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.

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.

Assessment of Serum Uric Acid, Urea, and Glucose Levels and Associated Factors among Breast Cancer Patients Attending A Tertiary Hospital in Bahirdar, Ethiopia: A Comparative Cross-Sectional Study.

Background: Breast cancer is currently become a major public health problem in both developed and developing regions, it is one of the most common surgical problems in Ethiopia. Therefore, this study assessed serum uric acid, urea, and glucose levels and associated factors among benign, malignant breast cancer patients and apparently healthy women attending at Felege-Hiwot comprehensive Specialized Hospital. Methods: Hospital based comparative cross-sectional study was conducted among benign, malignant breast cancer patients and apparently healthy women attending at Felege-Hiwot Comprehensive Specialized Hospital. Out of 178 study participants 66 benign and 23 malignant fine needle aspirate cytology confirmed breast cancer patients and 89 apparently healthy women, included. Multivariable logistic regression models used to measure the strength of associations. A P value of < 0.05 was considered statistically significant. Results: Majority of the study participants, 81(91%) controls, 55(83.3%) benign, and 17(73.9%) malignant cases were premenopausal. Serum glucose 144.47±74.35 and uric acid 6.84±2.54 levels were significantly elevated in malignant cases than control (p-value< 0.05). Patients with malignant status were 4.38 times more likely to have hyperglycemia (AOR=4.38, 95%CI: 1.98-19.97) and 5.53 times more likely have hyperuricemia (AOR=20.43-95% CI: 6.80-61.23), 4 times more likely to have uremia (AOR=4.09, 95% CI: 1.06-15.91) compared to apparently healthy women. Conclusion: Serum glucose, and uric acid levels were significantly higher in malignant and benign cases compared with apparently healthy women. Family history of breast cancer, body mass index, systolic hypertension, comorbidity, residence and menopausal status were significantly associated with hyperglycemia, uremia and hyperuricemia.

Hydrogel Nanoarchitectonics: An Evolving Paradigm for Ultrasensitive Biosensing.

The integration of nanoarchitectonics and hydrogel into conventional biosensing platforms offers the opportunities to design physically and chemically controlled and optimized soft structures with superior biocompatibility, better immobilization of biomolecules, and specific and sensitive biosensor design. The physical and chemical properties of 3D hydrogel structures can be modified by integrating with nanostructures. Such modifications can enhance their responsiveness to mechanical, optical, thermal, magnetic, and electric stimuli, which in turn can enhance the practicality of biosensors in clinical settings. This review describes the synthesis and kinetics of gel networks and exploitation of nanostructure-integrated hydrogels in biosensing. With an emphasis on different integration strategies of hydrogel with nanostructures, this review highlights the importance of hydrogel nanostructures as one of the most favorable candidates for developing ultrasensitive biosensors. Moreover, hydrogel nanoarchitectonics are also portrayed as a promising candidate for fabricating next-generation robust biosensors.

Opportunities for Early Cancer Detection: The Rise of ctDNA Methylation-Based Pan-Cancer Screening Technologies.

The efficiency of conventional screening programs to identify early-stage malignancies can be limited by the low number of cancers recommended for screening as well as the high cumulative false-positive rate, and associated iatrogenic burden, resulting from repeated multimodal testing. The opportunity to use minimally invasive liquid biopsy testing to screen asymptomatic individuals at-risk for multiple cancers simultaneously could benefit from the aggregated diseases prevalence and a fixed specificity. Increasing both latter parameters is paramount to mediate high positive predictive value-a useful metric to evaluate a screening test accuracy and its potential harm-benefit. Thus, the use of a single test for multi-cancer early detection (stMCED) has emerged as an appealing strategy for increasing early cancer detection rate efficiency and benefit population health. A recent flurry of these stMCED technologies have been reported for clinical potential; however, their development is facing unique challenges to effectively improve clinical cost-benefit. One promising avenue is the analysis of circulating tumour DNA (ctDNA) for detecting DNA methylation biomarker fingerprints of malignancies-a hallmark of disease aetiology and progression holding the potential to be tissue- and cancer-type specific. Utilizing panels of epigenetic biomarkers could potentially help to detect earlier stages of malignancies as well as identify a tumour of origin from blood testing, useful information for follow-up clinical decision making and subsequent patient care improvement. Overall, this review collates the latest and most promising stMCED methodologies, summarizes their clinical performances, and discusses the specific requirements multi-cancer tests should meet to be successfully implemented into screening guidelines.

Characterizing the Heterogeneity of Small Extracellular Vesicle Populations in Multiple Cancer Types via an Ultrasensitive Chip.

Identifying small extracellular vesicle (sEV) subpopulations based on their different molecular signatures could potentially reveal the functional roles in physiology and pathology. However, it is a challenge to achieve this aim due to the nano-sized dimensions of sEVs, low quantities of biological cargo each sEV carries, and our incomplete knowledge of identifying features capable of separating heterogeneous sEV subpopulations. Here, a sensitive, multiplexed, and nano-mixing-enhanced sEV subpopulation characterization platform (ESCP) is proposed to precisely determine the sEV phenotypic heterogeneity and understand the role of sEV heterogeneity in cancer progression and metastasis. The ESCP utilizes spatially patterned anti-tetraspanin-functionalized micro-arrays for sEV subpopulation sorting and nanobarcode-based surface-enhanced Raman spectroscopy for multiplexed read-outs. An ESCP has been used for investigating sEV phenotypic heterogeneity in terms of canonical sEV tetraspanin molecules and cancer-associated protein biomarkers in both cancer cell line models and cancer patient samples. Our data explicitly demonstrate the selective enrichment of tetraspanins and cancer-associated protein biomarkers, in particular sEV subpopulations. Therefore, it is believed that the ESCP could enable the evaluation and broader application of sEV subpopulations as potential diagnostic disease biomarkers.

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.

Nutritional Composition and Bioactive Compounds in Tomatoes and Their Impact on Human Health and Disease: A Review.

Tomatoes are consumed worldwide as fresh vegetables because of their high contents of essential nutrients and antioxidant-rich phytochemicals. Tomatoes contain minerals, vitamins, proteins, essential amino acids (leucine, threonine, valine, histidine, lysine, arginine), monounsaturated fatty acids (linoleic and linolenic acids), carotenoids (lycopene and ß-carotenoids) and phytosterols (ß-sitosterol, campesterol and stigmasterol). Lycopene is the main dietary carotenoid in tomato and tomato-based food products and lycopene consumption by humans has been reported to protect against cancer, cardiovascular diseases, cognitive function and osteoporosis. Among the phenolic compounds present in tomato, quercetin, kaempferol, naringenin, caffeic acid and lutein are the most common. Many of these compounds have antioxidant activities and are effective in protecting the human body against various oxidative stress-related diseases. Dietary tomatoes increase the body's level of antioxidants, trapping reactive oxygen species and reducing oxidative damage to important biomolecules such as membrane lipids, enzymatic proteins and DNA, thereby ameliorating oxidative stress. We reviewed the nutritional and phytochemical compositions of tomatoes. In addition, the impacts of the constituents on human health, particularly in ameliorating some degenerative diseases, are also discussed.

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.

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.

Tracking extracellular vesicle phenotypic changes enables treatment monitoring in melanoma.

Monitoring targeted therapy in real time for cancer patients could provide vital information about the development of drug resistance and improve therapeutic outcomes. Extracellular vesicles (EVs) have recently emerged as a promising cancer biomarker, and EV phenotyping shows high potential for monitoring treatment responses. Here, we demonstrate the feasibility of monitoring patient treatment responses based on the plasma EV phenotypic evolution using a multiplex EV phenotype analyzer chip (EPAC). EPAC incorporates the nanomixing-enhanced microchip and the multiplex surface-enhanced Raman scattering (SERS) nanotag system for direct EV phenotyping without EV enrichment. In a preclinical model, we observe the EV phenotypic heterogeneity and different phenotypic responses to the treatment. Furthermore, we successfully detect cancer-specific EV phenotypes from melanoma patient plasma. We longitudinally monitor the EV phenotypic evolution of eight melanoma patients receiving targeted therapy and find specific EV profiles involved in the development of drug resistance, reflecting the potential of EV phenotyping for monitoring treatment responses.

Dual platform based sandwich assay surface-enhanced Raman scattering DNA biosensor for the sensitive detection of food adulteration.

Surface enhanced Raman scattering (SERS) DNA biosensing is an ultrasensitive, selective, and rapid detection technique with the ability to produce molecule-specific distinct fingerprint spectra. It supersedes the long amplicon based PCR assays, the fluorescence and spectroscopic techniques with their quenching and narrow spectral bandwidth, and the electrochemical detection techniques using multiplexing. However, the performance of the SERS DNA biosensor relies on the DNA probe length, platform composition, both the presence and position of Raman tags and the chosen sensing strategy. In this context, we herein report a SERS biosensor based on dual nanoplatforms with a uniquely designed Raman tag (ATTO Rho6G) intercalated short-length DNA probe for the sensitive detection of the pig species Sus scrofa. In the design of the signal probe (SP), a Raman tag was incorporated adjacent to the spacer arm, followed by a terminal thiol modifier, which consequently had a strong influence on the SERS signal enhancement. The detection strategy involves the probe-target DNA hybridization mediated coupling of the two platforms, i.e., the graphene oxide-gold nanorod (GO-AuNR) functionalized capture probe (CP) and SP-conjugated gold nanoparticles (AuNPs), consequently enhancing the SERS intensity by both the electromagnetic hot spots generated at the junctions or interstices of the two platforms and the chemical enhancement between the AuNPs and the adsorbed intercalated Raman tag. This dual platform based SERS DNA biosensor exhibited outstanding sensitivity in detecting pork DNA with a limit of detection (LOD) of 100 aM validated with DNA extracted from a pork sample (LOD 1 fM). Moreover, the fabricated SERS biosensor showed outstanding selectivity and specificity for differentiating the DNA sequences of six closely related non-target species from the target DNA sequences with single and three nucleotide base-mismatches. Therefore, the developed short-length DNA linked dual platform based SERS biosensor could replace the less sensitive traditional methods of pork DNA detection and be adopted as a universal detection approach for the qualitative and quantitative detection of DNA from any source.

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.

DNA Methylation-Based Point-of-Care Cancer Detection: Challenges and Possibilities.

Eukaryotic cell DNA conserves a distinct genomic methylation pattern, which acts as a molecular switch to control the transcriptional machinery of the cell. However, pathological processes can alter this methylation pattern, leading to the onset of diseases such as cancer. Recent advances in methylation analysis provide a more precise understanding of the consequence of DNA methylation changes towards cancer progression. Consequently, the discoveries of numerous methylation-based biomarkers have inspired the development of simple tests for cancer detection. In this opinion article, we systematically discuss the benefits and challenges associated with the promising methylation-based approaches and develop a point-of-care index to evaluate their potential in terms of point-of-care cancer diagnostics.

Graphene oxide and gold nanoparticle based dual platform with short DNA probe for the PCR free DNA biosensing using surface-enhanced Raman scattering.

Surface-enhanced Raman scattering (SERS) based DNA biosensors have considered as excellent, fast and ultrasensitive sensing technique which relies on the fingerprinting ability to produce molecule specific distinct spectra. Unlike conventional fluorescence based strategies SERS provides narrow spectral bandwidths, fluorescence quenching and multiplexing ability, and fitting attribute with short length probe DNA sequences. Herein, we report a novel and PCR free SERS based DNA detection strategy involving dual platforms and short DNA probes for the detection of endangered species, Malayan box turtle (MBT) (Cuora amboinensis). In this biosensing feature, the detection is based on the covalent linking of the two platforms involving graphene oxide-gold nanoparticles (GO-AuNPs) functionalized with capture probe 1 and gold nanoparticles (AuNPs) modified with capture probe 2 and Raman dye (Cy3) via hybridization with the corresponding target sequences. Coupling of the two platforms generates locally enhanced electromagnetic field 'hot spot', formed at the junctions and interstitial crevices of the nanostructures and consequently provide significant amplification of the SERS signal. Therefore, employing the two SERS active substrates and short-length probe DNA sequences, we have managed to improve the sensitivity of the biosensors to achieve a lowest limit of detection (LOD) as low as 10 fM. Furthermore, the fabricated biosensor exhibited sensitivity even for single nucleotide base-mismatch in the target DNA as well as showed excellent performance to discriminate closely related six non-target DNA sequences. Although the developed SERS biosensor would be an attractive platform for the authentication of MBT from diverse samples including forensic and/or archaeological specimens, it could have universal application for detecting gene specific biomarkers for many diseases including cancer.

Label-free detection of exosomes using a surface plasmon resonance biosensor.

The development of a sensitive and specific detection platform for exosomes is highly desirable as they are believed to transmit vital tumour-specific information (mRNAs, microRNAs, and proteins) to remote cells for secondary metastasis. Herein, we report a simple method for the real-time and label-free detection of clinically relevant exosomes using a surface plasmon resonance (SPR) biosensor. Our method shows high specificity in detecting BT474 breast cancer cell-derived exosomes particularly from complex biological samples (e.g. exosome spiked in serum). This approach exhibits high sensitivity by detecting as low as 8280 exosomes/µL which may potentially be suitable for clinical analysis. We believe that this label-free and real-time method along with the high specificity and sensitivity may potentially be useful for clinical settings.

A SERS microfluidic platform for targeting multiple soluble immune checkpoints.

Immune checkpoint blockade therapies are promising next generation immunotherapeutic treatments for cancer. Whilst sequential solid biopsies are an invaluable source of prognostic information, they are not feasible for monitoring therapeutic outcomes over time. Monitoring soluble immune checkpoint markers expression in body fluids could potentially be a better alternative. Current methods (e.g. ELISA) for detecting immune-checkpoint proteins mostly rely on the use of monoclonal antibodies which are expensive and time-consuming to manufacture and isolate. Herein, we report an integrated surface enhanced Raman scattering (SERS)-microfluidics device for the detection of immune checkpoint proteins which involves the use of i) nano yeast single chain variable fragment (scFv) as a promising alternative to monoclonal antibodies providing high stability at relative low-cost and simplicity for production, ii) graphene oxide functionalised surface to reduces the bio functionalization steps, thus avoiding the general paradigm of biotin-streptavidin chemistry and iii) a microfluidic platform enabling alternating current electrohydrodynamics (ac-EHD) induced nanomixing to enhance the target scFv binding and minimize the non-specific interactions. Specific and multiplex detection of immune checkpoint biomarkers is achieved by SERS based spectral encoding. Using this platform, we successfully demonstrated the detection of clinically relevant soluble immune checkpoints PD-1, PD-L1 and LAG-3 from as low as 100 fg/mL of analytes spiked in human serum.

Epigenetically reprogrammed methylation landscape drives the DNA self-assembly and serves as a universal cancer biomarker.

Epigenetic reprogramming in cancer genomes creates a distinct methylation landscape encompassing clustered methylation at regulatory regions separated by large intergenic tracks of hypomethylated regions. This methylation landscape that we referred to as Methylscape is displayed by most cancer types, thus may serve as a universal cancer biomarker. To-date most research has focused on the biological consequences of DNA Methylscape changes whereas its impact on DNA physicochemical properties remains unexplored. Herein, we examine the effect of levels and genomic distribution of methylcytosines on the physicochemical properties of DNA to detect the Methylscape biomarker. We find that DNA polymeric behaviour is strongly affected by differential patterning of methylcytosine, leading to fundamental differences in DNA solvation and DNA-gold affinity between cancerous and normal genomes. We exploit these Methylscape differences to develop simple, highly sensitive and selective electrochemical or colorimetric one-step assays for the detection of cancer. These assays are quick, i.e., analysis time ≤10 minutes, and require minimal sample preparation and small DNA input.

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.