1,721,017 research outputs found
Miniaturized Aptamer-Based Assays for Protein Detection
No full textThe 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
Atomic Force Microscopy and Spectroscopic Ellipsometry combined analysis of Small Ubiquitin-like Modifier adsorption on functional monolayers
No full textThe comprehension of mechanisms of interaction between functional layers and proteins is relevant for the development of sensitive and precise biosensors. Here we report our study which combines Atomic Force Microscopy and Spectroscopic Ellipsometry to investigate the His-Ni-NTA mediated interaction between 6His-tagged Small Ubiquitin-like Modifier (SUMO) protein with self assembled monolayers of NTA terminated alkanethiols. The use of AFM-based nanolithograhic tools and the analysis of ellipsometric spectra in situ and ex situ provided us a solid method to disentangle the effects of Ni(II)-mediated interaction between the NTA layer and the 6His-tagged SUMO and to accurately determine in physiological condition the thickness value of the SUMO layer. This investigation is a first step towards the study of layered systems of greater complexity of which the NTA/6His-tagged SUMO is a prototypical example
Functionalizing gold with single strand DNA: novel insight into optical properties via combined spectroscopic ellipsometry and nanolithography measurements
No full textWe have studied the self-assembly of 22-base oligonucleotides bound by a short alkyl thiol linker (C 6 -ssDNA) on flat Au films. The self-assembled monolayer (SAM) was modified by addition of a spacer (mercaptohexanol, MCH). Molecular depositions were monitored in situ by spectroscopic ellipsometry (SE). SAMs were characterized in a liquid environment by coupling SE (difference spectra method) with Atomic Force Microscope (AFM) measurements. We exploited the biofilm thickness obtained by AFM nanolithography and imaging to solve the refractive index/thickness correlation in optical measurements on ultrathin molecular layers. The combined SE/AFM analysis provided reliable estimates of the thickness and the refractive index of the biofilm in the NIR region (650-1300 nm) and revealed new aspects of DNA molecular organization: exposure to MCH leads to an increase of both film thickness and refractive index, which points to a reorganization of C 6 -ssDNA film. We show that the contribution of the thiol/Au interface has to be included in the optical model to obtain a more reliable determination of the refractive index of the biofilm in a liquid. The careful, correlative characterization of the mixed C 6 -ssDNA/MCH SAM represents a key step towards the optimization of a robust detection scheme based on helix-helix hybridization
Spectroscopic ellipsometry meets AFM nanolithography: About hydration of bio-inert oligo(ethylene glycol)-terminated self assembled monolayers on gold
No full textEasy fabrication of aligned PLLA nanofibers-based 2D scaffolds suitable for cell contact guidance studies
No full textAn easy, low-cost and fast wet processing-based method named ASB-SANS (Auxiliary Solvent-Based Sublimation-Aided NanoStructuring) has been used to fabricate poly(L-lactic acid) (PLLA) highly ordered and hierarchically organized 2D fibrillar patterns,with fiber widths between 40 and 500 nmand lengths exceeding tens of microns. A clear contact guidance effect of these nanofibrillar scaffolds with respect to HeLa and NIH-3T3 cells growth has been observed, on top of an overall good viability. For NIH-3T3 pronounced elongation of the cells was observed, as well as a remarkable ability of the patterns to guide the extension of pseudopodia. Moreover, SEM imaging revealed filopodia stemming from both sides of the pseudopodia and aligned with the secondary PLLA nanofibrous structures created by the ASB-SANS procedure. These results validate ASB-SANS as a technique capable to provide biocompatible 2D nanofibrillar patterns suitable for studying phenomena of contact guidance (and,more in general, the behavior of cells onto nanofibrous scaffolds), at very lowcosts and in an extremely easy way, accessible to virtually any laboratory
Mismatch detection in DNA monolayers by atomic force microscopy and electrochemical impedance spectroscopy
No full textThe 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 (μL range) assay conditions
Investigating organic multilayers by spectroscopic ellipsometry: specific and non-specific interactions of polyhistidine with NTA self-assembled monolayers
No full textBackground: A versatile strategy for protein–surface coupling in biochips exploits the affinity for polyhistidine of the nitrilotriacetic acid (NTA) group loaded with Ni(II). Methods based on optical reflectivity measurements such as spectroscopic ellipsometry (SE) allow for label-free, non-invasive monitoring of molecule adsorption/desorption at surfaces.Results: This paper describes a SE study about the interaction of hexahistidine (His6) on gold substrates functionalized with a thiolate self-assembled monolayer bearing the NTA end group. By systematically applying the difference spectra method, which emphasizes the small changes of the ellipsometry spectral response upon the nanoscale thickening/thinning of the molecular film, we characterized different steps of the process such as the NTA-functionalization of Au, the adsorption of the His6 layer and its eventual displacement after reaction with competitive ligands. The films were investigated in liquid, and ex situ in ambient air. The SE investigation has been complemented by AFM measurements based on nanolithography methods (nanografting mode).Conclusion: Our approach to the SE data, exploiting the full spectroscopic potential of the method and basic optical models, was able to provide a picture of the variation of the film thickness along the process. The combination of δΔi+1,i(λ), δΨi+1,i(λ) (layer-addition mode) and δΔ†i',i+1(λ), δΨ†i',i+1(λ) (layer-removal mode) difference spectra allowed us to clearly disentangle the adsorption of His6 on the Ni-free NTA layer, due to non specific interactions, from the formation of a neatly thicker His6 film induced by the Ni(II)-loading of the NTA SAM
Atomic Force Microscopy Application for the Measurement of Infliximab Concentration in Healthy Donors and Pediatric Patients with Inflammatory Bowel Disease
Full text linkThe 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 infliximab 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
Conformational properties of variable density DNA nanobrushes
Full text link2013/2014Advanced nanotechnologies allow the manipulation of molecules with nanoscale precision, and can be used for the production of sensitive devices for protein or nucleic acids detection for clinical use. DNA nano-assemblies are an excellent route for ultrasensitive DNA/RNA detection and for DNA-protein conjugated immobilization, for bio interaction studies, through the careful detection of single strand DNA (ssDNA) hybridization with complementary target sequences. For DNA nanoscale devices, the control of DNA surface density and conformation is crucial in order to achieve the highest reproducibility and to optimize the sensitivity. An improved understanding of the chemical and physical properties of the nanoscale DNA assemblies and of the recognition process is necessary for device performance optimization.
In this framework, we first focused on the understanding of the mechanisms that optimize and limit hybridization efficiency in variable density DNA monolayers. We performed Atomic Force Microscopy (AFM) assisted-Nanografting and AFM measurements to realize reference patches into a DNA self-assembled monolayer, and to carefully monitoring DNA hybridization. We then performed molecular dynamics (MD) simulations, in collaboration with a theoretical group, to capture the energetic hybridization limit in high dense DNA monolayers. We found that no more than 44% of the substrate ssDNA can be successfully hybridized, limited by molecular and electrostatic crowding effect connected to the highly charged nature of DNA.
To further capture the conformational properties of DNA monolayers, and their relation to biorecognition, we characterized the ionic strength effect on ssDNA nano-assembled of different density by careful AFM topography measurements in liquid environment. We confined ssDNA brushes with controlled surface densities within a bio-repellent self-assembled monolayer. We then monitored the topographic brush height variation upon changing salt type (NaCl, KCl, CaCl2 and MgCl2 ) and concentration inside the liquid cell. We showed that the measured height is related to scaling law of salt concentration, in agreement with the theory of polyelectrolyte brush. Using this scaling model to fit our experimental data, we quantified structural parameters such as the average internucleotide distance (d) for ssDNA brushes of different, estimated surface density σ, featuring a strong dependence of d on different salts species. This result is crucial for the structural designing of synthetic nucleic acids and, more generally, nucleic acid-based devices with controlled physical behaviors. In the last part of the work, we apply all knowledge learned on hybridization mechanism to a clinical problem. We studied the hybridization mechanism to distinguish single base mismatch and to detect at high sensitivity, without any labeling and amplification, microRNAs (miRNAs) connected to hearth failure disease. Our results demonstrate that the AFM nanolithography can serve as a sensitive and selective readout system to discriminate single nucleotide polymorphism. Also, our device allows for the detection of more than one sequence of miRNAs on a same assay with target in picomolar (100pM) range concentration.I recenti sviluppi delle nanotecnologie permettono di manipolare singole molecole con precisione nanometrica, e possono essere utilizzati per la produzione di dispositivi innovativi ad alta sensitivita` per la rivelazione di proteine e acidi nucleici, per usi clinici. Nanostrutture di DNA a singolo filamento rappresentano una eccellente soluzione per la rivelazione ultrasensibile di frammenti di DNA/RNA e per l’immobilizzazione di coniugati DNA-proteina per studi di bioriconoscimento, attaverso lo studio dell’ibridazione del DNA con le sequenze target complementari. Nello sviluppo di dipositivi alle nanoscale basati sul DNA, il controllo di parametri quali la densita` di superficie e la conformazione del DNA, risulta cruciale per raggiungere gli alti livelli di riproducibilita` richiesti e per ottimizzare la sensitivita`. Studiare e capire in dettaglio le proprieta` chimico -fisiche di strutture alle nanoscale di DNA a singolo filamento, e del relativo processo di bioriconoscimento risulta quindi fondamentale per ottimizzare le prestazioni del dispositivo associato.
In questo contesto, ci siamo dapprima focalizzati sullo studio dei meccanismi che ottimizzano e limitano l’efficienza di ibridazione in monolayer di DNA. Usando il microscopio a forza atomica (AFM) e una tecnica di nanolitografia basata sull’AFM, il nanografting, abbiamo costruito delle nanostrutture di riferimento in film di DNA autoassemblati, ad alta densita`, ed abbiamo accuratamente monitorato con l’AFM e con simulazioni di dinamica molecolare, il limite di ibridazione in tali film. In collaborazione con un gruppo di fisici teorici, abbiamo trovato un limite di ibridazione pari a circa il 44% delle sequenze probe, collegandolo a effetti di repulsione elettrostatica dovuta all’ alta densita` a di carica nei monolayer di DNA, un polielettrolita altamente carico in soluzione.
In un secondo tempo, per cogliere le proprieta` conformazionali dei monolayer di DNA, e la loro relazione con la capacita` di bioriconoscimento, abbiamo creato delle nanostrutture di DNA a singolo filamento, a densit variabile, in un monostrato autoassemblato di molecole bio-repellenti, e caratterizzato l’effetto della forza ionica della soluzione a mezzo di misure topografiche fatte con l’ AFM, in liquido. Da misure di variazione dell’ altezza topografica delle nanostrutture di DNA in funzione dei diversi sali usati in soluzione (NaCl, KCl, CaCl2 and MgCl2 ) e della loro concentrazione, abbiamo dimostrato che, per ogni sale, l’ altezza` legata alla concentrazione da una legge di scala, in accordo con la teoria dei polyelectrolyte brush. Utilizzando questa legge di scala, abbiamo fatto un fit dei dati sperimentali, quantificando un importante parametro strutturale, la distanza media tra nucleotidi nel filamento (d), per nanostrutture di DNA con divesra densita`, anch’essa stimata dal nostro fit. Questo risultato e` fondamentale per il disegno di acidi nucleici sintetici e piu` in generale per la progettazione di dispositivi miniaturizzati per la rivelazione di acidi nucleici.
Nella parte finale di questo lavoro di tesi, abbiamo applicato le conoscenze acquisite sui meccanismi di ibridazione del DNA su scale nanometriche, per realizzare dispositivi utili a scopi clinici. Abbiamo studiato il meccanismo di ibridazione per distinguere un mismatch tra due filamenti complementari di DNA relativo a una singola base e alla rivelazione di micro-RNA, biomarcatori rilevanti per monitorare specifiche malattie quali, nel presente caso, malattie cardiovascolari. Abbiamo dimostrato che i nostri nanodispositivi dimostrano un’ottima risoluzione (100 pM o meglio) e che possono essere utilizzati
senza bisogno di amplificazione del materiale genetico originale, o di altre modificazioni, in estratti provenienti da plasmi umani. Queste piattaforme possono essere ulteriormente sviluppate per il monitoraggio di polimorfismi di singolo nucleotide, estremamente rilevanti dal punto di vista clinicoXXVII Ciclo198
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