Nano-immuno assay development for the detection of cancer biomarkers

The quantitative analysis of protein markers is a promising strategy in diagnosis, prognosis and therapy monitoring of malignant tumors. The optimization of the clinical detection of these markers is directed towards the use of a small volume system. We developed a multiplexing nano-immuno array for proteomic analysis in low sample volumes with potential capability of pathological screening of cancer biomarkers. Nanografting, a tip assisted Atomic Force Microscopy (AFM) nanolithography technique is used to fabricate DNA nanoarrays. DNA nanospots created by nanografting are exploited in order to immobilize DNA-antibody conjugates that recognize specific proteins of interest. The determination of the antigen content of a biological sample was obtained from the analysis of AFM topographic profiles of the nanopatches before and after the incubation. As a proof of principle, we focused on the biomarker Human Epidermal Growth Factor Receptor 2 (Her2), relevant antigen found in some human cancers such as breast, lung and gastric ones. By measuring spot height variation we performed the detection of low concentrations of the biomarker and we were able to optimize the device sensitivity by correlating the density of the DNA-antibody conjugates on the surface and their capability to bind the Extra Cellular Domain (ECD) of Her2. As future perspectives we are also exploring the use of new binders, as aptamers and nanobodies (VHH), as new tool for the recognition of specific biomarkers with higher affinity

Miniaturized Aptamer-Based Assays for Protein Detection

The availability of devices for cancer biomarker detection at early stages of the disease is one of the most critical issues in biomedicine. Towards this goal, to increase the assay sensitivity, device miniaturization strategies empowered by the employment of high affinity protein binders constitute a valuable approach. In this work we propose two different surface-based miniaturized platforms for biomarker detection in body fluids: the first platform is an atomic force microscopy (AFM)-based nanoarray, where AFM is used to generate functional nanoscale areas and to detect biorecognition through careful topographic measurements; the second platform consists of a miniaturized electrochemical cell to detect biomarkers through electrochemical impedance spectroscopy (EIS) analysis. Both devices rely on robust and highly-specific protein binders as aptamers, and were tested for thrombin detection. An active layer of DNA-aptamer conjugates was immobilized via DNA directed immobilization on complementary single-stranded DNA self-assembled monolayers confined on a nano/micro area of a gold surface. Results obtained with these devices were compared with the output of surface plasmon resonance (SPR) assays used as reference. We succeeded in capturing antigens in concentrations as low as a few nM.We put forward ideas to push the sensitivity further to the pM range, assuring low biosample volume (L range) assay conditions

Two-dimensional enzyme diffusion in laterally confined DNA monolayers

Addressing the effects of confinement and crowding on biomolecular function may provide insight into molecular mechanisms within living organisms, and may promote the development of novel biotechnology tools. Here, using molecular manipulation methods, we investigate restriction enzyme reactions with double-stranded (ds)DNA oligomers confined in relatively large (and flat) brushy matrices of monolayer patches of controlled, variable density. We show that enzymes from the contacting solution cannot access the dsDNAs from the top-matrix interface, and instead enter at the matrix sides to diffuse two-dimensionally in the gap between top- and bottom-matrix interfaces. This is achieved by limiting lateral access with a barrier made of high-density molecules that arrest enzyme diffusion. We put forward, as a possible explanation, a simple and general model that relates these data to the steric hindrance in the matrix, and we briefly discuss the implications and applications of this strikingly new phenomenon

Atomic Force Microscopy Application for the Measurement of Infliximab Concentration in Healthy Donors and Pediatric Patients with Inflammatory Bowel Disease

The use of infliximab has completely changed the therapeutic landscape in inflammatory bowel disease. However, despite its proven efficacy to induce and maintain clinical remission, increasing evidence suggests that treatment failure may be associated with inadequate drug blood concentrations. The introduction of biosensors based on different nanostructured materials for the rapid quantification of drugs has been proposed for therapeutic drug monitoring. This study aimed to apply atomic force microscopy (AFM)-based nanoassay for the measurement of infliximab concentration in serum samples of healthy donors and pediatric IBD patients. This assay measured the height signal variation of a nanostructured gold surface covered with a self-assembled monolayer of alkanethiols. Inside this monolayer, we embedded the DNA conjugated with a tumor necrosis factor able to recognize the drug. The system was initially fine-tuned by testing known infliximab concentrations (0, 20, 30, 40, and 50 nM) in buffer and then spiking the same concentrations of influximab into the sera of healthy donors, followed by testing pediatric IBD patients. A good correlation between height variation and drug concentration was found in the buffer in both healthy donors and pediatric IBD patients (p-value < 0.05), demonstrating the promising use of AFM nanoassay in TDM

Lipid bilayer fluidity and degree of order regulates small EVs adsorption on model cell membrane

Small extracellular vesicles (sEVs) are known to play an important role in the communication between distant cells and to deliver biological information throughout the body. To date, many studies have focused on the role of sEVs characteristics such as cell origin, surface composition, and molecular cargo on the resulting uptake by the recipient cell. Yet, a full understanding of the sEV fusion process with recipient cells and in particular the role of cell membrane physical properties on the uptake are still lacking. Here we explore this problem using sEVs from a cellular model of triple-negative breast cancer fusing to a range of synthetic planar lipid bilayers both with and without cholesterol, and designed to mimic the formation of ‘raft’-like nanodomains in cell membranes. Using time-resolved Atomic Force Microscopy we were able to track the sEVs interaction with the different model membranes, showing the process to be strongly dependent on the local membrane fluidity. The strongest interaction and fusion is observed over the less fluid regions, with sEVs even able to disrupt ordered domains at sufficiently high cholesterol concentration. Our findings suggest the biophysical characteristics of recipient cell membranes to be crucial for sEVs uptake regulation

spectroscopic ellipsometry meets afm nanolithography about hydration of bio inert oligo ethylene glycol terminated self assembled monolayers on gold

An accurate thickness determination provides insight into the complex vertical morphology of OEG-terminated SAMs

Quantification of Circulating Cancer Biomarkers via Sensitive Topographic Measurements on Single Binder Nanoarrays

Early detection of cancer plays a crucial role in disease prognosis. It requires the recognition and quantification of low amounts of specific molecular biomarkers, either free or transported inside nanovesicles, through the development of novel sensitive diagnostic technologies. In this context, we have developed a nanoarray platform for the noninvasive quantification of cancer biomarkers circulating in the bloodstream. The assay is based on molecular manipulation to create functional spots of surface-immobilized binders and differential topography measurements. It is label-free and requires just a single binder per antigen, and when it is implemented with fluorescence labeling/readout, it can be used for epitope mapping. As a benchmark, we focused on the plasma release of Her2 extracellular domain (ECD), a proposed biomarker for the progression of Her2-positive tumors and response to anticancer therapies. By employing robust, easily engineered camelid nanobodies as binders, we measured ECD-Her2 concentrations in the range of the actual clinical cutoff value for Her2-positive breast cancer. The specificity for Her2 detection was preserved when it was measured in parallel with other potential biomarkers, demonstrating a forthcoming implementation of this approach for multiplexing analysis. Prospectively, this nanorarray platform may be customized to allow for the detection of promising new classes of circulating biomarkers, such as exosomes and microvesicles

Subcellular elements responsive to the biomechanical activity of triple-negative breast cancer-derived small extracellular vesicles

Abstract Triple-negative breast cancer (TNBC) stands out for its aggressive, fast spread, and highly metastatic behavior and for being unresponsive to the classical hormonal therapy. It is considered a disease with a poor prognosis and limited treatment options. Among the mechanisms that contribute to TNBC spreading, attention has been recently paid to small extracellular vesicles (sEVs), nano-sized vesicles that by transferring bioactive molecules to recipient cells play a crucial role in the intercellular communication among cancer, healthy cells, and tumor microenvironment. In particular, TNBC-derived sEVs have been shown to alter proliferation, metastasis, drug resistance, and biomechanical properties of target cells. To shed light on the molecular mechanisms involved in sEVs mediation of cell biomechanics, we investigated the effects of sEVs on the main subcellular players, i.e., cell membrane, cytoskeleton, and nuclear chromatin organization. Our results unveiled that TNBC-derived sEVs are able to promote the formation and elongation of cellular protrusions, soften the cell body, and induce chromatin decondensation in recipient cells. In particular, our data suggest that chromatin decondensation is the main cause of the global cell softening. The present study added new details and unveiled a novel mechanism of activity of the TNBC-derived sEVs, providing information for the efficient translation of sEVs to cancer theranostics

Shedding Plasma Membrane Vesicles Induced by Graphene Oxide Nanoflakes in Brain Cultured Astrocytes

Microvesicles (MVs) generated and released by astrocytes, the brain prevalent cells, crucially contribute to intercellular communication, representing key vectorized systems able to spread and actively transfer signaling molecules from astrocytes to neurons, ultimately modulating target cell functions. The increasing clinical relevance of these signaling systems requires a deeper understanding of MV features, currently limited by both their nanoscale dimensions and the low rate of their constituent release. Hence, to investigate the features of such glial signals, nanotechnology-based approaches and the applications of unconventional, cost-effective tools in generating MVs are needed. Here, small graphene oxide (s-GO) nanoflakes are used to boost MVs shedding from astrocytes in cultures and s-GO generated MVs are compared with those generated by a natural stimulant, namely ATP, by atomic force microscopy, light scattering, attenuated total reflection\u2013fourier transform infra-red and ultraviolet resonance Raman spectroscopy. We also report the ability of both types of MVs, upon acute and transient exposure of patch clamped cultured neurons, to modulate basal synaptic transmission, inducing a stable increase in synaptic activity accompanied by changes in neuronal plasma membrane elastic features

Graphene Oxide Nanosheets Reshape Synaptic Function in Cultured Brain Networks

Graphene offers promising advantages for biomedical applications. However, adoption of graphene technology in biomedicine also poses important challenges in terms of understanding cell responses, cellular uptake, or the intracellular fate of soluble graphene derivatives. In the biological microenvironment, graphene nanosheets might interact with exposed cellular and subcellular structures, resulting in unexpected regulation of sophisticated biological signaling. More broadly, biomedical devices based on the design of these 2D planar nanostructures for interventions in the central nervous system require an accurate understanding of their interactions with the neuronal milieu. Here, we describe the ability of graphene oxide nanosheets to down-regulate neuronal signaling without affecting cell viability