Biosensors based on nucleic acid interaction

DNA sensing is an emerging technology based on hybridisation reaction between an immobilised DNA probe and a molecular target, consisting of a probe complementary sequence in solution. Many application have been developed in the field of environmental, food and clinical analysis.Surface plasmon resonance and piezoelectric sensing are reported as transduction principles for DNA-based devices. These techniques are able to monitor in real-time and without the use of any label the hybridisation reaction between nucleic acids. Particular attention is given to Genetically Modified Organism detection

Surface plasmon resonance imaging for affinity-based biosensors

SPR imaging (SPRi) is at the forefront of optical label-free and real-time detection. It offers the possibility of monitoring hundreds of biological interactions simultaneously and from the binding profiles, allows the estimation of the kinetic parameters of the interactions between the immobilised probes and the ligands in solution. We review the current state of development of SPRi technology and its application including commercially available SPRi instruments. Attention is also given to surface chemistries for biochip functionalisation and suitable approaches to improve sensitivity

Cardiac Troponin T capture and detection in real-time via epitope-imprinted polymer and optical biosensing

Millions of premature deaths per year from cardiovascular diseases represent a global threat urging governments to increase global initiatives, as advised by World Health Organization. In particular, together with prevention and management of risk factors, the development of portable platforms for early diagnosis of cardiovascular disorders appears a fundamental task to carry out. Contemporary assays demonstrated very good accuracy for diagnosis of acute myocardial infarction (AMI), but they are based on expensive and fragile capture antibodies. Accordingly, also considering the massive demand from developing countries, we have devoted our study to an affinity-based biosensor for detection of troponin T (TnT), a preferred biomarker of AMI. This combines a stable and inexpensive molecularly imprinted polymer (MIP) based on polydopamine (PDA) with surface plasmon resonance (SPR) transduction. Herein we report the fast and specific answer upon TnT binding onto an epitope- imprinted surface that strongly encourages the further development toward antibody-free point-of-care testing for cardiac injury

Bio)Sensor Approach in the Evaluation of Polyphenols in Vegetal Matrices

Polyphenols are compounds widely distributed in the plant kingdom and have attracted much attention, because of their health benefits and important properties such as radical scavenging, metal chelating agents, inhibitors of lipoprotein oxidation, anti-inflammatory and anti-allergic activities. Due to their important role in the diet and in therapy, it is important to estimate their content in the different matrices of interest. Besides classical analytical methods, new emerging technologies have also appeared in the last decade aiming for simple and eventually cheap detection of polyphenols. This review focused on the recent applications of biosensing-based technologies for polyphenol estimation in vegetal matrices, using different transduction principles. These analytical tools are generally fast, giving responses in the order of a few seconds/minutes, and also very sensitive and generally selective (mainly depending on the enzyme used). Direct measurements in most of the investigated matrices were possible, both in aqueous and organic phases

Tunable growth of gold nanostructures at a PDMS surface to obtain plasmon rulers with enhanced optical features

Innovative optical devices for (bio)sensing based on Localized Surface Plasmon Resonance (LSPR) transduction allow to exploit the sensitivity of traditional SPR on miniaturized spectrometers suitable for point-of-care testing. In fact, LSPR reading can be realized by transmittance through interrogation with white light. In this framework, the effective coupling of plasmonic nanomaterials to optically transparent polymers is of great interest for having cheap, versatile, and sensitive optical devices of new concept. Some effort in fabricating gold and silver spherical nanoparticles through their in situ synthesis on PDMS has been reported, displaying moderate bulk sensitivity up to 70 nm/RIU. Herein we report that simple modifications to the general preparation of these composites (AuNPs@PDMS) can result in several improvements of the optical features. In particular, a two-steps growth of AuNPs@PDMS including PDMS surface chemical treatment before the second growth results in the formation of well-exposed and densely-packed 3D conglomerates of spheroids with enhanced bulk sensitivity and plasmon rulers character. Differently from available data, the obtained structures achieve sensitivity to refractive index change of about 250 nm/RIU, 3.5 higher than spherical nanoparticles with similar protocols and near the optical performances of anisotropic NPs. Due to the strong 3D character of the obtained structures, plasmon coupling effects are realized and investigated at PDMS surface. In particular, we show that these nanocomposite substrates subjected to biological stimuli exhibit red/blue-shifts typical of induced plasmon coupling/uncoupling, providing a step forward in composite plasmonic nanomaterials for application to (bio)sensing

Colorimetric determination of p-nitrophenol by using ELISA microwells modified with an adhesive polydopamine nanofilm containing catalytically active gold nanoparticles

A microplate method is described for the quantification of p-nitrophenol (p-NPh) in urine samples where it can be found after exposure to certain insecticides such as methyl parathion or paraoxon. The assay is based on the use of a polydopamine (PDA) film doped with gold nanoparticles (AuNPs). The latter exerts a catalytic effect on the reduction of nitrophenols by NaBH4. PDA has adhesive properties and can be used to fix the AuNPs on several solid substrates, here ELISA polystyrene microwells. The optical and catalytic properties of different populations of AuNPs spontaneously grown on PDA films were investigated, mainly in terms of the relationship between AuNPs@PDA nanocomposite preparation and its catalytic activity and stability. The reduction of o-, m-, and p-nitrophenols by NaBH4 in aqueous solution was exploited as model study. The approach demonstrates that useful kinetic information on the catalytic effect can be obtained on 96-wells simultaneously by a conventional ELISA reader at a fixed wavelength of 415 nm. The method was successfully applied to the quantification of p-NPh in (spiked) urine samples and gave high reproducibility (RSD = 3.5%) and a 6.30 μM (836 μg/L) detection limit. [Figure not available: see fulltext.]. © 2019, Springer-Verlag GmbH Austria, part of Springer Nature

Real-time tau protein detection by sandwich-based piezoelectric biosensing: Exploring tubulin as a mass enhancer

Human tau protein is one of the most advanced and accepted biomarkers for AD and tauopathies diagnosis in general. In this work, a quartz crystal balance (QCM) immunosensor was developed for the detection of human tau protein in buffer and artificial cerebrospinal fluid (aCSF), through both direct and sandwich assays. Starting from a conventional immuno-based sandwich strategy, two monoclonal antibodies recognizing different epitopes of tau protein were used, achieving a detection limit for the direct assay in nanomolar range both in HBES-EP and aCSF. Afterward, for exploring alternative specific receptors as secondary recognition elements for tau protein biosensing, we tested tubulin and compared its behavior to a conventional secondary antibody in the sandwich assay. Tau–tubulin binding has shown an extended working range coupled to a signal improvement in comparison with the conventional secondary antibody-based approach, showing a dose–response trend at lower tau concentration than is usually investigated and closer to the physiological levels in the reference matrix for protein tau biomarker. Our results open up new and encouraging perspectives for the use of tubulin as an alternative receptor for tau protein with interesting features due to the possibility of taking advantage of its polymerization and reversible binding to this key hallmark of Alzheimer’s disease

Structurally Constrained MUC1-Tn Mimetic Antigen as Template for Molecularly Imprinted Polymers (MIPs): A Promising Tool for Cancer Diagnostics

Abnormal glycoconjugates have distinctly been recognized as potential biomarkers for cancer diagnosis. A great deal of attention has been focused on Tn antigen, an oversimplified mucin-1 O-glycan, over-expressed in different cancers. Herein, we investigate the possibility to replace the use of anti-Tn monoclonal antibodies with an innovative class of catecholamine-based Molecularly Imprinted Polymers (MIPs), emerging in recent years as promising tools for bioanalytical applications. MIPs are synthetic receptors characterized by high sensitivity and specificity towards the imprinted target. Here, original polynorepinephrine-based MIPs coupled to Surface Plasmon Resonance biosensing for Tn antigen recognition are reported. We have verified the imprinting and binding capacity of these MIPs towards very small antigenic entities, represented by the natural Tn antigen and the TnThr mimetic 1 (conjugated to BSA or linked to a MUC1 hexapeptide analogue), and compared the biosensor performances with an anti-Tn monoclonal antibody. The results clearly display the effectiveness of the pursued imprinting strategies

DEVELOPMENT OF AN ELECTROCHEMICAL SENSOR BASED ON SCREEN-PRINTED ELECTRODES FOR OCHRATOXIN A IN PORK MEAT SAMPLES

Ochratoxin A (OTA) is a nephrotoxic, immunosuppressive and teratogenic mycotoxin produced by As- pergillus and Penicillium spp. fungi during food storage. OTA can be detected in cereal products, coffee, wine, beer, cheese and in poultry and pork meat. Many detection techniques, such as liquid chromatography coupled with immunoaffinity column or solid phase extraction cleanup, have been used for OTA determina- tion in different samples (1). In recent years electrochemical techniques have been used for the rapid and accurate detection of OTA (2). The aim of the present study was to develop a new analytical method for OTA quantitative detection in pork meat based on electrochemical sensing, using graphite-based screen-printed electrodes and differential pulse voltammetry (DPV) as detection technique. Experiments were performed with an electrochemical transducer Palmsens, monitored with a personal computer using PSTrace software (Palm Instrument BV, Houten, The Netherlands) for data acquisition and subsequent analysis. The electrochemical assays were performed with miniaturized disposable graphite based screen-printed electrodes (EcoBioServices & Researches s.r.l., Florence, Italy). The effect of pH (range 2-7) and of ionic strength (KCl concentration range 10-200 mM) of the supporting electrolyte solution (acetate buffer) on the DPV peak current and potentials was investigated to optimize the DPV method. The effect of the DPV parameters on OTA oxidation peak was studied. Potential pulse amplitude (Epulse) was evaluated in the range of 10-100 mV. Step height was evaluated in the range of 2-10 mV. The influence of the scan rate was examined in the range of 0.005-0.1V/s. Standard addition method was applied for quantitative analysis. The method was applied for OTA determination in spiked pork meat samples. Results were compared with those provided by a reference HPLC method. The OTA peak current increased with increasing acetate buffer pH (from 2.0 to 7), thus pH of 7.0 for the supporting electrolyte solution was chosen. Concentrations of 75 mM KCl in the supporting electrolyte was selected. The optimization of DPV parameters indicated that best results for voltammograms were obtained from 0 to 1.1 V by using 5 mV potential step, 50 mV potential pulse, 0.01 V/sec scan rate and 0.07 sec time pulse; each scan was performed after an equilibrium time of 30 sec. Calibration graphs of peak height against concentration for OTA by DPV were plotted over the range 25-1000 μg/l in the supporting electrolyte with a LOQ of 25 μg/l. The findings obtained with voltammetric-based sensing were in good agreement with results obtained by HPLC analysis but matrix effects have been detected at lower OTA concentrations indicating the need of more selective extraction procedure. The proposed method is more rapid and inexpensive in comparison with the classical methods for OTA analysis, and can be considered a promising alternative for the evaluation of OTA in meat. 1) Turner et al, Anal Chim Acta, 2009, 632 ,168-180. 2) Prieto-Simón et al, TrAC, 2007, 26, 689-702

A LysLysLys-tag as trigger in polynorepinephrine epitope imprinting: The case study of soluble PD-L1 detection in serum by optical-based sensing

Polycatecholamines (pCAs)-based molecularly imprinted polymers (MIPs) represent the new performing generation of biocompatible ligand/receptor mimetics. In this context, dealing with MIPs synthesis for bio-macromolecules detection/extraction, one of the critical steps in ensuring effective binding affinity for the parent molecule is the selection of suitable epitopes for pCAs imprinting. To address this challenge, here we investigated the ability of lysine (K) residues to trigger the epitope imprinting process into a polynorepinephrine (PNE) matrix. To this aim, we first designed a set of model epitopes composed of three K and six alanine (A) residues to investigate the influence of each 'KA' combination on the imprinting process and the resulting binding performance by Surface Plasmon Resonance (SPR). Only the case of three flanking K residues in N-terminus arose as an excellent trigger for epitope imprinting. The efficacy of the 3K-tag strategy was then evaluated on two peptide templates belonging to soluble programmed cell death protein 1 ligand (PD-L1), which is of great interest as a cancer biomarker in liquid biopsies. These templates were selected due to their negligible natural ability to be imprinted into the PNE matrix and were modified with 3K-tags, in N-, C-, and N/C- positions, respectively. The SPR sensor developed by exploiting the N-3K tag strategy allowed us to achieve excellent sensitivity (0.31 ± 0.04 ng mL-1) and repeatability (avCV% = 4.5) in human serum samples. This strategy opens new insights both for epitopes' design for pCAs-based mimetics and as triggering tags when native epitopes display negligible imprinting capabilities