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.

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 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.

Amplification-Free Detection of Gene Fusions in Prostate Cancer Urinary Samples Using mRNA-Gold Affinity Interactions.

A crucial issue in present-day prostate cancer (PCa) detection is the lack of specific biomarkers for accurately distinguishing between benign and malignant cancer forms. This is causing a high degree of overdiagnosis and overtreatment of otherwise clinically insignificant cases. As around half of all malignant PCa cases display a detectable gene fusion mutation between the TMPRSS2 promoter sequence and the ERG coding sequence (TMPRSS2:ERG) in urine, noninvasive screening of TMPRSS2:ERG mRNA in patient urine samples could improve the specificity of current PCa diagnosis. However, current gene fusion detection methodologies are largely dependent on RNA enzymatic amplification, which requires extensive sample manipulation, costly labels for detection, and is prone to bias/artifacts. Herein we introduce the first successful amplification-free electrochemical assay for direct detection of TMPRSS2:ERG mRNA in PCa urinary samples by selectively isolating and adsorbing TMPRSS2:ERG mRNA onto bare gold electrodes without requiring any surface modification. We demonstrated excellent limit-of-detection (10 cells) and specificity using PCa cell line models, and showcased clinical utility by accurately detecting TMPRSS2:ERG in a collection of 17 urinary samples obtained from PCa patients. Furthermore, these results were validated with the current gold standard reverse transcription (RT)-PCR approach with 100% concordance.

Poly(A) Extensions of miRNAs for Amplification-Free Electrochemical Detection on Screen-Printed Gold Electrodes.

Current amplification-based microRNA (miRNA) detection approaches are limited by the small sizes of miRNAs as well as amplification bias/artifacts. Herein, we report on an amplification-free miRNA assay based on elevated affinity interaction between polyadenylated miRNA and bare gold electrode. The poly(A) extension on the 3' ends of magnetically isolated miRNA targets facilitated high adsorption efficiency onto gold electrode surfaces for electrochemical detection without any cumbersome electrode surface functionalization procedures. The assay showed excellent detection sensitivity (10 fM) and specificity and was demonstrated for quantitative miR-107 detection in human cancer cell lines and clinical urine samples. We believe our assay could be useful as an amplification-free alternative for miRNA detection.

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.

Detecting exosomes specifically: a multiplexed device based on alternating current electrohydrodynamic induced nanoshearing.

Exosomes show promise as noninvasive biomarkers for cancer, but their effective capture and specific detection is a significant challenge. Herein, we report a multiplexed microfluidic device for highly specific capture and detection of multiple exosome targets using a tunable alternating current electrohydrodynamic (ac-EHD) methodology, referred to as nanoshearing. In our system, electrical body forces generated by ac-EHD act within nanometers of an electrode surface (i.e., within the electrical layer) to generate nanoscaled fluid flow that enhances the specificity of capture and also reduce nonspecific adsorption of weakly bound molecules from the electrode surface. This approach demonstrates the analysis of exosomes derived from cells expressing human epidermal growth factor receptor 2 (HER2) and prostate specific antigen (PSA), and is also capable of specifically isolating exosomes from breast cancer patient samples. The device also exhibited a 3-fold enhancement in detection sensitivity in comparison to hydrodynamic flow based assays (LOD 2760 exosomes/µL for ac-EHD vs LOD 8300 exosomes/µL for hydrodynamic flow; (n = 3)). We propose this approach can potentially have relevance as a simple and rapid quantification tool to analyze exosome targets in biological applications.

Methylsorb: a simple method for quantifying DNA methylation using DNA-gold affinity interactions.

The analysis of DNA methylation is becoming increasingly important both in the clinic and also as a research tool to unravel key epigenetic molecular mechanisms in biology. Current methodologies for the quantification of regional DNA methylation (i.e., the average methylation over a region of DNA in the genome) are largely affected by comprehensive DNA sequencing methodologies which tend to be expensive, tedious, and time-consuming for many applications. Herein, we report an alternative DNA methylation detection method referred to as "Methylsorb", which is based on the inherent affinity of DNA bases to the gold surface (i.e., the trend of the affinity interactions is adenine > cytosine ≥ guanine > thymine).1 Since the degree of gold-DNA affinity interaction is highly sequence dependent, it provides a new capability to detect DNA methylation by simply monitoring the relative adsorption of bisulfite treated DNA sequences onto a gold chip. Because the selective physical adsorption of DNA fragments to gold enable a direct read-out of regional DNA methylation, the current requirement for DNA sequencing is obviated. To demonstrate the utility of this method, we present data on the regional methylation status of two CpG clusters located in the EN1 and MIR200B genes in MCF7 and MDA-MB-231 cells. The methylation status of these regions was obtained from the change in relative mass on gold surface with respect to relative adsorption of an unmethylated DNA source and this was detected using surface plasmon resonance (SPR) in a label-free and real-time manner. We anticipate that the simplicity of this method, combined with the high level of accuracy for identifying the methylation status of cytosines in DNA, could find broad application in biology and diagnostics.

eMethylsorb: rapid quantification of DNA methylation in cancer cells on screen-printed gold electrodes.

Simple, sensitive and inexpensive regional DNA methylation detection methodologies are imperative for routine patient diagnostics. Herein, we describe eMethylsorb, an electrochemical assay for quantitative detection of regional DNA methylation on a single-use and cost-effective screen-printed gold electrode (SPE-Au) platform. The eMethylsorb approach is based on the inherent differential adsorption affinity of DNA bases to gold (i.e. adenine > cytosine ≥ guanine > thymine). Through bisulfite modification and asymmetric PCR of DNA, methylated and unmethylated DNA in the sample becomes guanine-enriched and adenine-enriched respectively. Under optimized conditions, adenine-enriched unmethylated DNA (higher affinity to gold) adsorbs more onto the SPE-Au surface than methylated DNA. Higher DNA adsorption causes stronger coulombic repulsion and hinders reduction of ferricyanide [Fe(CN)6](3-)ions on the SPE-Au surface to give a lower electrochemical response. Hence, the response level is directly proportional to the methylation level in the sample. The applicability of this methodology was tested by detecting the regional methylation status in a cluster of eight CpG sites within the engrailed (EN1) gene promoter of the MCF7 breast cancer cell line. A 10% methylation level sensitivity with good reproducibility (RSD = 5.8%, n = 3) was achieved rapidly in 10 min. Furthermore, eMethylsorb also has advantages over current methylation assays such as being inexpensive, rapid and does not require any electrode surface modification. We thus believe that the eMethylsorb assay could potentially be a rapid and accurate diagnostic assay for point-of-care DNA methylation analysis.

Sensitive and label-free biosensing of RNA with predicted secondary structures by a triplex affinity capture method.

A novel biosensing approach for the label-free detection of nucleic acid sequences of short and large lengths has been implemented, with special emphasis on targeting RNA sequences with secondary structures. The approach is based on selecting 8-aminoadenine-modified parallel-stranded DNA tail-clamps as affinity bioreceptors. These receptors have the ability of creating a stable triplex-stranded helix at neutral pH upon hybridization with the nucleic acid target. A surface plasmon resonance biosensor has been used for the detection. With this strategy, we have detected short DNA sequences (32-mer) and purified RNA (103-mer) at the femtomol level in a few minutes in an easy and level-free way. This approach is particularly suitable for the detection of RNA molecules with predicted secondary structures, reaching a limit of detection of 50 fmol without any label or amplification steps. Our methodology has shown a marked enhancement for the detection (18% for short DNA and 54% for RNA), when compared with the conventional duplex approach, highlighting the large difficulty of the duplex approach to detect nucleic acid sequences, especially those exhibiting stable secondary structures. We believe that our strategy could be of great interest to the RNA field.

Suitable combination of noble/ferromagnetic metal multilayers for enhanced magneto-plasmonic biosensing.

We present a theoretical and experimental study on the biosensing sensitivity of Au/Co/Au multilayers as transducers of the magneto-optic surface-plasmon-resonance (MOSPR) sensor. We demonstrate that the sensing response of these magneto-plasmonic (MP) transducers is a trade-off between the optical absorption and the magneto-optical activity, observing that the MP multilayer with larger MO effect does not provide the best sensing response. We show that it is possible to design highly-sensitive MP transducers able to largely surpass the limit of detection of the conventional surface-plasmon-resonance (SPR) sensor. This was proved comparing the biosensing performance of both sensors for the label-free detection of short DNA chains hybridization. For this purpose, we used and tested a novel label-free biofunctionalization protocol based on polyelectrolytes, which increases the resistance of MP transducers in aqueous environments.

Understanding the role of thiol and disulfide self-assembled DNA receptor monolayers for biosensing applications.

A detailed study of the immobilization of three differently sulfur-modified DNA receptors for biosensing applications is presented. The three receptors are DNA-(CH)n-SH-, DNA-(CH)n-SS-(CH)n-DNA, and DNA-(CH)n-SS-DMTO. Nanomechanical and surface plasmon resonance biosensors and fluorescence and radiolabelling techniques were used for the experimental evaluation. The results highlight the critical role of sulfur linker type in DNA self-assembly, affecting the kinetic adsorption and spatial distribution of DNA chains within the monolayer and the extent of chemisorption and physisorption. A spacer (mercaptohexanol, MCH) is used to evaluate the relative efficiencies of chemisorption of the three receptors by analysing the extent to which MCH can remove physisorbed molecules from each type of monolayer. It is demonstrated that -SH derivatization is the most suitable for biosensing purposes as it results in densely packed monolayers with the lowest ratio of physisorbed probes.

Influence of the linker type on the Au-S binding properties of thiol and disulfide-modified DNA self-assembly on polycrystalline gold.

We investigate the Au-S binding properties of thiol and disulfide-modified DNA on polycrystalline gold by means of X-ray photoelectron spectroscopy in conditions close to dynamic processes of biosensors. The dependence of the immobilisation period on the quality and density of the self-assembly process of thiol (SH-(CH(2))(6)-DNA), disulfide (DNA-CH(2))(6)-SS-(CH(2))(6)-DNA and DMTO-SS-(CH(2))(6)-DNA) sulfur-modified oligonucleotide solutions (1 microM) that are employed for bioreceptor immobilisation is analysed. Two electronic components are found in the analysis of the S 2p core levels. One of them is clearly associated to thiolate formation, while the other can be associated to different origins. In order to identify the origin of this last component, a quantification of the non-specifically adsorbed species has been performed by rinsing the self-assembled monolayers (SAMs) with a mercapto hexanol (MCH) solution. It has been found that non-specifically adsorbed species contribute only partially to the appearance of this sulfur peak component in SAMs formed from disulfides. Electron bombardment was performed to study the evolution of this component as a consequence of surface degradation due to radiation effects. The results are also correlated with the possible presence of disulfides. We found that MCH is not stable during the measurements. The evolution of this compound and the possible causes for this behaviour are discussed.

Phosphoprotein Biosensors for Monitoring Pathological Protein Structural Changes.

Current biotechnological developments are driving a significant shift towards integrating proteomic analysis with landmark genomic, methylomic, and transcriptomic data to elucidate functional effects. For the majority of proteins, structure and function are closely intertwined. Post-translational protein modifications (e.g., phosphorylation) leading to aberrantly active structures can originate a wide variety of pathological conditions, including cancer. Analysis of protein structure variants is thus integral to the identification of clinically actionable targets and the design of novel disease diagnosis and therapy approaches. However, it is still challenging to interrogate subtle structural changes of proteins in a rapid and cost-effective manner with current tools. This review primarily compiles the latest biosensing techniques for protein structural analysis.

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.

Biosensors based on cantilevers.

Microcantilevers based-biosensors are a new label-free technique that allows the direct detection of biomolecular interactions in a label-less way and with great accuracy by translating the biointeraction into a nanomechanical motion. Low cost and reliable standard silicon technologies are widely used for the fabrication of cantilevers with well-controlled mechanical properties. Over the last years, the number of applications of these sensors has shown a fast growth in diverse fields, such as genomic or proteomic, because of the biosensor flexibility, the low sample consumption, and the non-pretreated samples required. In this chapter, we report a dedicated design and a fabrication process of highly sensitive microcantilever silicon sensors. We will describe as well an application of the device in the environmental field showing the immunodetection of an organic toxic pesticide as an example. The cantilever biofunctionalization process and the subsequent pesticide determination are detected in real time by monitoring the nanometer-scale bending of the microcantilever due to a differential surface stress generated between both surfaces of the device.

Reading Conformational Changes in Proteins with a New Colloidal-Based Interfacial Biosensing System.

Many biological events such as mutations or aberrant post-translational modifications can alter the conformation and/or folding stability of proteins and their subsequent biological function, which may trigger the onset of diseases like cancer. Evaluating protein folding is hence crucial for the diagnosis of these diseases. Yet, it is still challenging to detect changes in protein folding, especially if they are subtle, in a simple and highly sensitive manner with the current assays. Herein, we report a new colloidal-based interfacial biosensing approach for qualitative and quantitative profiling of various types of changes in protein folding; from denaturation to variant conformations in native proteins, such as protein activation via mutations or phosphorylation. The approach is based on the direct interfacial interaction of proteins freely available in solution with added tannic-acid-capped gold nanoparticles, to interrogate their folding status in their solubilized form. We found that under the optimized conditions, proteins can modulate colloids solvation according to their folding or conformational status, which can be visualized in a single step, by the naked eye, with minimal protein input requirements (limit of detection of 1 ng/µL). Protein folding detection was achieved regardless of protein topology and size without using conformation-specific antibodies and mutational analysis, which are the most common assays for sensing malfunctioning proteins. The approach showed excellent sensitivity, superior to circular dichroism, for the detection of the very subtle conformational changes induced by activating mutations and phosphorylation in epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) proteins. This enabled their detection even in complex samples derived from lung cancer cells, which contained up to 95% excess of their wild-type forms. A broader clinical translation was shown via monitoring the action of conformation-restoring drugs, such as tyrosine kinase inhibitors, on EGFR conformation and its downstream protein network, using the ERK protein as a surrogate.

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.

An exosomal- and interfacial-biosensing based strategy for remote monitoring of aberrantly phosphorylated proteins in lung cancer cells.

It is a well-known phenomenon that cancer cells release key biological information such as DNA, RNA or proteins into body fluids (e.g., blood, urine or saliva). The analysis of these molecules-often encapsulated within nanovesicules called exosomes-is highly attractive, because it could replace current surgical biopsies, which are painful, costly and potentially risky for patients. For example, current strategies in lung cancer diagnosis involve genetic analyses from tumour tissues to detect the presence of underlying DNA mutations, known to alter the phosphorylation status and function of proteins. This information is used to direct therapy, as aberrantly phosphorylated proteins are the main targets of current drugs such as Tyrosine Kinase Inhibitors (TKIs). An alternative and less invasive strategy would be the remote analysis of these phospho-proteins by isolating them from cancer-derived exosomes. This would allow evaluating not only their phosphorylation status at diagnosis, but also the timely restoration of protein phosphorylation levels during therapy with TKIs. Yet, this proteomic approach remains vastly unexplored. Herein, we demonstrate that key lung cancer phosphoproteins, such as EGFR and ERK, are expressed in lung cancer exosomes and we outline a new exosomal proteomic-based approach for their fast and convenient detection. This approach, which could complement current genetic analysis for lung cancer detection, easily detects the phosphorylation status of lung cancer exosomal proteins within minutes after their extraction, bringing hope of circumventing the need for tissue biopsy and costly and cumbersome DNA sequencing techniques. It exploits the fact that phosphorylation induces protein conformational changes, which in turn alter protein's ability to effectively interact with bare gold surfaces. This leads to phosphorylated and non-phosphorylated protein isoforms displaying different gold-adsorption profiles. Using single-use and inexpensive, gold (Au) screen-printed electrodes (SPEs), we demonstrate the successful detection of aberrantly phosphorylated EGFR and ERK protein isoforms derived from lung cancer cell exosomes with a sensitivity down to 15 ng µL-1 in samples with up to 90% excess of their non-phosphorylated (wild-type) forms. We further show the applicability of this strategy for monitoring the action of Tyrosine Kinase Inhibitors over time. We believe that this non-invasive technique will open up new avenues for facilitating cancer diagnosis and time-point monitoring of therapeutic responses.

Interfacial nano-mixing in a miniaturised platform enables signal enhancement and in situ detection of cancer biomarkers.

Interfacial biosensing performs the detection of biomolecules at the bare-metal interface for disease diagnosis by comparing how biological species derived from patients and healthy individuals interact with bare metal surfaces. This technique retrieves clinicopathological information without complex surface functionalisation which is a major limitation of conventional techniques. However, it is still challenging to detect subtle molecular changes by interfacial biosensing, and the detection often requires prolonged sensing times due to the slow diffusion process of the biomolecules towards the sensor surface. Herein, we report on a novel strategy for interfacial biosensing which involves in situ electrochemical detection under the action of an electric field-induced nanoscopic flow at nanometre distance to the sensing surface. This nanomixing significantly increases target adsorption, reduces sensing time, and enables the detection of small molecular changes with enhanced sensitivity. Using a multiplex electrochemical microdevice that enables nanomixing and in situ label-free electrochemical detection, we demonstrate the detection of multiple cancer biomarkers on the same device. We present data for the detection of aberrant phosphorylation in the EGFR protein and hypermethylation in the EN1 gene region. Our method significantly shortens the assay period (from 40 min and 20 min to 3 minutes for protein and DNA, respectively), increases the sensitivity by up to two orders of magnitude, and improves detection specificity.