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.

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.

A Study on the Interfacial Compatibility, Microstructure and Physico-Chemical Properties of Polyimide/Organically Modified Silica Nanocomposite Membrane.

Polyimide-silica (PI-Silica) composites are of tremendous research interest as high-performance materials because of their excellent thermal and mechanical properties and chemical resistance to organic solvents. Particularly, the sol-gel method of fabricating such composites is popular for manipulating their properties. In this work, PI-silica composite films are synthesized by the sol-gel method and thermal imidization from the solution mixtures of hydrolyzed tetraethoxysilane (TEOS) (or glycidoxypropyltrimethoxysilane (GPMS)) modified silica and an aromatic polyamic acid (PAA) based on 3,3',4,4'-biphenyl tetracarboxylic dianhydride (BPDA)-p-phenylenediamine (PDA). The phase morphology of composites is found to be controlled by the substitution of TEOS with GPMS. Solid-state NMR spectroscopy is used to confirm the structural components of silica and GPMS-modified silica, whereas FT-IR results confirm the complete imidization of polyimide and composite film and suggest successful incorporation of Si-O-Si bonds into polyimide. The thermal, optical transmittance, and dielectric constant characterizations of pure polyimide and composite films are also carried out. Thermal stability of pure polyimide is found to be increased significantly by the addition of silica, whereas the partial substitution of TEOS with GPMS decreases the thermal stability of the composite, due to the presence of the alkyl organic segment of GPMS. The optical transmittance and dielectric constant of the composite films are controlled by manipulating the GPMS content.

Simultaneous BRAFV600E Protein and DNA Aberration Detection in Circulating Melanoma Cells Using an Integrated Multimolecular Sensor.

Liquid biopsy has emerged as the next generation target for diagnostics and therapeutic monitoring of many diseases including cancer. Liquid biopsy offers noninvasive analysis of aberrant biomolecular changes (e.g., aberrant protein expression, DNA mutation) which can provide crucial information on disease stages and therapy responses. As a diagnostically important biomarker for melanoma, the detection of the BRAFV600E aberration at the DNA and protein level in liquid biopsies confers an attractive option. This method describes the preparation and operation of an integrated multimolecular sensor (IMMS) for simultaneous detection of the BRAFV600E aberration in both molecular forms from circulating melanoma cells in liquid biopsy. IMMS integrates specific melanoma cell capture, cell release, cell lysis, and electrochemical BRAFV600E detection on a single device. IMMS is demonstrated for a sample-to-answer workflow of plasma spiked with melanoma cells.

A digital single-molecule nanopillar SERS platform for predicting and monitoring immune toxicities in immunotherapy.

The introduction of immune checkpoint inhibitors has demonstrated significant improvements in survival for subsets of cancer patients. However, they carry significant and sometimes life-threatening toxicities. Prompt prediction and monitoring of immune toxicities have the potential to maximise the benefits of immune checkpoint therapy. Herein, we develop a digital nanopillar SERS platform that achieves real-time single cytokine counting and enables dynamic tracking of immune toxicities in cancer patients receiving immune checkpoint inhibitor treatment - broader applications are anticipated in other disease indications. By analysing four prospective cytokine biomarkers that initiate inflammatory responses, the digital nanopillar SERS assay achieves both highly specific and highly sensitive cytokine detection down to attomolar level. Significantly, we report the capability of the assay to longitudinally monitor 10 melanoma patients during immune inhibitor blockade treatment. Here, we show that elevated cytokine concentrations predict for higher risk of developing severe immune toxicities in our pilot cohort of patients.

An integrated multi-molecular sensor for simultaneous BRAFV600E protein and DNA single point mutation detection in circulating tumour cells.

The analysis of circulating cancer biomarkers in the form of liquid biopsies confers several potential benefits as compared to traditional surgical tissue sampling. As a common key anomaly strongly implicated across several cancer types, the BRAFV600E mutation is one of the most valuable oncogenic biomarkers available in liquid biopsies. Crucially, BRAFV600E is also an actionable mutation which could be arrested by clinically beneficial drug inhibitors. Yet, as is true for most single base disease mutations, current BRAFV600E detection in either its DNA or protein molecular state is still liable to false positive/negative outcomes, thus impacting patient treatment benefit. Here we present an integrated multi-molecular sensor (IMMS) for an entire sample-to-answer workflow from melanoma cell capture to simultaneous quantification of both intracellular BRAFV600E DNA and protein levels on a single platform. The IMMS combines (i) specific capture and release of circulating melanoma cells; (ii) electric field-induced cell lysis; (iii) simultaneous quantification of BRAFV600E DNA and protein levels. We investigated the IMMS system's analytical performance in cell capture, release and lysis, and intracellular BRAFV600E detection by ligase-mediated DNA amplification and antibody-based protein hybridization. As a proof-of-concept, we successfully demonstrated circulating BRAFV600E detection at both DNA and protein molecular levels in simulated melanoma plasma samples. With its capabilities in integrated and miniaturized analysis, the IMMS could lead the emergence of a new generation of multi-molecular lab-on-chip biosensors for enabling more accurate and extensive analysis of powerful circulating biomarkers in patient liquid biopsies.

Electrochemical detection of protein glycosylation using lectin and protein-gold affinity interactions.

We report a new method for the electrochemical detection of glycosylation on proteins, which relies on lectin-protein interaction on a bare gold electrode. The target protein isolated by immunoaffinity is directly adsorbed onto a gold surface and its glycosylation status is retrieved by subsequent addition of specific lectins. The adsorption and subsequent recognition process is monitored electrochemically in the presence of [Fe(CN)6](3-/4-) redox system. By decoupling target protein capture from glycosylation read-out steps, this approach circumvents unwanted antibody-lectin crosstalk while enabling specific glycosylation detection of a glycoprotein in serum-spiked samples in less than 1 h.