limited to use in a laboratory setting. Herein, we presented a portable surface-enhanced Raman spectroscopy (SERS) assay for multiplex cell surface immunophenotyping. We synthesized and functionalized customizable SERS nanotags for cell labeling and subsequent signal measurement using a portable Raman spectrometer. We extensively evaluated and validated the analytical assay performance of the portable SERS immunophenotyping assay in two different cellular models (red blood cells and breast cancer cells). In terms of analytical specificity, the cell surface immunophenotyping of both red blood cells and breast cancer cells correlated well with flow cytometry. The portable SERS immunophenotyping assay also has comparable analytical repeatability to flow cytometry, with coefficient of variation values of 21.89-23.33% and 6.88-17.32% for detecting red blood cells and breast cancer cells, respectively. The analytical detection limits were 77 cells/mL for red blood cells and 1-17 cells/mL for breast cancer cells. As an alternative to flow cytometry, the portable SERS immunophenotyping assay demonstrated excellent analytical assay performance and possessed advantages such as quick sample-to-result turnaround time, multiplexing capability, and small equipment footprint.

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.

Nucleic Acid Hybridization-Based Noise Suppression for Ultraselective Multiplexed Amplification of Mutant Variants.

The analysis of mutant nucleic acid (NA) variants can provide crucial clinical and biological insights for many diseases. Yet, existing analysis techniques are generally constrained by nonspecific "noise" signals from excessive wildtype background sequences, especially under rapid isothermal multiplexed target amplification conditions. Herein, the molecular hybridization chemistry between NA bases is manipulated to suppress noise signals and achieve ultraselective multiplexed detection of cancer gene fusion NA variants. Firstly, modified locked NA (LNA) bases are rationally introduced into oligonucleotide sequences as designed "locker probes" for high affinity hybridization to wildtype sequences, leading to enrichment of mutant variants for multiplexed isothermal amplification. Secondly, locker probes are coupled with a customized "proximity-programmed" (SERS) readout which allows precise control of hybridization-based plasmonic signaling to specifically detect multiple target amplicons within a single reaction. Moreover, the use of triple bond Raman reporters endows NA noise signal-free quantification in the Raman silent region (≈1800-2600 cm-1 ). With this dual molecular hybridization-based strategy, ultraselective multiplexed detection of gene fusion NA variants in cancer cellular models is actualized with successful noise suppression of native wildtype sequences. The distinct benefits of isothermal NA amplification and SERS multiplexing ability are simultaneously harnessed.

Correction: Nucleic acid purification from plants, animals and microbes in under 30 seconds.

[This corrects the article DOI: 10.1371/journal.pbio.2003916.].

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.

Nucleic acid purification from plants, animals and microbes in under 30 seconds.

Nucleic acid amplification is a powerful molecular biology tool, although its use outside the modern laboratory environment is limited due to the relatively cumbersome methods required to extract nucleic acids from biological samples. To address this issue, we investigated a variety of materials for their suitability for nucleic acid capture and purification. We report here that untreated cellulose-based paper can rapidly capture nucleic acids within seconds and retain them during a single washing step, while contaminants present in complex biological samples are quickly removed. Building on this knowledge, we have successfully created an equipment-free nucleic acid extraction dipstick methodology that can obtain amplification-ready DNA and RNA from plants, animals, and microbes from difficult biological samples such as blood and leaves from adult trees in less than 30 seconds. The simplicity and speed of this method as well as the low cost and availability of suitable materials (e.g., common paper towelling), means that nucleic acid extraction is now more accessible and affordable for researchers and the broader community. Furthermore, when combined with recent advancements in isothermal amplification and naked eye DNA visualization techniques, the dipstick extraction technology makes performing molecular diagnostic assays achievable in limited resource settings including university and high school classrooms, field-based environments, and developing countries.

Nanostructured mesoporous gold electrodes detect protein phosphorylation in cancer with electrochemical signal amplification.

Protein phosphorylation is a post-translational modification of kinase proteins that changes a protein's conformation to regulate crucial biological functions. However, the phosphorylation of protein is significantly altered during cancer progression which triggers abnormal cellular pathways and this phosphorylation can serve as an emergent diagnostic and prognostic biomarker for cancer. Herein, we develop a nanostructured mesoporous gold electrode (NMGE)-based biosensor that enables a highly sensitive detection of protein phosphorylation with electrochemical signal amplification. The biosensor comprises nanostructured mesoporous gold electrodes whose electro-conductive framework is superior to that of the nonporous electrodes. We characterize our developed nano/mesoporous gold electrode with various electrochemical methods in the presence of the [Fe(CN)6]3-/4- redox system. We find that the mesoporous gold electrode catalyzes both the oxidation and reduction processes of the [Fe(CN)6]3-/4- system and generates a current signal that is 3 times higher than that of the nonporous gold electrode. This superior signal transduction of our nano/mesoporous gold electrode is enabled through a pore-induced (i) high electrochemically active surface area and (ii) reduced impedance with a high signal to noise ratio. The assay utilizes direct adsorption of an immunoprecipitated purified BRAF protein towards the mesoporous gold electrode and thus avoids the cumbersome sensor surface functionalization. Our developed biosensor detects the phosphorylated BRAF protein with a 2.5-fold increase in sensitivity and an ≈10-fold increase in the limit of detection (LOD) in comparison with the nonporous gold electrodes. The assay also works on a wide dynamic range from 0.5 to 20 ng µL-1 of the protein which further shows its potential for clinical application. We envisage that this nanostructured mesoporous gold biosensor will be of high interest for clinical application.

Optimizing Size Exclusion Chromatography for Extracellular Vesicle Enrichment and Proteomic Analysis from Clinically Relevant Samples.

The field of extracellular vesicle (EV) research has rapidly expanded in recent years, with particular interest in their potential as circulating biomarkers. Proteomic analysis of EVs from clinical samples is complicated by the low abundance of EV proteins relative to highly abundant circulating proteins such as albumin and apolipoproteins. To overcome this, size exclusion chromatography (SEC) has been proposed as a method to enrich EVs whilst depleting protein contaminants; however, the optimal SEC parameters for EV proteomics have not been thoroughly investigated. Here, quantitative evaluation and optimization of SEC are reported for separating EVs from contaminating proteins. Using a synthetic model system followed by cell line-derived EVs, it is found that a 10 mL Sepharose 4B column in PBS produces optimal resolution of EVs from background protein. By spiking-in cancer cell-derived EVs to healthy plasma, it is shown that some cancer EV-associated proteins are detectable by nano-LC-MS/MS when as little as 1% of the total plasma EV number are derived from a cancer cell line. These results suggest that an optimized SEC and nanoLC-MS/MS workflow may be sufficiently sensitive for disease EV protein biomarker discovery from patient-derived clinical samples.

Native MicroRNA Targets Trigger Self-Assembly of Nanozyme-Patterned Hollowed Nanocuboids with Optimal Interparticle Gaps for Plasmonic-Activated Cancer Detection.

The modernized use of nucleic acid (NA) sequences to drive nanostructure self-assembly has given rise to a new class of designed nanomaterials with controllable plasmonic functionalities for broad surface-enhanced Raman scattering (SERS)-based bioanalysis applications. Herein, dual usage of microRNAs (miRNAs) as both valuable cancer biomarkers and direct self-assembly triggers is identified and capitalized upon for custom-designed plasmonic nanostructures. Through strict NA hybridization of miRNA targets, Au nanospheres selectively self-assemble onto hollowed Au/Ag alloy nanocuboids with ideal interparticle distances (≈2.3 nm) for optimal SERS signaling. The intrinsic material properties of the self-assembled nanostructures further elevate miRNA detection performance via nanozyme catalytic SERS signaling cascades. This enables fM-level miR-107 detection limit within a clinically-relevant range without any molecular target amplification. The miRNA-triggered nanostructure self-assembly approach is further applied in clinical patient samples, and showcases the potential of miR-107 as a non-invasive prostate cancer diagnostic biomarker. The use of miRNA targets to drive nanostructure self-assembly holds great promise as a practical tool for miRNA detection in disease applications.