Bioreceptors' immobilization by hydrogen bonding interactions and differential pulse voltammetry for completely label-free electrochemical biosensors.

Label-free electrochemical biosensors show great potential for the development of point-of-care devices (POCDs) for environmental and clinical applications. These sensors operate with shorter analysis times and are more economic than the labelled ones. Here, four completely label-free biosensors without electron transfer mediators were developed for hepatitis B virus (HBV) detection. The approach consisted in (i) the modification of gold surfaces with cysteamine (CT) or cysteine (CS) linkers, (ii) the subsequent antibody (Ab) immobilization, either directly by hydrogen bonding (HB) interactions or by covalent bonds (CB) using additional reagents, and (iii) measuring the biosensor response by electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). The electrode surfaces at each stage of the modification process were characterised by X-ray photo-electron spectroscopy (XPS) and atomic force microscopy (AFM). The combination of Ab immobilization by HB with the DPV analysis displayed improved repeatability, lower interference to serum matrix and similar limits of detection and quantification than the traditional biosensors that immobilize the Ab via CB and use EIS as readout technique. The Ab immobilization by HB is shown as a simple, efficient and low-cost alternative to CB ones, while DPV was faster and showed better performance than EIS. The CT-HB biosensor displayed the lowest limits of detection and quantification of 0.14 and 0.46 ng/mL, respectively, a 0.46-12.5 ng/mL linear analytical range, and 100% of recovery for 1/10 human serum media during HBV surface antigen detection by DPV. Even, it preserved the initial sensing capability after 7 days of its fabrication.

Flexible Nanoarchitectonics for Biosensing and Physiological Monitoring Applications.

Flexible and implantable electronics hold tremendous promises for advanced healthcare applications, especially for physiological neural recording and modulations. Key requirements in neural interfaces include miniature dimensions for spatial physiological mapping and low impedance for recognizing small biopotential signals. Herein, a bottom-up mesoporous formation technique and a top-down microlithography process are integrated to create flexible and low-impedance mesoporous gold (Au) electrodes for biosensing and bioimplant applications. The mesoporous architectures developed on a thin and soft polymeric substrate provide excellent mechanical flexibility and stable electrical characteristics capable of sustaining multiple bending cycles. The large surface areas formed within the mesoporous network allow for high current density transfer in standard electrolytes, highly suitable for biological sensing applications as demonstrated in glucose sensors with an excellent detection limit of 1.95 µm and high sensitivity of 6.1 mA cm-2  µM-1 , which is approximately six times higher than that of benchmarking flat/non-porous films. The low impedance of less than 1 kΩ at 1 kHz in the as-synthesized mesoporous electrodes, along with their mechanical flexibility and durability, offer peripheral nerve recording functionalities that are successfully demonstrated in vivo. These features highlight the new possibilities of our novel flexible nanoarchitectonics for neuronal recording and modulation applications.

All-Solid-State Interdigitated Micro-Supercapacitors Based on Porous Gold Electrodes.

Recent developments in embedded electronics require the development of micro sources of energy. In this paper, the fabrication of an on-chip interdigitated all-solid-state supercapacitor, using porous gold electrodes and a PVA/KOH quasisolid electrolyte, is demonstrated. The fabrication of the interdigitated porous gold electrode is performed using an original bottom-up approach. A templating method is used for porosity, using a wet chemistry process followed by microfabrication techniques. This paper reports the first example of an all-gold electrode micro-supercapacitor. The supercapacitor exhibits a specific capacitance equal to 0.28 mF·cm-2 and a specific energy of 0.14 mJ·cm-2. The capacitance value remains stable up to more than 8000 cycles.

Design of Pyrrole-Based Gate-Controlled Molecular Junctions Optimized for Single-Molecule Aflatoxin B1 Detection.

Food contamination by aflatoxins is an urgent global issue due to its high level of toxicity and the difficulties in limiting the diffusion. Unfortunately, current detection techniques, which mainly use biosensing, prevent the pervasive monitoring of aflatoxins throughout the agri-food chain. In this work, we investigate, through ab initio atomistic calculations, a pyrrole-based Molecular Field Effect Transistor (MolFET) as a single-molecule sensor for the amperometric detection of aflatoxins. In particular, we theoretically explain the gate-tuned current modulation from a chemical-physical perspective, and we support our insights through simulations. In addition, this work demonstrates that, for the case under consideration, the use of a suitable gate voltage permits a considerable enhancement in the sensor performance. The gating effect raises the current modulation due to aflatoxin from 100% to more than 103÷104%. In particular, the current is diminished by two orders of magnitude from the µA range to the nA range due to the presence of aflatoxin B1. Our work motivates future research efforts in miniaturized FET electrical detection for future pervasive electrical measurement of aflatoxins.

Adsorption of rhodamine 6G and choline on gold electrodes: a molecular dynamics study.

The adsorption of analyte molecules on nano-optoelectrodes (e.g. a combined nanoantenna and nanoelectrode device) significantly affects the signal characteristics in surface-enhanced Raman scattering (SERS) measurements. Understanding how different molecules adsorb on electrodes and their electrical potential modulation helps interpret SERS measurements better. We use molecular dynamics simulations to investigate the adsorption of prototypical analyte molecules (rhodamine 6G and choline) on gold electrodes with negative, neutral, and positive surface charges. We show that both molecules can readily adsorb on gold surfaces at all surface charge densities studied. Nevertheless, the configurations of the adsorbed molecules can differ for different surface charge densities, and adsorption can also change a molecule's conformation. Rhodamine 6G molecules adsorb more strongly than choline molecules, and the adsorption of both molecules is affected by electrode charge in 0.25 M NaCl solutions. The mechanisms of these observations are elucidated, and their implications for voltage-modulated SERS measurements are discussed.

Spotlight on the Effect of Electrolyte Composition on the Potential of Maximum Entropy: Supporting Electrolytes Are Not Always Inert.

The influence of electrolyte pH, the presence of alkali metal cations (Na+ , K+ ), and the presence of O2 on the interfacial water structure of polycrystalline gold electrodes has been experimentally studied in detail. The potential of maximum entropy (PME) was determined by the laser-induced current transient (LICT) technique. Our results demonstrate that increasing the electrolyte pH and introducing O2 shift the PME to more positive potentials. Interestingly, the PME exhibits a higher sensitivity to the pH change in the presence of K+ than Na+ . Altering the pH of the K2 SO4 solution from 4 to 6 can cause a drastic shift in the PME. These findings reveal that, for example, K2 SO4 and Na2 SO4 cannot be considered as equal supporting electrolytes: it is not a viable assumption. This can likely be extrapolated to other common "inert" supporting electrolytes. Beyond this, knowledge about the near-ideal electrolyte composition can be used to optimize electrochemical devices such as electrolyzers, fuel cells, batteries, and supercapacitors.

Electrochemical Immunosensor for the Quantification of S100B at Clinically Relevant Levels Using a Cysteamine Modified Surface.

Neuronal damage secondary to traumatic brain injury (TBI) is a rapidly evolving condition, which requires therapeutic decisions based on the timely identification of clinical deterioration. Changes in S100B biomarker levels are associated with TBI severity and patient outcome. The S100B quantification is often difficult since standard immunoassays are time-consuming, costly, and require extensive expertise. A zero-length cross-linking approach on a cysteamine self-assembled monolayer (SAM) was performed to immobilize anti-S100B monoclonal antibodies onto both planar (AuEs) and interdigitated (AuIDEs) gold electrodes via carbonyl-bond. Surface characterization was performed by atomic force microscopy (AFM) and specular-reflectance FTIR for each functionalization step. Biosensor response was studied using the change in charge-transfer resistance (Rct) from electrochemical impedance spectroscopy (EIS) in potassium ferrocyanide, with [S100B] ranging 10-1000 pg/mL. A single-frequency analysis for capacitances was also performed in AuIDEs. Full factorial designs were applied to assess biosensor sensitivity, specificity, and limit-of-detection (LOD). Higher Rct values were found with increased S100B concentration in both platforms. LODs were 18 pg/mL(AuES) and 6 pg/mL(AuIDEs). AuIDEs provide a simpler manufacturing protocol, with reduced fabrication time and possibly costs, simpler electrochemical response analysis, and could be used for single-frequency analysis for monitoring capacitance changes related to S100B levels.

pH-Activated Single Molecule Conductance and Binding Mechanism of Imidazole on Gold.

We identify imidazole as a pH-activated linker for forming stable single molecule-gold junctions with several distinct configurations and reproducible electrical characteristics. Using a scanning tunneling microscope break junction (STMBJ) technique, we find multiple robust conductance signatures at integer multiples of 1.9 × 10-2G0 and 1.2 × 10-4G0 and determine that this molecule bridges the electrodes in its deprotonated form through the nitrogen atoms in basic conditions only, with several molecules able to bind in parallel and in series. The elongation these junctions can sustain is longer than the length of the molecule, suggesting that plastic deformation of gold electrodes occurs during stretching. Density functional theory calculations confirm that the imidazolate-linked junctions exhibit bond strengths of ∼2 eV, which can allow for plastic extraction of gold atoms. On the basis of these results, we hypothesize that lower conductance peaks correspond to chains of repeating molecule-gold units that we form and measure in situ.

Fractal Gold Nanoframework for Highly Stretchable Transparent Strain-Insensitive Conductors.

Percolation networks of one-dimensional (1D) building blocks (e.g., metallic nanowires or carbon nanotubes) represent the mainstream strategy to fabricate stretchable conductors. One of the inherent limitations is the control over junction resistance between 1D building blocks in natural and strained states of conductors. Herein, we report highly stretchable transparent strain-insensitive conductors using fractal gold (F-Au) nanoframework based on a one-pot templateless wet chemistry synthesis method. The monolayered F-Au nanoframework (∼20 nm in thickness) can be obtained from the one-pot synthesis without any purification steps involved and can be transferred directly to arbitrary substrates like polyethylene terephthalate, food-wrap, polydimethylsiloxane (PDMS), and ecoflex. The F-Au thin film with no capping agents leads to a highly conductive thin film without any post-treatment and can be stretched up to 110% strain without significantly losing conductivity yet with the optical transparency of 70% at 550 nm. Remarkably, the F-Au thin film shows the strain-insensitive behavior up to 20% stretching strain. This originates from the unique fractal nanomesh-like structure which can absorb external mechanical forces, thus maintaining electron pathways throughout the nanoframework. In addition, a semitransparent bilayered F-Au film on 100% prestrained PDMS could achieve to a high stretchability of 420% strain with negligible resistance changes under low-level strains.

A Portable Setup for the Voltammetric Determination of Total Mercury in Fish with Solid and Nanostructured Gold Electrodes.

A simple procedure for field fish sample pretreatment was developed. This treatment in combination with square wave anodic stripping voltammetry (SW-ASV) with solid gold electrodes (SGE) and gold nanoparticle-modified glassy carbon electrodes (AuNPs-GCE) was applied for the determination of total mercury content. A certified reference material (CRM, Tuna Fish BCR 463), ten freeze-dried samples of canned tuna and two fresh fish samples were analysed both with a bench-top voltammetric analyser after microwave digestion and with a portable potentiostat after mild eating using a small commercial food warmer. The results obtained by the two SW-ASV approaches and by a Direct Mercury Analyser (DMA), the official method for mercury determination, were in very good agreement. In particular, (i) the results obtained with in field procedure are consistent with those obtained with the conventional microwave digestion; (ii) the presence of gold nanoparticles on the active electrode surface permits an improvement of the analytical performance in comparison to the SGE: the Limit of Quantification (LOQ) for mercury in fish-matrix was 0.1 µg L-1 (Hg cell concentration), corresponding to 0.06 mg kg-1 wet fish, which is a performance comparable to that of DMA. The pretreatment proposed in this study is very easy and applicable to fresh fish; in combination with a portable potentiostat, it proved to be an interesting procedure for on-site mercury determination.

Directional Preference of DNA-Mediated Electron Transfer in Gold-Tethered DNA Duplexes: Is DNA a Molecular Rectifier?

Electrical properties of self-assembling DNA nanostructures underlie the paradigm of nanoscale bioelectronics, and as such require clear understanding. DNA-mediated electron transfer (ET) from a gold electrode to DNA-bound Methylene Blue (MB) shows directional preference, and it is sequence-specific. During the electrocatalytic reduction of [Fe(CN)6 ]3- catalyzed by DNA-bound MB, the ET rate constant for DNA-mediated reduction of MB reaches (1.32±0.2)103 and (7.09±0.4)103  s-1 for (dGdC)20 and (dAdT)25 duplexes. The backward oxidation process is less efficient, making the DNA duplex a molecular rectifier. Lower rates of ET via (dGdC)20 agree well with its disturbed π-stacked sub-molecular structure. Such direction- and sequence-specific ET may be implicated in DNA oxidative damage and repair, and be relevant to other polarized surfaces, such as cell membranes and biomolecular interfaces.

Conductometric Sensor for PAH Detection with Molecularly Imprinted Polymer as Recognition Layer.

A conductometric sensor based on screen-printed interdigital gold electrodes on glass substrate coated with molecularly imprinted polyurethane layers was fabricated to detect polycyclic aromatic hydrocarbons (PAHs) in water. The results prove that screen-printed interdigital electrodes are very suitable transducers to fabricate low-cost sensor systems for measuring change in resistance of PAH-imprinted layers while exposing to different PAHs. The sensor showed good selectivity to its templated molecules and high sensitivity with a detection limit of 1.3 nmol/L e.g., for anthracene in water which is lower than WHO's permissible limit.

N-Heterocyclic-Carbene-Treated Gold Surfaces in Pentacene Organic Field-Effect Transistors: Improved Stability and Contact at the Interface.

N-Heterocyclic carbenes (NHCs), which react with the surface of Au electrodes, have been successfully applied in pentacene transistors. With the application of NHCs, the charge-carrier mobility of pentacene transistors increased by five times, while the contact resistance at the pentacene-Au interface was reduced by 85 %. Even after annealing the NHC-Au electrodes at 200 °C for 2 h before pentacene deposition, the charge-carrier mobility of the pentacene transistors did not decrease. The distinguished performance makes NHCs as excellent alternatives to thiols as metal modifiers for the application in organic field-effect transistors (OFETs).

Electrochemical Oxidation of l-selenomethionine and Se-methylseleno-l-cysteine at a Thiol-Compound-Modified Gold Electrode: Its Application in a Flow-Through Voltammetric Sensor.

A flow-electrolytic cell that consists of a bare gold wire or of different thiol-compound-modified gold electrodes (such as 2,4-thiazolidinedione, 2-mercapto-5-thiazoline, 2-mercaptothiazoline, l-cysteine, thioglycolic acid) was designed to be used in a voltammetric detector to identify l-selenomethionine and Se-methylseleno-l-cysteine using high-performance liquid chromatography. Both l-selenomethionine and Se-methylseleno-l-cysteine are more efficiently electrochemically oxidized on a thiol/gold than on a bare gold electrode. For the DC mode, and for measurements with suitable experimental parameters, a linear concentration from 10 to 1600 ng·mL-1 was found. The limits of quantification for l-selenomethionine and Se-methylseleno-l-cysteine were below 10 ng·mL-1. The method can be applied to the quantitative determination of l-selenomethionine and Se-methylseleno-l-cysteine in commercial selenium-containing supplement products. Findings using high-performance liquid chromatography with a flow-through voltammetric detector and ultraviolet detector are comparable.

Design, Synthesis, and Use of Peptides Derived from Human Papillomavirus L1 Protein for the Modification of Gold Electrode Surfaces by Self-Assembled Monolayers.

In order to obtain gold electrode surfaces modified with Human Papillomavirus L1 protein (HPV L1)-derived peptides, two sequences, SPINNTKPHEAR and YIK, were chosen. Both have been recognized by means of sera from patients infected with HPV. The molecules, Fc-Ahx-SPINNTKPHEAR, Ac-C-Ahx-(Fc)KSPINNTKPHEAR, Ac-C-Ahx-SPINNTKPHEAR(Fc)K, C-Ahx-SPINNTKPHEAR, and (YIK)2-Ahx-C, were designed, synthesized, and characterized. Our results suggest that peptides derived from the SPINNTKPHEAR sequence, containing ferrocene and cysteine residues, are not stable and not adequate for electrode surface modification. The surface of polycrystalline gold electrodes was modified with the peptides C-Ahx-SPINNTKPHEAR or (YIK)2-Ahx-C through self-assembly. The modified polycrystalline gold electrodes were characterized via infrared spectroscopy and electrochemical measurements. The thermodynamic parameters, surface coverage factor, and medium pH effect were determined for these surfaces. The results indicate that surface modification depends on the peptide sequence (length, amino acid composition, polyvalence, etc.). The influence of antipeptide antibodies on the voltammetric response of the modified electrode was evaluated by comparing results obtained with pre-immune and post-immune serum samples.

Methylene blue covalently attached to single stranded DNA as electroactive label for potential bioassays.

Methylene blue is an electroactive molecule that has been employed for the detection of the DNA hybridization event in electrochemical sensors. However, its use as a covalent label is very scarce and in most of the cases, non-covalent interactions (hydrophobic, electrostatic) are employed. Although it has advantages as simplicity and fewer number of procedure steps, the covalent attachment is less exploited in the development of these sensors. In this article, the electrochemical behavior of methylene blue attached to different DNA-strands is studied. Several lengths (15- and 30-mer) and different degree of DNA modification (MB-DNA, MB-DNA-MB and MB-DNA-SH) have been studied. The highest signals were obtained for longer strands with two MB molecules. In all the cases the signal is enhanced by CNT-nanostructuration of the electrode. Adsorption on these modified screen-printed electrodes allowed the amplification by employing an accumulation time. In this way, a sensitivity of -0.2864 µA µM-1 and a limit of detection of 800 nM for a 120 s accumulation time were obtained.

Evaluation of Transducer Elements Based on Different Material Configurations for Aptamer-Based Electrochemical Biosensors.

The selection of an appropriate transducer is a key element in biosensor development. Currently, a wide variety of substrates and working electrode materials utilizing different fabrication techniques are used in the field of biosensors. In the frame of this study, the following three specific material configurations with gold-finish layers were investigated regarding their efficacy to be used as electrochemical (EC) biosensors: (I) a silicone-based sensor substrate with a layer configuration of 50 nm SiO/50 nm SiN/100 nm Au/30-50 nm WTi/140 nm SiO/bulk Si); (II) polyethylene naphthalate (PEN) with a gold inkjet-printed layer; and (III) polyethylene terephthalate (PET) with a screen-printed gold layer. Electrodes were characterized using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) to evaluate their performance as electrochemical transducers in an aptamer-based biosensor for the detection of cardiac troponin I using the redox molecule hexacyanoferrade/hexacyaniferrade (K3[Fe (CN)6]/K4[Fe (CN)6]. Baseline signals were obtained from clean electrodes after a specific cleaning procedure and after functionalization with the thiolate cardiac troponin I aptamers "Tro4" and "Tro6". With the goal of improving the PEN-based and PET-based performance, sintered PEN-based samples and PET-based samples with a carbon or silver layer under the gold were studied. The effect of a high number of immobilized aptamers will be tested in further work using the PEN-based sample. In this study, the charge-transfer resistance (Rct), anodic peak height (Ipa), cathodic peak height (Ipc) and peak separation (∆E) were determined. The PEN-based electrodes demonstrated better biosensor properties such as lower initial Rct values, a greater change in Rct after the immobilization of the Tro4 aptamer on its surface, higher Ipc and Ipa values and lower ∆E, which correlated with a higher number of immobilized aptamers compared with the other two types of samples functionalized using the same procedure.

Cell-SELEX for aptamer discovery and its utilization in constructing electrochemical biosensor for rapid and highly sensitive detection of Legionella pneumophila serogroup 1.

This study introduces an innovative electrochemical aptasensor designed for the highly sensitive and rapid detection of Legionella pneumophila serogroup 1 (L. pneumophila SG1), a particularly virulent strain associated with Legionellosis. Employing a rigorous selection process utilizing cell-based systematic evolution of ligands by exponential enrichment (cell-SELEX), we identified new high-affinity aptamers specifically tailored for L. pneumophila SG1. The selection process encompassed ten rounds of cell-SELEX cycles with live L. pneumophila, including multiple counter-selection steps against the closely related Legionella sub-species. The dissociation constant (Kd) of the highest affinity sequence to L. pneumophila SG1 was measured at 14.2 nM, representing a ten-fold increase in affinity in comparison with the previously reported aptamers. For the development of electrochemical aptasensor, a gold electrode was modified with the selected aptamer through the formation of self-assembled monolayers (SAMs). The newly developed aptasensor exhibited exceptional sensitivity, and specificity in detecting and differentiating various Legionella sp., with a detection limit of 5 colony forming units (CFU)/mL and an insignificant/negligible cross-reactivity with closely related sub-species. Furthermore, the aptasensor effectively detected L. pneumophila SG1 in spiked water samples, demonstrating an appreciable recovery percentage. This study shows the potential of our aptamer-based electrochemical biosensor as a promising approach for detecting L. pneumophila SG1 in diverse environments.

Recombinase polymerase amplification in combination with electrochemical readout for sensitive and specific detection of PIK3CA point mutations.

As part of the ongoing evolution towards personalized anticancer therapy, mutation screening is becoming increasingly important and, therefore, also alternative detection strategies that allow for fast genetic diagnostics at the point of care. In the case of breast cancer, detecting cancer-associated point mutations in the PIK3CA gene is of particular importance for treatment decisions. We developed a recombinase polymerase amplification assay combined with an enzyme-linked electrochemical assay on multi-channel screen-printed gold sensors for specific and highly sensitive detection of three PIK3CA point mutations (H1047R, E545K, and E542K). Recombinase polymerase amplification (RPA) of the target sequences was optimized and characterized with a real-time RPA assay. Comparison with real-time PCR reveals that RPA is slightly inferior in terms of efficiency and sensitivity. However, the desired target DNA is successfully amplified at initial concentrations down to 100 copies µL-1. For electrochemical readout, biotinylated dCTP is used to label the target DNA during RPA. Single-stranded target DNA is produced with either asymmetric RPA or symmetric RPA followed by lambda exonuclease digestion. Characterization of the two different approaches in terms of sensitivity results in comparable detection limits (229 copies µL-1 and 224 copies µL-1, respectively), though RPA followed by lambda exonuclease digestion yields significantly higher currents. Finally, this method, together with a designed wild-type blocking oligo that inhibits binding of the wild-type target DNA during probe-target hybridization, allows for detecting the PIK3CA point mutations H1047R, E545K, and E542K in the presence of wild-type target DNA when the proportion of mutant target DNA is >20%.

Application of egg yolk IgY on carboxylated polypyrrole films for impedimetric detection of PfHRP2 antigen.

This paper described an impedimetric immunosensor for detecting Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Antibodies from egg yolk (Ab-PfHRP2, IgY type) were linked covalently to the screen-printed gold electrodes (SPGE) surface modified with a thin film of Poly-pyrrole-pyrrole 3 carboxylic acid (P(Py-Py3COOH) to develop the sensing platform. The fabrication steps were followed by microscopic (scanning electron microscopy), spectroscopic (RAMAN spectroscopy and Energy-dispersive X-ray spectroscopy), and electrochemical (electrochemical impedance spectroscopy (EIS) and cyclic voltammetry) techniques. The determination of Ag-PfHRP2 was performed by EIS, and the BSA(bovine serum albumin)/Ab-PfHRP2(IgY)/P(Py-Py3COOH)/SPGE immunosensor recorded a linear response at 100-1000 ng mL-1 concentration range, with a limit of detection (LOD) of 27.47 ng mL-1. Its performance was confirmed by Enzyme-Linked Immuno-Sorbent Assay. The fabricated device uses a simple strategy of IgY immobilization, showing high sensitivity and good selectivity, and can be considered an alternative for carrying out malaria tests.