Table of Contents

Abstract: In this article, we report on the formation and mode-of-operation of an affinity biosensor, where alternate layers of biotin/streptavidin/biotinylated-CRP-antigen/anti-CRP antibody are grown on printed gold electrodes on disposable paper-substrates. We have successfully demonstrated and detected the formation of consecutive layers of supra-molecular protein assembly using an electrical (impedimetric) technique. The formation process is also supplemented and verified using conventional surface plasmon resonance (SPR) measurements and surface sensitive characterization techniques, such as X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). The article provides a possible biosensor development scheme, where—(1) fabrication of paper substrate (2) synthesis of gold nanoparticle inks (3) inkjet printing of gold electrodes on paper (4) formation of the biorecognition layers on the gold electrodes and (5) electrical (impedimetric) analysis of growth—all are coupled together to form a test-structure for a recyclable and inexpensive point-of-care diagnostic platform.

Abstract: Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed.

Abstract: Barrier tissue protects the body against external factors by restricting the passage of molecules. The gastrointestinal epithelium is an example of barrier tissue with the primary purpose of allowing the passage of ions and nutrients, while restricting the passage of pathogens and toxins. It is well known that the loss of barrier function can be instigated by a decrease in extracellular calcium levels, leading to changes in protein conformation and an increase in paracellular transport. In this study, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid (EGTA), a calcium chelator, was used to disrupt the gastrointestinal epithelial barrier. The effect of EGTA on barrier tissue was monitored by a novel label-free method based on an organic electrochemical transistor (OECT) integrated with living cells and validated against conventional methods for measuring barrier tissue integrity. We demonstrate that the OECT can detect breaches in barrier tissue upon exposure to EGTA with the same sensitivity as existing methods but with increased temporal resolution. Due to the potential of low cost processing techniques and the flexibility in design associated with organic electronics, the OECT has great potential for high-throughput, disposable sensing and diagnostics.

Abstract: Polyquinone derivatives are widely recognized in the literature for their remarkable properties, their biocompatibility, simple synthesis, and easy bio-functionalization. We have shown that polyquinones present very stable electroactivity in neutral aqueous medium within the cathodic potential domain avoiding side oxidation of interfering species. Besides, they can act as immobilized redox transducers for probing biomolecular interactions in sensors. Our group has been working on devices based on such modified electrodes with a view to applications for proteins, antibodies and organic pollutants using a reagentless label-free electrochemical immunosensor format. Herein, these developments are briefly reviewed and put into perspective.

Abstract: A nanoplasmonic biosensor for highly-sensitive, single-step detection of protein biomarkers is presented. The principle is based on the utilization of the optical scattering properties of gold nanorods (GNRs) conjugated to bio-recognition molecules. The nanoplasmonic properties of the GNRs were utilized to detect proteins using near-infrared light interferometry. We show that the antibody-conjugated GNRs can specifically bind to our model analyte, Glucose Transporter-1 (Glut-1). The signal intensity of back-scattered light from the GNRs bound after incubation, correlated well to the Glut-1 concentration as per the calibration curve. The detection range using this nanoplasmonic immunoassay ranges from 10 ng/mL to 1 ug/mL for Glut-1. The minimal detectable concentration based on the lowest discernable concentration from zero is 10 ng/mL. This nanoplasmonic immunoassay can act as a simple, selective, sensitive strategy for effective disease diagnosis. It offers advantages such as wide detection range, increased speed of analysis (due to fewer incubation/washing steps), and no label development as compared to traditional immunoassay techniques. Our future goal is to incorporate this detection strategy onto a microfluidic platform to be used as a point-of-care diagnostic tool.

Abstract: The starting point of modern biosensing was the application of actual biological species for recognition. Increasing understanding of the principles underlying such recognition (and biofunctionality in general), however, has triggered a dynamic field in chemistry and materials sciences that aims at joining the best of two worlds by combining concepts derived from nature with the processability of manmade materials, e.g., sensitivity and ruggedness. This review covers different biomimetic strategies leading to highly selective (bio)chemical sensors: the first section covers molecularly imprinted polymers (MIP) that attempt to generate a fully artificial, macromolecular mold of a species in order to detect it selectively. A different strategy comprises of devising polymer coatings to change the biocompatibility of surfaces that can also be used to immobilized natural receptors/ligands and thus stabilize them. Rationally speaking, this leads to self-assembled monolayers closely resembling cell membranes, sometimes also including bioreceptors. Finally, this review will highlight some approaches to generate artificial analogs of natural recognition materials and biomimetic approaches in nanotechnology. It mainly focuses on the literature published since 2005.

Abstract: We present an immunoassay for Interferon-γ (IFN-γ) with a limit of detection of 1.9 pM (30 pg/mL) and a linear concentration range spanning three orders of magnitude. The developed one-step assay takes only 12 min and can replace the time-consuming and labor-intensive enzyme-linked immunosorbent assay (ELISA). The solid-phase sandwich assay is performed on a new measurement system comprising single-use test tubes and a compact fluorescence reader. The polymer tubes contain an optical configuration for the detection of supercritical angle fluorescence, allowing for highly sensitive real-time binding measurements.

Abstract: The aim of the present editorial is to briefly summarize the current scientific and technological accomplishments in the field of organic electronic biosensors as described in the articles published in this Special Issue. By definition, a biosensor is a robust analytical device that combines a biological recognition element (e.g., antibodies, enzymes, cells) with a transducer. Organic electronic bio-devices are considered as potentially reliable substitutes of conventional and rather expensive analytical techniques employed for several applications such as medical diagnosis, food safety and environment pollution monitoring. Some insights into the selection and immobilization of recognition elements, signal amplification, fabrication techniques and analytical performance of biosensing devices will be presented.

Abstract: Dynamic fluoroimmunoassay with a flow-through system using optical fiber probes consisting of polystyrene was developed and applied to a quantitative detection of E. coli O157:H7. The system measures E. coli as fluorescence of sandwich-type immune complexes formed by capture antibodies immobilized on the surface of the probe, E. coli cells, and fluorescently labeled detection antibodies. Excitation was carried out using an evanescent wave from the probe. Resulting fluorescence recoupled into the probe was detected by a photodiode. The assay system was constructed with a flow cell which was available for sequential injection of experimental reagents. In vitro characterization was performed using the flow cell, and the calibration range of E. coli O157:H7 was from 10³ to 10⁷ cells/mL. The measurement for each sample was completed within 12 min. Furthermore, it was also possible to estimate the concentrations of E. coli O157:H7 by the increasing rate of fluorescence during binding reaction of detection antibodies to antigens. This minimized the time for measurement down to 6 min. The system is suitable for rapid and direct determination for microorganisms or bacteria in food, clinical, and environmental sources.

Abstract: The use of label-free technologies based on electrical impedance is becoming more and more popular in drug discovery. Indeed, such a methodology allows the continuous monitoring of diverse cellular processes, including proliferation, migration, cytotoxicity and receptor-mediated signaling. The objective of the present study was to further assess the usefulness of the real-time cell analyzer (RTCA) and, in particular, the xCELLigence platform, in the context of early drug development for pharmacology and toxicology investigations. In the present manuscript, four cellular models were exposed to 50 compounds to compare the cell index generated by RTCA and cell viability measured with a traditional viability assay. The data revealed an acceptable correlation (ca. 80%) for both cell lines (i.e., HepG2 and HepaRG), but a lack of correlation (ca. 55%) for the primary human and rat hepatocytes. In addition, specific RTCA profiles (signatures) were generated when HepG2 and HepaRG cells were exposed to calcium modulators, antimitotics, DNA damaging and nuclear receptor agents, with a percentage of prediction close to 80% for both cellular models. In a subsequent experiment, HepG2 cells were exposed to 81 proprietary UCB compounds known to be genotoxic or not. Based on the DNA damaging signatures, the RTCA technology allowed the detection of ca. 50% of the genotoxic compounds (n = 29) and nearly 100% of the non-genotoxic compounds (n = 52). Overall, despite some limitations, the xCELLigence platform is a powerful and reliable tool that can be used in drug discovery for toxicity and pharmacology studies.

Abstract: An immobilization protocol was developed to attach receptors on smooth silver thin films. Dense and packed 11-mercaptoundecanoic acid (11-MUA) was used to avoid uncontrolled sulfidization and harmful oxidation of silver nanolayers. N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were added to make the silver surfaces reactive. A comparative study was carried out with different immersion times of silver samples in 11-MUA solutions with different concentrations to find the optimum conditions for immobilization. The signals, during each step of the protocol, were analyzed with a refractometer based on the surface plasmon resonance (SPR) effect and luminescence techniques. Molecular interactions at the surfaces between the probe and target at the surface nanolayer shift the SPR signal, thus indicating the presence of the substance. To demonstrate specific biosensing, rabbit anti-estrone polyclonal immunoglobulin G (IgG) antibody was immobilized through a linker on 47 nm silver layer deposited on SF11 glass. At the final stage, the representative endocrine disruptor—estrone—was attached and detected in deionized water with a diverging beam SPR imaging sensor.

Abstract: The mechanosensitive channel of large conductance, MscL, has been proposed as a triggered nanovalve to be used in drug release and other nanodevices. It is a small homopentameric bacterial protein that has the largest gated pore known: greater than 30 Å. Large molecules, even small proteins can be released through MscL. Although MscL normally gates in response to membrane tension, early studies found that hydrophilic or charged residue substitutions near the constriction of the channel leads to pore opening. Researchers have successfully changed the modality of MscL to open to stimuli such as light by chemically modifying a single residue, G22, within the MscL pore. Here, by utilizing in vivo, liposome efflux, and patch clamp assays we compared modification of G22 with that of another neighboring residue, G26, and demonstrate that modifying G26 may be a better choice for triggered nanovalves used for triggered vesicular release of compounds.