Tetraspanins are unevenly distributed across single extracellular vesicles and bias sensitivity to multiplexed cancer biomarkers.

BACKGROUND: Tetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their detection and classification, a practice that typically assumes their consistent expression across EV sources. RESULTS: Here we demonstrate that there are distinct patterns in colocalization of tetraspanin expression of EVs enriched from a variety of in vitro and in vivo sources. We report an optimized method for the use of single particle antibody-capture and fluorescence detection to identify subpopulations according to tetraspanin expression and compare our findings with nanoscale flow cytometry. We found that tetraspanin profile is consistent from a given EV source regardless of isolation method, but that tetraspanin profiles are distinct across various sources. Tetraspanin profiles measured by flow cytometry do not totally agree, suggesting that limitations in subpopulation detection significantly impact apparent protein expression. We further analyzed tetraspanin expression of single EVs captured non-specifically, revealing that tetraspanin capture can bias the apparent multiplexed tetraspanin profile. Finally, we demonstrate that this bias can have significant impact on diagnostic sensitivity for tumor-associated EV surface markers. CONCLUSION: Our findings may reveal key insights into protein expression heterogeneity of EVs that better inform EV capture and detection platforms for diagnostic or other downstream use.

Homogenous high enhancement surface-enhanced Raman scattering (SERS) substrates by simple hierarchical tuning of gold nanofoams.

Surface enhanced Raman scattering (SERS) is a powerful tool for vibrational spectroscopy, providing orders of magnitude increase in chemical sensitivity compared to spontaneous Raman scattering. Yet it remains a challenge to synthesize robust, uniform SERS substrates quickly and easily. Lithographic approaches to produce substrates can achieve high, uniform sensitivity but are expensive and complex, thus difficult to scale. Facile solution-phase chemical approaches often result in unreliable SERS substrates due to heterogeneous arrangement of "hot spots" throughout the material. Here we demonstrate the synthesis and characterization of a homogeneous gold nanofoam (AuNF) substrate produced by a rapid, one-pot, four-ingredient synthetic approach. AuNFs are rapidly nucleated with macroscale porosity and then chemically roughened to produce nanoscale features that confer homogeneous and high signal enhancement (~109) across large areas, a comparable performance to lithographically produced substrates.

Fused Raman spectroscopic analysis of blood and saliva delivers high accuracy for head and neck cancer diagnostics.

As a rapid, label-free, non-destructive analytical measurement requiring little to no sample preparation, Raman spectroscopy shows great promise for liquid biopsy cancer detection and diagnosis. We carried out Raman analysis and mass spectrometry of plasma and saliva from more than 50 subjects in a cohort of head and neck cancer patients and benign controls (e.g., patients with benign oral masses). Unsupervised data models were built to assess diagnostic performance. Raman spectra collected from either biofluid provided moderate performance to discriminate cancer samples. However, by fusing together the Raman spectra of plasma and saliva for each patient, subsequent analytical models delivered an impressive sensitivity, specificity, and accuracy of 96.3%, 85.7%, and 91.7%, respectively. We further confirmed that the metabolites driving the differences in Raman spectra for our models are among the same ones that drive mass spectrometry models, unifying the two techniques and validating the underlying ability of Raman to assess metabolite composition. This study bolsters the relevance of Raman to provide additive value by probing the unique chemical compositions across biofluid sources. Ultimately, we show that a simple data augmentation routine of fusing plasma and saliva spectra provided significantly higher clinical value than either biofluid alone, pushing forward the potential of clinical translation of Raman spectroscopy for liquid biopsy cancer diagnostics.

Surface enhanced Raman scattering of extracellular vesicles for cancer diagnostics despite isolation dependent lipoprotein contamination.

Given the emerging diagnostic utility of extracellular vesicles (EVs), it is important to account for non-EV contaminants. Lipoprotein present in EV-enriched isolates may inflate particle counts and decrease sensitivity to biomarkers of interest, skewing chemical analyses and perpetuating downstream issues in labeling or functional analysis. Using label free surface enhanced Raman scattering (SERS), we confirm that three common EV isolation methods (differential ultracentrifugation, density gradient ultracentrifugation, and size exclusion chromatography) yield variable lipoprotein content. We demonstrate that a dual-isolation method is necessary to isolate EVs from the major classes of lipoprotein. However, combining SERS analysis with machine learning assisted classification, we show that the disease state is the main driver of distinction between EV samples, and largely unaffected by choice of isolation. Ultimately, this study describes a convenient SERS assay to retain accurate diagnostic information from clinical samples by overcoming differences in lipoprotein contamination according to isolation method.

Hybrid Nanoplasmonic Porous Biomaterial Scaffold for Liquid Biopsy Diagnostics Using Extracellular Vesicles.

For more effective early-stage cancer diagnostics, there is a need to develop sensitive and specific, non- or minimally invasive, and cost-effective methods for identifying circulating nanoscale extracellular vesicles (EVs). Here, we report the utilization of a simple plasmonic scaffold composed of a microscale biosilicate substrate embedded with silver nanoparticles for surface-enhanced Raman scattering (SERS) analysis of ovarian and endometrial cancer EVs. These substrates are rapidly and inexpensively produced without any complex equipment or lithography. We extensively characterize the substrates with electron microscopy and outline a reproducible methodology for their use in analyzing EVs from in vitro and in vivo biofluids. We report effective chemical treatments for (i) decoration of metal surfaces with cysteamine to nonspecifically pull down EVs to SERS hotspots and (ii) enzymatic cleavage of extraluminal moieties at the surface of EVs that prevent localization of complementary chemical features (lipids/proteins) to the vicinity of the metal-enhanced fields. We observe a major loss of sensitivity for ovarian and endometrial cancer following enzymatic cleavage of EVs' extraluminal domain, suggesting its critical significance for diagnostic platforms. We demonstrate that the SERS technique represents an ideal tool to assess and measure the high heterogeneity of EVs isolated from clinical samples in an inexpensive, rapid, and label-free assay.

Nanoplasmonic Approaches for Sensitive Detection and Molecular Characterization of Extracellular Vesicles.

All cells release a multitude of nanoscale extracellular vesicles (nEVs) into circulation, offering immense potential for new diagnostic strategies. Yet, clinical translation for nEVs remains a challenge due to their vast heterogeneity, our insufficient ability to isolate subpopulations, and the low frequency of disease-associated nEVs in biofluids. The growing field of nanoplasmonics is poised to address many of these challenges. Innovative materials engineering approaches based on exploiting nanoplasmonic phenomena, i.e., the unique interaction of light with nanoscale metallic materials, can achieve unrivaled sensitivity, offering real-time analysis and new modes of medical and biological imaging. We begin with an introduction into the basic structure and function of nEVs before critically reviewing recent studies utilizing nanoplasmonic platforms to detect and characterize nEVs. For the major techniques considered, surface plasmon resonance (SPR), localized SPR, and surface enhanced Raman spectroscopy (SERS), we introduce and summarize the background theory before reviewing the studies applied to nEVs. Along the way, we consider notable aspects, limitations, and considerations needed to apply plasmonic technologies to nEV detection and analysis.