Glycan Profiling in Small Extracellular Vesicles with a SERS Microfluidic Biosensor Identifies Early Malignant Development in Lung Cancer.

Glycosylation is the most common post-translational modification of proteins and regulates a myriad of fundamental biological processes under normal, and pathological conditions. Altered protein glycosylation is linked to malignant transformation, showing distinct glycopatterns that are associated with cancer initiation and progression by regulating tumor proliferation, invasion, metastasis, and therapeutic resistance. The glycopatterns of small extracellular vesicles (sEVs) released by cancer cells are promising candidates for cancer monitoring since they exhibit glycopatterns similar to their cell-of-origin. However, the clinical application of sEV glycans is challenging due to the limitations of current analytical technologies in tracking the trace amounts of sEVs specifically derived from tumors in circulation. Herein, a sEV GLYcan PHenotype (EV-GLYPH) assay that utilizes a microfluidic platform integrated with surface-enhanced Raman scattering for multiplex profiling of sEV glycans in non-small cell lung cancer is clinically validated. For the first time, the EV-GLYPH assay effectively identifies distinct sEV glycan signatures between non-transformed and malignantly transformed lung cells. In a clinical study evaluated on 40 patients, the EV-GLYPH assay successfully differentiates patients with early-stage malignant lung nodules from benign lung nodules. These results reveal the potential to profile sEV glycans for noninvasive diagnostics and prognostics, opening up promising avenues for clinical applications and understanding the role of sEV glycosylation in lung cancer.

A Mesoporous Gold Sensor Unveils Phospho PD-L1 in Extracellular Vesicles as a Proxy for PD-L1 Expression in Lung Cancer Tissue.

Immune checkpoint inhibitors (ICIs) targeting programmed cell death ligand 1 (PD-L1), or its receptor, PD-1 have improved survival in patients with non-small-cell lung cancer (NSCLC). Assessment of PD-L1 expression requires tissue biopsy or fine needle aspiration that are currently used to identify patients most likely to respond to single agent anti-PD-1/PD-L1 therapy. However, obtaining sufficient tissue to generate a PD-L1 tissue proportion score (TPS) ≥ 50% using immunohistochemistry remains a challenge that potentially may be overcome by liquid biopsies. This study utilized a mesoporous gold sensor (MGS) assay to examine the phosphorylation status of PD-L1 in plasma extracellular vesicles (EV pPD-L1) and PD-L1 levels in plasma from NSCLC patient samples and their association with tumor PD-L1 TPS. The 3-dimensional mesoporous network of the electrodes provides a large surface area, high signal-to-noise ratio, and a superior electro-conductive framework, thereby significantly improving the detection sensitivity of PD-L1 nanosensing. Test (n = 20) (Pearson's r = 0.99) and validation (n = 45) (Pearson's r = 0.99) cohorts show that EV pPD-L1 status correlates linearly with the tumor PD-L1 TPS assessed by immunohistochemistry irrespective of the tumor stage, with 64% of patients overall showing detectable EV pPD-L1 levels in plasma. In contrast to the EV pPD-L1 results, plasma PD-L1 levels did not correlate with the tumor PD-L1 TPS score or EV pPD-L1 levels. These data demonstrate that EV pPD-L1 levels may be used to select patients for appropriate PD-1 and PD-L1 ICI therapy regimens in early, locally advanced, and advanced NSCLC and should be tested further in randomized controlled trials. Most importantly, the assay used has a less than 24h turnaround time, facilitating adoption of the test into the routine diagnostic evaluation of patients prior to therapy.

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.

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.

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.

Correction: Plasma extracellular vesicle phenotyping for the differentiation of early-stage lung cancer and benign lung diseases.

Correction for 'Plasma extracellular vesicle phenotyping for the differentiation of early-stage lung cancer and benign lung diseases' by Liwen Yuan et al., Nanoscale Horiz., 2023, 8, 746-758, https://doi.org/10.1039/d2nh00570k.

A universal reagent for detection of emerging diseases using bioengineered multifunctional yeast nanofragments.

Accurate and early detection of biomarkers provides the molecular evidence for disease management, allowing prompt actions and timely treatments to save lives. Multivalent biomolecular interactions between the probe and biomarker as well as controlled probe orientation on material surfaces are keys for highly sensitive detection. Here we report the bioengineering of programmable and multifunctional nanoprobes, which can provide rapid, specific and highly sensitive detection of emerging diseases in a range of widely used diagnostic systems. These nanoprobes composed of nanosized cell wall fragments, termed as synthetic bionanofragments (SynBioNFs), are generated by the fragmentation of genetically programmed yeast cells. SynBioNFs display multiple copies of biomolecules for high-affinity target binding and molecular handles for the precisely orientated attachment on surfaces used in diagnostic platforms. SynBioNFs are demonstrated for the capture and detection of SARS-CoV-2 virions using multiple diagnostic platforms, including surface-enhanced Raman scattering, fluorescence, electrochemical and colorimetric-based lateral flow systems with sensitivity comparable with the gold-standard reverse-transcription quantitative polymerase chain reaction.

A Microfluidic SERS Assay to Characterize the Phenotypic Heterogeneity in Cancer-Derived Small Extracellular Vesicles.

Small extracellular vesicles (sEVs) are nanoscopic bioparticles that transport biomolecular cargoes between cells. sEVs have been implicated in many pathological processes such as cancer, rendering them as promising targets for therapeutics and diagnostics. Characterizing phenotypic differences in sEV biomolecular cargos could support understanding their roles in cancer. However, this is difficult due to similar physical properties of sEVs and requirement for highly sensitive analysis. Our method describes the preparation and operation of a microfluidic immunoassay with surface-enhanced Raman scattering (SERS) readouts, termed sEV subpopulation characterization platform (ESCP). ESCP applies an alternating current induced electrohydrodynamic flow to enhance collisions of sEVs with the antibody-functionalized sensor surface. Captured sEVs are labeled with plasmonic nanoparticles to facilitate multiplexed and highly sensitive phenotypic characterization of sEVs by SERS. ESCP is demonstrated for characterizing the expression of three tetraspanins (CD9, CD63, CD81) and four cancer-associated biomarkers (MCSP, MCAM, ErbB3, LNGFR) in sEVs derived from cancer cell lines and plasma samples.

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.

A Multiplexed SERS Microassay for Accurate Detection of SARS-CoV-2 and Variants of Concern.

Severe acute respiratory syndrome coronavirus 2 variants play an important role in predicting patient outcome during postinfection, and with growing fears of COVID-19 reservoirs in domestic and wild animals, it is necessary to adapt detection systems for variant detection. However, variant-specific detection remains challenging. Surface-enhanced Raman scattering is a sensitive and multiplexing technique that allows the simultaneous detection of multiple targets for accurate identification. Here we propose the development of a multiplex SERS microassay to detect both the spike and nucleocapsid structural proteins of SARS-CoV-2. The designed SERS microassay integrates gold-silver hollow nanobox barcodes and electrohydrodynamically induced nanomixing which in combination enables highly specific and sensitive detection of SARS-CoV-2 and the S-protein epitopes to delineate between ancestral prevariant strains with the newer variants of concern, Delta and Omicron. The microassay allows detection from as low as 20 virus/µL and 50 pg/mL RBD protein and can clearly identify the virus among infected versus healthy nasopharyngeal swabs, with the potential to identify between variants. The detection of both S- and N-proteins of SARS-CoV-2 and the differentiation of variants on the SERS microassay can aid the early detection of COVID-19 to reduce transmission rates and lead into adequate treatments for those severely affected by the virus.

Plasma extracellular vesicle phenotyping for the differentiation of early-stage lung cancer and benign lung diseases.

The development of a minimally invasive technique for early-stage lung cancer detection is crucial to reducing mortality. Phenotyping of tumor-associated extracellular vesicles (EVs) has the potential for early-stage lung cancer detection, yet remains challenging due to the lack of sensitive, integrated techniques that can accurately detect rare tumor-associated EV populations in blood. Here, we integrated gold core-silver shell nanoparticles and nanoscopic mixing in a microfluidic assay for sensitive phenotypic analysis of EVs directly in plasma without EV pre-isolation. The assay enabled multiplex detection of lung cancer-associated markers PTX3 and THBS1 and canonical EV marker CD63 by surface-enhanced Raman spectroscopy, providing a squared correlation coefficient of 0.97 in the range of 103-107 EVs mL-1 and a limit of detection of 19 EVs mL-1. Significantly, our machine learning-based nanostrategy provided 92.3% sensitivity and 100% specificity in differentiating early-stage lung cancer from benign lung diseases, superior to the CT scan-based lung cancer diagnosis (92.3% sensitivity and 71.4% specificity). Overall, our integrated nanostrategy achieved an AUC value of 0.978 in differentiating between early-stage lung cancer patients (n = 28) and controls consisting of patients with benign lung diseases (n = 23) and healthy controls (n = 26), which showed remarkable diagnostic performance and great clinical potential for detecting the early occurrence of lung cancer.

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.

Nurturing the marriages of urinary liquid biopsies and nano-diagnostics for precision urinalysis of prostate cancer.

Prostate cancer remains the second-most common cancer diagnosed in men, despite the increasingly widespread use of serum prostate-specific antigen (PSA) screening. The controversial clinical implications and cost benefits of PSA screening have been highlighted due to its poor specificity, resulting in a high rate of overdiagnosis and underdiagnosis. Thus, the development of novel biomarkers for prostate cancer detection remains an intriguing challenge. Urine is emerging as a source for prostate cancer biomarker discovery. Currently, new urine biomarkers already outperform serum PSA in clinical diagnosis. Meanwhile, the advances in nanotechnology have provided a suite of diagnostic tools to study prostate cancer in more detail, sparking a new era of biomarker discoveries. In this review, we envision that future prostate cancer diagnosis will probably integrate multiplex nano-diagnostic approaches to detect novel urinary biomarkers. However, challenges remain in differentiating indolent from aggressive cancers to better inform treatment decisions, and clinical translation still needs to be overcome.

Digital Decoding of Single Extracellular Vesicle Phenotype Differentiates Early Malignant and Benign Lung Lesions.

Accurate identification of malignant lung lesions is a prerequisite for rational clinical management to reduce morbidity and mortality of lung cancer. However, classification of lung nodules into malignant and benign cases is difficult as they show similar features in computer tomography and sometimes positron emission tomography imaging, making invasive tissue biopsies necessary. To address the challenges in evaluating indeterminate nodules, the authors investigate the molecular profiles of small extracellular vesicles (sEVs) in differentiating malignant and benign lung nodules via a liquid biopsy-based approach. Aiming to characterize phenotypes between malignant and benign groups, they develop a single-molecule-resolution-digital-sEV-counting-detection (DECODE) chip that interrogates three lung-cancer-associated sEV biomarkers and a generic sEV biomarker to create sEV molecular profiles. DECODE capturessEVs on a nanostructured pillar chip, confines individual sEVs, and profiles sEV biomarker expression through surface-enhanced Raman scattering barcodes. The author utilize DECODE to generate a digitally acquired sEV molecular profiles in a cohort of 33 people, including patients with malignant and benign lung nodules, and healthy individuals. Significantly, DECODE reveals sEV-specific molecular profiles that allow the separation of malignant from benign (area under the curve, AUC = 0.85), which is promising for non-invasive characterisation of lung nodules found in lung cancer screening and warrants further clinincal validaiton with larger cohorts.

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.

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.

C5b-9 Membrane Attack Complex Formation and Extracellular Vesicle Shedding in Barrett's Esophagus and Esophageal Adenocarcinoma.

The early complement components have emerged as mediators of pro-oncogenic inflammation, classically inferred to cause terminal complement activation, but there are limited data on the activity of terminal complement in cancer. We previously reported elevated serum and tissue C9, the terminal complement component, in esophageal adenocarcinoma (EAC) compared to the precursor condition Barrett's Esophagus (BE) and healthy controls. Here, we investigate the level and cellular fates of the terminal complement complex C5b-9, also known as the membrane attack complex. Punctate C5b-9 staining and diffuse C9 staining was detected in BE and EAC by multiplex immunohistofluorescence without corresponding increase of C9 mRNA transcript. Increased C9 and C5b-9 staining were observed in the sequence normal squamous epithelium, BE, low- and high-grade dysplasia, EAC. C5b-9 positive esophageal cells were morphologically intact, indicative of sublytic or complement-evasion mechanisms. To investigate this at a cellular level, we exposed non-dysplastic BE (BAR-T and CP-A), high-grade dysplastic BE (CP-B and CP-D) and EAC (FLO-1 and OE-33) cell lines to the same sublytic dose of immunopurified human C9 (3 µg/ml) in the presence of C9-depleted human serum. Cellular C5b-9 was visualized by immunofluorescence confocal microscopy. Shed C5b-9 in the form of extracellular vesicles (EV) was measured in collected conditioned medium using recently described microfluidic immunoassay with capture by a mixture of three tetraspanin antibodies (CD9/CD63/CD81) and detection by surface-enhanced Raman scattering (SERS) after EV labelling with C5b-9 or C9 antibody conjugated SERS nanotags. Following C9 exposure, all examined cell lines formed C5b-9, internalized C5b-9, and shed C5b-9+ and C9+ EVs, albeit at varying levels despite receiving the same C9 dose. In conclusion, these results confirm increased esophageal C5b-9 formation during EAC development and demonstrate capability and heterogeneity in C5b-9 formation and shedding in BE and EAC cell lines following sublytic C9 exposure. Future work may explore the molecular mechanisms and pathogenic implications of the shed C5b-9+ EV.

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.

Generation of Nanoyeast Single-Chain Variable Fragments as High-Avidity Biomaterials for Dengue Virus Detection.

Bioengineered yeast bio-nanomaterials termed nanoyeasts displaying antibody single-chain variable fragments (scFvs) against diagnostic targets are a promising alternative to monoclonal antibodies (mAbs). A potential limitation for translating nanoyeasts into diagnostic tools is batch-to-batch variability. Herein, we demonstrate a systematic approach for cost-efficient production of highly specific nanoyeasts that enabled accurate dengue virus (DENV) detection by immunoassay (2.5% CV). Yeasts bioengineered to surface express DENV-specific scFvs (up to 66% of the total cell population) were fragmented into nanoyeast fractions trialing sonication, bead beating, and high-pressure disruption methods. Nanoyeast fractions from sonication had optimal target binding, uniform particle size (±89 nm), were stable, and retained diagnostic activity for 7 days at 37 °C compared to traditional mAbs that lost activity after 1 day at 37 °C. We engineered a panel of nanoyeast scFvs targeting DENV nonstructural protein 1 (NS1): (i) specific for serotyping DENV 1-4 and (ii) cross-reactive anti-DENV scFvs that are suitable for "yes/no" diagnostic applications. We demonstrate highly specific nanoyeast scFvs for serotyping DENV. We show that nanoyeast scFvs specifically detect NS1 in simulated patient plasma with a limit of detection of 250 ng/mL, the concentration found in infected patients.