Nanochannels for electrical biosensing

This review shows the recent trends on the use of both single and array nanochannels for electrical biosensing applications. Some general considerations on the principles of the stochastic sensing, together with an overview about the common routes for nanochannels preparation before focusing on the applications for DNA, protein, virus, toxin and other analytes detection are given. Emerging materials used to obtain nanochannels, such as graphene and its analogues as well as novel systems based on the use of nanoparticles in combination with nanochannels are discussed. Aspects related to the analytical performance of the developed devices are also discussed. Finally prospects for future improvements and applications of this technology are included

Electrochemical detection of plant virus using gold nanoparticle-modified electrodes

Tristeza is one of the destructive diseases of citrus causing by citrus tristeza virus (CTV). Historically, CTV has been associated with serious outbreaks of quick decline of citrus, therefore CTV monitoring is important aspect for avoiding such re-emerging epidemics, which would threat citrus production through the world. In this context, we have designed for the first time a label-free impedimetric biosensor for the detection of nucleic acid of CTV. The sensing platform based on a screen-printed carbon electrode (SPCE) was modified by electrodeposited gold nanoparticles (AuNPs), which allowed to efficiently immobilizing thiolated ssDNA probes as well to enhance the electrode conductivity. The growth of AuNPs was optimized and characterized using scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). We investigated the behavior of thiolated ssDNA probe layer and its hybridization with target DNA onto AuNP surfaces by EIS measurements in Fe(CN₆)⁴⁻)/Fe(CN₆)³⁻ red-ox system. The main sensor design aspects such as AuNPs size, probe DNA concentration and immobilization time together with DNA hybridization time were optimized so as to achieve the best performance. Impedance values of DNA hybridization increased with Citrus tristeza-related synthetic DNA concentration, showing a logarithmic relation in the range of 0.1-10 μM. The results also indicate that the biosensor was able to selectively detect CTV nucleic acids in the presence of other non-specific DNAs. Moreover, we have demonstrated the good performance of the system in a real plant sample matrix. In addition, the sensor reproducibility enhanced after the hybridization onto MCH/poly (AT) thiolated DNA probes which was confirmed by intra- and inter-day variability assays

Alzheimer's disease biomarkers detection in human samples by efficient capturing through porous magnetic microspheres and labelling with electrocatalytic gold nanoparticles

A nanobiosensor based on the use of porous magnetic microspheres (PMM) as efficient capturing/pre-concentrating platform is presented for detection of Alzheimer's disease (AD) biomarkers. These PMMs prepared by a multistep swelling polymerization combined with iron oxide precipitation afford carboxyl functional groups suitable for immobilization of antibodies on the particle surface allowing an enhanced efficiency in the capturing of AD biomarkers from human serum samples. The AD biomarkers signaling is produced by gold nanoparticle (AuNP) tags monitored through their electrocatalytic effect towards hydrogen evolution reaction (HER). Novel properties of PMMs in terms of high functionality and high active area available for enhanced catalytic activity of the captured AuNPs electrocatalytic tags are exploited for the first time. A thorough characterization by scanning transmission electron microscope in high angle annular dark field mode (STEM-HAADF) demonstrates the enhanced ability of PMMs to capture a higher quantity of analyte and consequently of electrocatalytic label, when compared with commercially available microspheres. The optimized and characterized PMMs are also applied for the first time for the detection of beta amyloid and ApoE at clinical relevant levels in cerebrospinal fluid (CSF), serum and plasma samples of patients suffering from AD

Organic-based field effect transistors for protein detection fabricated by inkjet-printing

Altres ajuts: CERCA Programme/Generalitat de Catalunya.Biosensors based on Organic Field-Effect Transistors (OFETs) have attracted increasing attention due to the possibility of rapid, label-free, and inexpensive detection. Among all the different possibilities, inkjet-printed top-gate organic Field Effect Transistors-Based Biosensors (BioFETs) using a polymeric gate insulator have been seldom reported. In this work, a systematic investigation in terms of topographical and electrical characterization was carried out in order to find the optimal fabrication process for obtaining a reliable polymer insulator. Previous studies have demonstrated that the best electrical performance arises from the use of the perfluoropolymer Cytop™[12,13,14]. Consequently, a simple immobilization protocol was used to ensure the proper attachment of a model biomolecule onto the Cytop's hydrophobic surface whilst keeping its remarkable insulating properties with gate current in the range of dozens of pico-amperes. The top-gate inkjet-printed BioFETs presented in this study operate at threshold voltages in the range of 1-2 V and show durability even when exposed to oxygen plasma, wet amine functionalization treatments, and aqueous media. As a preliminary application, the inkjet-printed top-gate BioFETs is used for monitoring an immunoreaction by measuring changes in the drain current, paving the way for further use of this device in the immunosensing field

In situ monitoring of PTHLH secretion in neuroblastoma cells cultured onto nanoporous membranes

In this work, we propose for the first time the use of anodic aluminum oxide (AAO) nanoporous membranes for in situ monitoring of parathyroid hormone-like hormone (PTHLH) secretion in cultured human cells. The biosensing system is based on the nanochannels blockage upon immunocomplex formation, which is electrically monitored through the voltammetric oxidation of Prussian blue nanoparticles (PBNPs). Models evaluated include a neuroblastoma cell line (SK-N-AS) and immortalized keratinocytes (HaCaT) as a control of high PTHLH production. The effect of total number of seeded cells and incubation time on the secreted PTHLH levels is assessed, finding that secreted PTHLH levels range from approximately 60 to 400 ng/mL. Moreover, our methodology is also applied to analyse PTHLH production following PTHLH gene knockdown upon transient cell transfection with a specific silencing RNA (siRNA). Given that inhibition of PTHLH secretion reduces cell proliferation, survival and invasiveness in a number of tumors, our system provides a powerful tool for the preclinical evaluation of therapies that regulate PTHLH production. This nanoporous membrane - based sensing technology might be useful to monitor the active secretion of other proteins as well, thus contributing to characterize their regulation and function

Control of Electron-transfer in Immunonanosensors by Using Polyclonal and Monoclonal Antibodies

The design and operation of biosensors is not trivial. For instance, variation in the output signal during monitoring of analytes can not usually be controlled. Hence, if such control were possible, and could be triggered on demand, it would greatly facilitate system design and operation. Herein, we report the design of two types of voltamperometric immunosensors, in which the magnitude of the current output signal (differential pulse voltammetry [DPV]) can be increased or decreased as needed. The designed systems use monoclonal and polyclonal anti-human IgG antibodies, conjugated to monopodal ferrocene-modified gold nanoparticles that are casted onto screen-printed carbon electrodes (Ab/mFcL/AuNPs/SPCEs). Upon addition of human IgG as antigen, the systems exhibit opposite responses according to the Ab: the current decreases when monoclonal Ab is used, whereas it increases when polyclonal Ab is used. We attributed the former response to inhibition of electron-transfer (due to the formation of a protein layer), and the latter response, to a global increase in electron transfer (induced by the aggregation of gold nanoparticles). These effects were confirmed by studying a custom-made lipoic acid-based bipodal ligand, which confirmed that the increase in current is effectively induced by the aggregation of the modified nanoparticles (pAb/mFcL/AuNPs). Both sensors have large dynamic ranges, although the pAb-based one was found to be 3.3-times more sensitive. Tests of selectivity and specificity for ovalbumin, α-lactalbumin and serum bovine albumin showed that the immunosensors are highly selective and specific, even in the presence of up to 1000-fold levels of potentially competitive proteins. The limit of detection for human IgG using the pAb/mFcL/AuNP bioconjugate was estimated to be 0.85 ng/mL. The pAb/mFcL/AuNPs-based biosensor has used to determine amounts of human IgG in real sample

Electrical monitoring of infection biomarkers in chronic wounds using nanochannels

Chronic wounds represent an important healthcare challenge in developed countries, being wound infection a serious complication with significant impact on patients’ life conditions. However, there is a lack of methods allowing an early diagnosis of infection and a right decision making for a correct treatment. In this context, we propose a novel methodology for the electrical monitoring of infection biomarkers in chronic wound exudates, using nanoporous alumina membranes. Lysozyme, an enzyme produced by the human immune system indicating wound infection, is selected as a model compound to prove the concept. Peptidoglycan, a component of the bacterial layer and the native substrate of lysozyme, is immobilized on the inner walls of the nanochannels, blocking them both sterically and electrostatically. The steric blocking is dependent on the pore size (20 - 100 nm) and the peptidoglycan concentration, whereas the electrostatic blocking depends on the pH. The proposed analytical method is based on the electrical monitoring of the steric/electrostatic nanochannels unblocking upon the specific degradation of peptidoglycan by lysozyme, allowing to detect the infection biomarker at 280 ng/mL levels, which are below those expected in wounds. The low protein adsorption rate and thus outstanding filtering properties of the nanoporous alumina membranes allowed us to discriminate wound exudates from patients with both sterile and infected ulcers without any sample pre-treatment usually indispensable in most diagnostic devices for analysis of physiological fluids. Although size and charge effects in nanochannels have been previously approached for biosensing purposes, as far as we know, the use of nanoporous membranes for monitoring enzymatic cleavage processes, leading to analytical systems for the specific detection of the enzymes has not been deeply explored so far. Compared with previously reported methods, our methodology presents the advantages of no need of neither bioreceptors (antibodies or aptamers) nor competitive assays, low matrix effects and quantitative and rapid analysis at the point-of-care, being also of potential application for the determination of other protease biomarkers.Peer ReviewedPostprint (published version

Iridium oxide (IV) nanoparticle-based lateral flow immunoassay

Lateral flow biosensors are paper-based devices that allow the detection of different types of analytes with quickness, robustness and selectivity, without leaving behind paper sensors benefits as low-cost, recyclability and sustainability. Nanomaterials have been widely reported in lateral flow biosensors, offering new sensing strategies based on optical or electrical detection techniques. Looking for other advantageous nanomaterials, we propose for the first time the use of iridium oxide (IV) nanoparticles in lateral flow assays for the detection of human immunoglobulin as a model protein. These nanoparticles can be easily prepared and conjugated with biomarkers. Their dark blue color gives a high contrast against the white background of the strips being in this way excellent labels

Fully printed one-step biosensing device using graphene/AuNPs composite

Driven by the growing need of simple, cost efficient and flexible sensing systems, we have designed here a fully printed Reduced Graphene Oxide (rGO) based impedimetric sensor for one step sensing of DNA. The DNA sensor was fabricated by stamping of layered rGO and rGO/gold nanoparticles/single stranded DNA (rGO/AuNPs/ssDNA) composites over PET substrates using wax-printing technique. rGO works as an excellent working electrode, while the AuNPs create a suitable environment for ssDNA immobilization. Counter and reference electrodes were previously screen-printed on the plastic substrate, making thus a compact and highly integrated sensing platform. The change in electron transfer resistance after hybridization with a target ssDNA specific of Coxsackie B3 virus was monitored using electrochemical impedance spectroscopy (EIS), finding a linear response in the range of concentrations 0.01-20 µM. The novel, simple and straightforward one-step printing process for fabrication of a biosensing device developed keeps in mind the growing need of large scale device manufacturing. The successful proof-of-concept for the detection of DNA hybridization can be extended to other affinity biosensors, taking advantage of the integration of the bioreceptor on the sensor surface. Such ready-to-use biosensor would lead to a one-step electrochemical detection

Signal enhancement on gold nanoparticle-based lateral flow tests using cellulose nanofibers

Lateral flow paper-based biosensors merge as powerful tools in point-of-care diagnostics since they are cheap, portable, robust, selective, fast and easy to use. However, the sensitivity of this type of biosensors is not always as high as required, often not permitting a clear quantification. To improve the colorimetric response of standard lateral flow strips (LFs), we have applied a new enhancement strategy that increases the sensitivity of LFs based on the use of cellulose nanofibers (CNF). CNF penetrate inside the pores of LFs nitrocellulose paper, compacting the pore size only in the test line, particularly near the surface of the strip. This modification retains the bioreceptors (antibodies) close to the surface of the strips, and thus further increasing the density of selectively attached gold nanoparticles (AuNPs) in the top part of the membrane, in the test line area, only when the sample is positive. This effect boosts in average a 36.6% the sensitivity of the LFs. The optical measurements of the LFs were carried out with a mobile phone camera whose imaging resolution was improved by attaching microscopic lens on the camera objective. The characterization of CNF into paper and their effect was analyzed using atomic force microscope (AFM) and scanning electron microscope (SEM) imaging techniques