A mesoporous gold biosensor to investigate immune checkpoint protein heterogeneity in single lung cancer cells.

Immune checkpoint proteins (ICPs) play a major role in a patient's immune response against cancer. Tumour cells usually express those proteins to communicate with immune cells as a process of escaping the anti-cancer immune response. Detecting the major functional immune checkpoint proteins present on cancer cells (such as circulating tumor cells or CTCs) and examining the heterogeneity in their expression at the single-cell level could play a crucial role in both cancer diagnosis and the monitoring of therapy. In this study, we develop a mesoporous gold biosensor to precisely assess ICP heterogeneity in individual cancer cells within a lung cancer model. The platform utilizes a nanostructured mesoporous gold surface to capture CTCs and a Surface Enhanced Raman Scattering (SERS) readout to identify and monitor the expression of key ICP proteins (PD-L1, B7H4, CD276, CD80) in lung cancer cells. The homogeneous and abundant pores in mesoporous 3D gold nanostructures enable increased antibody loading on-chip and an enhanced SERS signal, which are key to our single cell capture, and accurate analysis of ICPs in cancer cells with high sensitivity. Our lung cancer cell line model data showed that our method can detect single cells and analyse the expression of four lung cancer associated ICPs on individual cell surfaces during treatment. To show the potential of our mesoporous gold biosensor in analysing clinical samples, we tested 9 longitudinal Peripheral Blood Mononuclear Cells (PBMC) samples from lung cancer patient before and after therapy. Our mesoporous biosensor successfully captured single CTCs and found that the expression of ICPs in CTCs is highly heterogeneous in both pre-treatment and treated PBMC samples isolated from lung cancer patient blood. We suggest that our findings will help clinicians in selecting the most appropriate therapy for patients.

Nanophotonic immunoarray with electrochemically roughened surfaces for handheld detection of secreted PD-L1 to predict immuno-oncology efficacy.

The analysis of secreted protein biomarkers can be a useful non-invasive method of predicting or monitoring cancer therapeutic response. The increased level of soluble programmed cell death protein ligand 1 (sPD-L1) is a promising predictive biomarker for selecting patients who are likely to respond to immune checkpoint immunotherapy. The current established immunoassay for secreted protein analysis is enzyme-linked immunosorbent assay (ELISA). Yet, ELISA is generally still liable to limited detection sensitivity and restricted to bulky chromogenic readout equipment. Herein, we present a designed nanophotonic immunoarray sensor which achieved sPD-L1 analysis at high-throughput, enhanced detection sensitivity and portability. The key benefits of our nanophotonic immunoarray sensor are (i) high-throughput surface-enhanced Raman scattering (SERS) analysis of multiple samples on a singular platform; (ii) improved sPD-L1 detection sensitivity at 1 pg mL-1 (by two orders of magnitude as compared to ELISA) via electrochemically roughened gold sensor surfaces; (iii) fit for handheld SERS detection with miniaturized equipment footprint. We evaluated the analytical performance of the nanophotonic immunoarray sensor and successfully demonstrated quantitative sPD-L1 detection in a cohort of contrived human plasma samples.

Estimation of Physiologic Pressures: Invasive and Non-Invasive Techniques, AI Models, and Future Perspectives.

The measurement of physiologic pressure helps diagnose and prevent associated health complications. From typical conventional methods to more complicated modalities, such as the estimation of intracranial pressures, numerous invasive and noninvasive tools that provide us with insight into daily physiology and aid in understanding pathology are within our grasp. Currently, our standards for estimating vital pressures, including continuous BP measurements, pulmonary capillary wedge pressures, and hepatic portal gradients, involve the use of invasive modalities. As an emerging field in medical technology, artificial intelligence (AI) has been incorporated into analyzing and predicting patterns of physiologic pressures. AI has been used to construct models that have clinical applicability both in hospital settings and at-home settings for ease of use for patients. Studies applying AI to each of these compartmental pressures were searched and shortlisted for thorough assessment and review. There are several AI-based innovations in noninvasive blood pressure estimation based on imaging, auscultation, oscillometry and wearable technology employing biosignals. The purpose of this review is to provide an in-depth assessment of the involved physiologies, prevailing methodologies and emerging technologies incorporating AI in clinical practice for each type of compartmental pressure measurement. We also bring to the forefront AI-based noninvasive estimation techniques for physiologic pressure based on microwave systems that have promising potential for clinical practice.

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.

Machine Learning-Based Classification of Abnormal Liver Tissues Using Relative Permittivity.

The search for non-invasive, fast, and low-cost diagnostic tools has gained significant traction among many researchers worldwide. Dielectric properties calculated from microwave signals offer unique insights into biological tissue. Material properties, such as relative permittivity (εr) and conductivity (σ), can vary significantly between healthy and unhealthy tissue types at a given frequency. Understanding this difference in properties is key for identifying the disease state. The frequency-dependent nature of the dielectric measurements results in large datasets, which can be postprocessed using artificial intelligence (AI) methods. In this work, the dielectric properties of liver tissues in three mouse models of liver disease are characterized using dielectric spectroscopy. The measurements are grouped into four categories based on the diets or disease state of the mice, i.e., healthy mice, mice with non-alcoholic steatohepatitis (NASH) induced by choline-deficient high-fat diet, mice with NASH induced by western diet, and mice with liver fibrosis. Multi-class classification machine learning (ML) models are then explored to differentiate the liver tissue groups based on dielectric measurements. The results show that the support vector machine (SVM) model was able to differentiate the tissue groups with an accuracy up to 90%. This technology pipeline, thus, shows great potential for developing the next generation non-invasive diagnostic tools.

In Situ Single Cell Proteomics Reveals Circulating Tumor Cell Heterogeneity during Treatment.

Cancer is a dynamic disease with heterogenic molecular signatures and constantly evolves during the course of the disease. Single cell proteomic analysis could offer a suitable pathway to monitor cancer cell heterogeneity and deliver critical information for the diagnosis, recurrence, and drug-resistant mechanisms in cancer. Current standard techniques for proteomic analysis such as ELISA, mass spectrometry, and Western blots are time-consuming, expensive, and often require fluorescence labeling that fails to provide accurate information about the multiple protein expression changes at the single cell level. Herein, we report a surface-enhanced Raman spectroscopy-based simple microfluidic device that enables the screening of single circulating tumor cells (CTC) in a dynamic state to precisely understand the heterogeneous expression of multiple protein biomarkers in response to therapy. It further enables identifying intercellular heterogeneous expression of CTC surface proteins which would be highly informative to identify the cancer cells surviving treatment and potentially responsible for drug resistance. Using a bead and cell line-based model system, we successfully detect single bead and single cell spectra when flowed through the device. Using SK-MEL-28 melanoma cells, we demonstrate that our system is capable of monitoring heterogeneous expressions of multiple surface protein markers (MCSP, MCAM, and LNGFR) before and during drug treatment. Integrating a label-free electrochemical system with the device, we also monitor the expression of an intracellular protein (here, BRAFV600E) under drug treatment. Finally, we perform a longitudinal study with 15 samples from five different melanoma patients who underwent therapy. We find that the average expression of receptor proteins in a patient fails to determine the therapy response particularly when the disease progresses. However, single CTC analysis with our device shows a high level of intercellular heterogeneity in the receptor expression profiles of patient-derived CTCs and identifies heterogeneity within CTCs. More importantly, we find that a fraction of CTCs still shows a high expression of these receptor proteins during and after therapy, indicating the presence of resistant CTCs which may evolve after a certain time and progress the disease. We believe this automated assay will have high clinical importance in disease diagnosis and monitoring treatment and will significantly advance the understanding of cancer heterogeneity on the single cell level.

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.

Design of novel super wide band antenna close to the fundamental dimension limit theory.

This paper investigates the design and practical implementation of a Super Wide Band (SWB) antenna along with the application of fundamental bandwidth limitation theory of small antennas in the proposed design. The antenna is designed on a material with permittivity, εr = 3 where the patch metallization height is maintained as 0.035 mm. The designed antenna is then modified by enhancing the copper patch with an additional layer of 28.5 mm thickness. The proposed antenna achieves a huge frequency range with a ratio bandwidth starting from 96.96:1 to as high as 115.10: 1. The designed antenna operating band with thinner height starts from 1.65 to 160 GHz while with the added patch metallic height, the antenna operates from a minimum of 1.39 to 160 GHz with an average nominal bandwidth of more than 158 GHz. By enhancing the patch height, the antenna spherical volume is utilized more efficiently. Using this principle, the antenna impedance bandwidth is augmented while a reduction in electrical size is achieved. A comparison with the fundamental theories by Chu and Mclean illustrates that the designed SWB antenna electrical size exceeds Mclean and nearly touches the Chu fundamental limit curve. This eventually offers the maximized bandwidth with the most compact size for an SWB antenna. The designed antenna with thinner patch metallization height is practically fabricated and measured up to 67 GHz using Vector Network Analyzer to provide experimental validation.

Methylation dependent gold adsorption behaviour identifies cancer derived extracellular vesicular DNA.

Extracellular vesicles (EV) play a major role in intercellular communication by transmitting cellular materials (e.g. protein, RNA) among distant cells. Recent evidence suggests that they could also contribute to carrying DNA which could inform on the mutational status of the parent tumour DNA. Thus, the fundamental analysis of evDNA could open a better understanding of tumour metastasis and provide new pathways for noninvasive detection and monitoring of cancer. To explore the potential of evDNA for diagnostics, the isolation of pure evDNA from body fluids free of cfDNA contamination is crucial. Herein, we use a liposome based model system to develop an improved evDNA isolation protocol free from cfDNA contamination and evaluate the methylation dependent physicochemical properties of evDNA to develop a simple test for detecting cancer evDNA. Using a highly sensitive multiplex microelectrode device, we demonstrate that serum-evDNA derived from cancer patients show different solution and surface based properties than normal evDNA due to their different methylation landscape (i.e. methylscape). This microdevice allows simultaneous analysis of multiple samples in a single platform from as low as 500 pg µL-1 of evDNA.

Surface-Enhanced Raman Spectroscopy for Cancer Immunotherapy Applications: Opportunities, Challenges, and Current Progress in Nanomaterial Strategies.

Cancer immunotherapy encompasses a variety of approaches which target or use a patient's immune system components to eliminate cancer. Notably, the current use of immune checkpoint inhibitors to target immune checkpoint receptors such as CTLA-4 or PD-1 has led to remarkable treatment responses in a variety of cancers. To predict cancer patients' immunotherapy responses effectively and efficiently, multiplexed immunoassays have been shown to be advantageous in sensing multiple immunomarkers of the tumor microenvironment simultaneously for patient stratification. Surface-enhanced Raman spectroscopy (SERS) is well-regarded for its capabilities in multiplexed bioassays and has been increasingly demonstrated in cancer immunotherapy applications in recent years. This review focuses on SERS-active nanomaterials in the modern literature which have shown promise for enabling cancer patient-tailored immunotherapies, including multiplexed in vitro and in vivo immunomarker sensing and imaging, as well as immunotherapy drug screening and delivery.

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.

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.

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.

Tracking antigen specific T-cells: Technological advancement and limitations.

Assessing T-cell mediated immune status can help to understand the body's response to disease and also provide essential diagnostic information. However, detection and characterization of immune response are challenging due to the rarity of signature biomolecules in biological fluid and require highly sensitive and specific assay technique for the analysis. Until now, several techniques spanning from flow cytometry to microsensors have been developed or under investigation for T-cell mediated immune response monitoring. Most of the current assays are designed to estimate average immune responses, i.e., total functional protein analysis and detection of total T-cells irrespective of their antigen specificity. Although potential, immune response analysis without detecting and characterizing the rare subset of T-cell population could lead to over or underestimation of patient's immune status. Addressing this limitation, recently a number of technological advancements in biosensing have been developed for this. The potential of simple and precise micro-technologies including microarray and microfluidic platforms for assessing antigen-specific T-cells will be highlighted in this review, together with a discussion on existing challenges and future aspects of immune-sensor development.

A Sample-to-Targeted Gene Analysis Biochip for Nanofluidic Manipulation of Solid-Phase Circulating Tumor Nucleic Acid Amplification in Liquid Biopsies.

The use of circulating tumor nucleic acids (ctNA) in patient liquid biopsies for targeted genetic analysis is rapidly increasing in clinical oncology. Still, the call for an integrated methodology, which is both rapid and sensitive for analyzing trace ctNA amount in liquid biopsies, has unfortunately not been fully realized. Herein, we performed complex liquid biopsy sample-to-targeted genetic analysis on a biochip with a 50 copies-detection limit within 30 min. Our biochip uniquely integrated the following: (1) electrical lysis and release of cellular targets with minimal processing; (2) nanofluidic manipulation to accelerate molecular kinetics of solid-phase isothermal amplification; and (3) single-step capture and amplification of multiple NA targets prior to nanozyme-mediated electrochemical detection. Using prostate cancer liquid biopsies, we successfully demonstrated multifunctionality for cancer risk prediction; correlation of serum and urine analyses; and cancer relapse monitoring.

Parallel profiling of cancer cells and proteins using a graphene oxide functionalized ac-EHD SERS immunoassay.

Circulating biomarkers have emerged as promising non-invasive, real-time surrogates for cancer diagnosis, prognosis and monitoring of the therapeutic response. Current bio-sensing techniques mostly involve detection of either circulating cells or proteins which are inadequate in unfolding complex pathologic transformations. Herein, we report parallel detection of cellular and molecular markers (protein) for cancer using a multiplex platform featuring (i) graphene oxide (GO) functionalization that increases the active surface area and more importantly reduces the functionalization steps for rapid detection, (ii) alternating-current electrohydrodynamic (ac-EHD) fluid flow that provides delicate micro-mixing to enhance target-sensor interactions thereby minimizing non-specific binding and (iii) surface enhanced Raman scattering (SERS) for multiplex detection. We find that our platform possesses high sensitivity for detecting both proteins and cells. More importantly, this platform not only detects the cancer cells but also can simultaneously monitor the heterogeneous expression of cell surface proteins which could be clinically useful to determine effective patient therapy. We demonstrate the specific and sensitive detection of breast cancer cells from a mixture of non-target cells and report the heterogeneous expression of human epidermal growth factor receptor 2 (HER2) proteins on the individual cancer cell surface. Concurrently, we detect as low as 100 fg mL-1 HER2 and Mucin 16 proteins spiked in blood serum.

Amplification-Free Multi-RNA-Type Profiling for Cancer Risk Stratification via Alternating Current Electrohydrodynamic Nanomixing.

Simultaneous analysis of messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNA (lncRNA)-multi-RNA-type profiling-is increasingly crucial in cancer diagnostics. Yet, rapid multi-RNA-type profiling is challenging due to enzymatic amplification reliance and RNA-type-dependent characteristics. Here, a nanodevice is reported to uniquely use alterable alternating current electrohydrodynamic (ac-EHD) forces to enhance probe-target hybridization prior to direct native RNA target detection, without target amplification or surface functionalization. To exemplify clinical applicability, noninvasive screening of next-generation prostate cancer (PCa) RNA biomarkers (of different types) in patient urine samples is performed. A strong correlation between multi-RNA-type expression and aggressive PCa is found, and the nanodevice performance is statistically evaluated. It is believed that this miniaturized system exhibits great potential for cancer risk stratification via multi-RNA-type profiling.

A multiplex microplatform for the detection of multiple DNA methylation events using gold-DNA affinity.

We report a new multiplexed strategy for the electrochemical detection of regional DNA methylation across multiple regions. Using the sequence dependent affinity of bisulfite treated DNA towards gold surfaces, the method integrates the high sensitivity of a micro-fabricated multiplex device comprising a microarray of gold electrodes, with the powerful multiplexing capability of multiplex-PCR. The synergy of this combination enables the monitoring of the methylation changes across several genomic regions simultaneously from as low as 500 pg µl-1 of DNA with no sequencing requirement.

Electrohydrodynamic-Induced SERS Immunoassay for Extensive Multiplexed Biomarker Sensing.

Cancer diagnosis and patient monitoring require sensitive and simultaneous measurement of multiple cancer biomarkers considering that single biomarker analysis present inadequate information on the underlying biological transformations. Thus, development of sensitive and selective assays for multiple biomarker detection might improve clinical diagnosis and expedite the treatment process. Herein, a microfluidic platform for the rapid, sensitive, and parallel detection of multiple cancer-specific protein biomarkers from complex biological samples is presented. This approach utilizes alternating current electrohydrodynamic-induced surface shear forces that provide exquisite control over fluid flow thereby enhancing target-sensor interactions and minimizing non-specific binding. Further, the use of surface-enhanced Raman scattering-based spectral encoding with individual barcodes for different targets enables specific and simultaneous detection of captured protein biomarkers. Using this approach, the specific and sensitive detection of clinically relevant biomarkers including human epidermal growth factor receptor 2 (HER2); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor; and Mucin 16, cell surface associated (MUC16) at concentrations as low as 10 fg mL-1 in patient serum is demonstrated. Successful target detection from patient samples further demonstrates the potential of this current approach for the clinical diagnosis, which envisages a clinical translation for a rapid and sensitive appraisal of clinical samples in cancer diagnostics.

Real time and label free profiling of clinically relevant exosomes.

Tumor-derived exosomes possess significant clinical relevance due to their unique composition of genetic and protein material that is representative of the parent tumor. Specific isolation as well as identification of proportions of these clinically relevant exosomes (CREs) from biological samples could help to better understand their clinical significance as cancer biomarkers. Herein, we present a simple approach for quantification of the proportion of CREs within the bulk exosome population isolated from patient serum. This proportion of CREs can potentially inform on the disease stage and enable non-invasive monitoring of inter-individual variations in tumor-receptor expression levels. Our approach utilises a Surface Plasmon Resonance (SPR) platform to quantify the proportion of CREs in a two-step strategy that involves (i) initial isolation of bulk exosome population using tetraspanin biomarkers (i.e., CD9, CD63), and (ii) subsequent detection of CREs within the captured bulk exosomes using tumor-specific markers (e.g., human epidermal growth factor receptor 2 (HER2)). We demonstrate the isolation of bulk exosome population and detection of as low as 10% HER2(+) exosomes from samples containing designated proportions of HER2(+) BT474 and HER2(-) MDA-MB-231 cell derived exosomes. We also demonstrate the successful isolation of exosomes from a small cohort of breast cancer patient samples and identified that approximately 14-35% of their bulk population express HER2.