http://www.mdpi.com/journal/biosensors Latest open access articles published in Biosensors at http://www.mdpi.com/journal/biosensors http://www.mdpi.com/2079-6374/3/2/185 DNA-templated few-atom silver nanoclusters (DNA/Ag NCs) are a new class of organic/inorganic composite nanomaterials whose fluorescence emission can be tuned throughout the visible and near-IR range by simply programming the template sequences. Compared to organic dyes, DNA/Ag NCs can be brighter and more photostable. Compared to quantum dots, DNA/Ag NCs are smaller, less prone to blinking on long timescales, and do not have a toxic core. The preparation of DNA/Ag NCs is simple and there is no need to remove excess precursors as these precursors are non-fluorescent. Our recent discovery of the fluorogenic and color switching properties of DNA/Ag NCs have led to the invention of new molecular probes, termed NanoCluster Beacons (NCBs), for DNA detection, with the capability to differentiate single-nucleotide polymorphisms by emission colors. NCBs are inexpensive, easy to prepare, and compatible with commercial DNA synthesizers. Many other groups have also explored and taken advantage of the environment sensitivities of DNA/Ag NCs in creating new tools for DNA/RNA detection and single-nucleotide polymorphism identification. In this review, we summarize the recent trends in the use of DNA/Ag NCs for developing DNA/RNA sensors. Biosensors 2013-04-23 3 2 Review 10.3390/bios3020185 185 200 2079-6374 2013-04-23 doi: 10.3390/bios3020185 Judy Obliosca Cong Liu Robert Batson Mark Babin James Werner Hsin-Chih Yeh http://www.mdpi.com/2079-6374/3/1/171 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. Biosensors 2013-03-19 3 1 Article 10.3390/bios3010171 171 184 2079-6374 2013-03-19 doi: 10.3390/bios3010171 Irene Iscla Christina Eaton Juandell Parker Robin Wray Zoltán Kovács Paul Blount http://www.mdpi.com/2079-6374/3/1/157 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. Biosensors 2013-03-19 3 1 Article 10.3390/bios3010157 157 170 2079-6374 2013-03-19 doi: 10.3390/bios3010157 Alina Karabchevsky Lev Tsapovsky Robert Marks Ibrahim Abdulhalim http://www.mdpi.com/2079-6374/3/1/132 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. Biosensors 2013-03-15 3 1 Article 10.3390/bios3010132 132 156 2079-6374 2013-03-15 doi: 10.3390/bios3010132 Franck Atienzar Helga Gerets Karen Tilmant Gaëlle Toussaint Stéphane Dhalluin http://www.mdpi.com/2079-6374/3/1/120 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. Biosensors 2013-03-08 3 1 Article 10.3390/bios3010120 120 131 2079-6374 2013-03-08 doi: 10.3390/bios3010120 Kumiko Miyajima Tomoyuki Koshida Takahiro Arakawa Hiroyuki Kudo Hirokazu Saito Kazuyoshi Yano Kohji Mitsubayashi http://www.mdpi.com/2079-6374/3/1/116 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. Biosensors 2013-02-28 3 1 Editorial 10.3390/bios3010116 116 119 2079-6374 2013-02-28 doi: 10.3390/bios3010116 Luisa Torsi http://www.mdpi.com/2079-6374/3/1/108 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. Biosensors 2013-02-08 3 1 Article 10.3390/bios3010108 108 115 2079-6374 2013-02-08 doi: 10.3390/bios3010108 Christian Winterflood Thomas Ruckstuhl Stefan Seeger http://www.mdpi.com/2079-6374/3/1/89 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. Biosensors 2013-02-06 3 1 Review 10.3390/bios3010089 89 107 2079-6374 2013-02-06 doi: 10.3390/bios3010089 Munawar Hussain Judith Wackerlig Peter Lieberzeit http://www.mdpi.com/2079-6374/3/1/77 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. Biosensors 2013-02-06 3 1 Article 10.3390/bios3010077 77 88 2079-6374 2013-02-06 doi: 10.3390/bios3010077 Shradha Prabhulkar Adam de la Zerda Amit Paranjape Richard Awdeh http://www.mdpi.com/2079-6374/3/1/58 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. Biosensors 2013-01-14 3 1 Article 10.3390/bios3010058 58 76 2079-6374 2013-01-14 doi: 10.3390/bios3010058 Benoit Piro Steeve Reisberg Guillaume Anquetin Huynh-Thien Duc Minh-Chau Pham http://www.mdpi.com/2079-6374/3/1/44 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. Biosensors 2013-01-08 3 1 Article 10.3390/bios3010044 44 57 2079-6374 2013-01-08 doi: 10.3390/bios3010044 Scherrine Tria Leslie Jimison Adel Hama Manuelle Bongo Róisín Owens http://www.mdpi.com/2079-6374/3/1/18 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. Biosensors 2012-12-27 3 1 Review 10.3390/bios3010018 18 43 2079-6374 2012-12-27 doi: 10.3390/bios3010018 Laura Zanoli Giuseppe Spoto http://www.mdpi.com/2079-6374/3/1/1 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. Biosensors 2012-12-27 3 1 Article 10.3390/bios3010001 1 17 2079-6374 2012-12-27 doi: 10.3390/bios3010001 Petri Ihalainen Himadri Majumdar Tapani Viitala Björn Törngren Tuomas Närjeoja Anni Määttänen Jawad Sarfraz Harri Härmä Marjo Yliperttula Ronald Österbacka Jouko Peltonen http://www.mdpi.com/2079-6374/2/4/465 An electrotextile with a biosensing focus composed of conductive polymer coated microfibers that contain functional attachment sites for biorecognition elements was developed. Experiments were conducted to select a compound with a pendant functional group for inclusion in the polymer, a fiber platform, and polymerization solvent. The effects of dopant inclusion and post-polymerization wash steps were also analyzed. Finally, the successful attachment of avidin, which was then used to capture biotin, to the electrotextile was achieved. The initial results show a nonwoven fiber matrix can be successfully coated in a conductive, functionalized polymer while still maintaining surface area and fiber durability. A polypropylene fiber platform with a conductive polypyrrole coating using iron (III) chloride as an oxidant, water as a solvent, and 5-sulfosalicylic acid as a dopant exhibited the best coating consistency, material durability, and lowest resistance. Biological attachment of avidin was achieved on the fibers through the inclusion of a carboxyl functional group via 3-thiopheneacetic acid in the monomer. The immobilized avidin was then successfully used to capture biotin. This was confirmed through the use of fluorescent quantum dots and confocal microscopy. A preliminary electrochemical experiment using avidin for biotin detection was conducted. This technology will be extremely useful in the formation of electrotextiles for use in biosensor systems. Biosensors 2012-11-29 2 4 Article 10.3390/bios2040465 465 478 2079-6374 2012-11-29 doi: 10.3390/bios2040465 Shannon McGraw Evangelyn Alocilja Andre Senecal Kris Senecal http://www.mdpi.com/2079-6374/2/4/448 Many cancer treatments rely on inhibition of epidermal growth factor (EGF)-induced cellular responses. Evaluating drug effects on such responses becomes critical to the development of new cancer therapeutics. In this report, we have employed a label-free acoustic sensor, the quartz crystal microbalance with dissipation monitoring (QCM-D), to track the EGF-induced response of mutant MCF10A cells under various inhibitory conditions. We have identified a complex cell de-adhesion process, which can be distinctly altered by inhibitors of signaling pathways and cytoskeleton formation in a dose-dependent manner. The dose dependencies of the inhibitors provide IC50 values which are in strong agreement with the values reported in the literature, demonstrating the sensitivity and reliability of the QCM-D as a screening tool. Using immunofluorescence imaging, we have also verified the quantitative relationship between the ΔD-response (change in energy dissipation factor) and the level of focal adhesions quantified with the areal density of immunostained vinculin under those inhibitory conditions. Such a correlation suggests that the dynamic restructuring of focal adhesions can be assessed based on the time-dependent change in ΔD-response. Overall, this report has shown that the QCM-D has the potential to become an effective sensing platform for screening therapeutic agents that target signaling and cytoskeletal proteins. Biosensors 2012-11-13 2 4 Article 10.3390/bios2040448 448 464 2079-6374 2012-11-13 doi: 10.3390/bios2040448 Marcela Garcia Ammar Shahid Jennifer Chen Jun Xi http://www.mdpi.com/2079-6374/2/4/433 This work illustrates the enhancement of the sensitivity of the ELISA titration for recombinant human and native prion proteins, while reducing other non-specific adsorptions that could increase the background signal and lead to a low sensitivity and false positives. It is achieved thanks to the association of plasma chemistry and coating with different amphiphilic molecules bearing either ionic charges and/or long hydrocarbon chains. The treated support by 3-butenylamine hydrochloride improves the signal detection of recombinant protein, while surface modification with the 3,7-dimethylocta-2,6-dien-1-diamine (geranylamine) enhances the sensitivity of the native protein. Beside the surface chemistry effect, these different results are associated with protein conformation. Biosensors 2012-10-24 2 4 Article 10.3390/bios2040433 433 447 2079-6374 2012-10-24 doi: 10.3390/bios2040433 Caroline Mille Dominique Debarnot Willy Zorzi Benaissa Moualij Isabelle Quadrio Armand Perret-Liaudet Arnaud Coudreuse Gilbert Legeay Fabienne Poncin-Epaillard http://www.mdpi.com/2079-6374/2/4/427 Globally, over hundreds of million people are infected with the hepatitis C virus: the global rate of death as a direct result of the hepatitis C virus has increased remarkably. For this reason, the development of efficient drug treatments for the biological effects of the hepatitis C virus is highly necessary. We have previously shown that quantum dots (QDs)-conjugated RNA oligonucleotide can recognize the hepatitis C virus NS3 protein specifically and sensitively. In this study, we elucidated that this biochip can analyze inhibitors to the hepatitis C virus NS3 protein using a nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, 7,8,4'-trihydroxyisoflavone and 6,7,4'-trihydroxyisoflavone demonstrated a remarkable inhibition activity on the hepatitis C virus NS3 protein. Both 7,8,4'-trihydroxyisoflavone and 6,7,4'-trihydroxyisoflavone attenuated the binding affinity in a concentrated manner as evidenced by QDs conjugated RNA oligonucleotide. At a concentration of 0.01 μg·mL−1, 7,8,4'-trihydroxyisoflavone and 6,7,4'-trihydroxyisoflavone showed more than a 30% inhibition activity of a nanoparticle-based RNA oligonucleotide biochip system. Biosensors 2012-10-24 2 4 Communication 10.3390/bios2040427 427 432 2079-6374 2012-10-24 doi: 10.3390/bios2040427 Changhyun Roh http://www.mdpi.com/2079-6374/2/4/417 The detection of pathogenic bacteria remains a challenge for the struggle against biological weapons, nosocomial diseases, and for food safety. In this research, our aim was to develop an easy-to-use electrochemical immunosensor for the detection of pathogenic Staphylococcus aureus ATCC25923. The biosensor was elaborated by the immobilization of anti-S. aureus antibodies using a self-assembled monolayer (SAMs) of 3-Mercaptopropionic acid (MPA). These molecular assemblies were spontaneously formed by the immersion of the substrate in an organic solvent containing the SAMs that can covalently bond to the gold surface. The functionalization of the immunosensor was characterized using two electrochemical techniques: cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Here, the analysis was performed in phosphate buffer with ferro/ferricyanide as the redox probe. The EIS technique was used for affinity assays: antibody-cell binding. A linear relationship between the increment in the electron transfer resistance (RCT) and the logarithmic value of S. aureus concentration was observed between 10 and 106 CFU/mL. The limit of detection (LOD) was observed at 10 CFU/mL, and the reproducibility was calculated to 8%. Finally, a good selectivity versus E. coli and S. epidermidis was obtained for our developed immunosensor demonstrating its specificity towards only S. aureus. Biosensors 2012-10-16 2 4 Article 10.3390/bios2040417 417 426 2079-6374 2012-10-16 doi: 10.3390/bios2040417 Mohamed Braiek Karima Rokbani Amani Chrouda Béchir Mrabet Amina Bakhrouf Abderrazak Maaref Nicole Jaffrezic-Renault http://www.mdpi.com/2079-6374/2/4/405 Although real-time PCR (RT-PCR) has become a diagnostic standard for rapid identification of bacterial species, typical methods remain time-intensive due to sample preparation and amplification cycle times. The assay described in this work incorporates on-chip dielectrophoretic capture and concentration of bacterial cells, thermal lysis, cell permeabilization, and nucleic acid denaturation and fluorescence resonance energy transfer assisted in situ hybridization (FRET-ISH) species identification. Combining these techniques leverages the benefits of all of them, allowing identification to be accomplished completely on chip less than thirty minutes after receipt of sample, compared to multiple hours required by traditional RT-PCR and its requisite sample preparation. Biosensors 2012-10-10 2 4 Article 10.3390/bios2040405 405 416 2079-6374 2012-10-10 doi: 10.3390/bios2040405 Michelle M. Packard Maxim Shusteff Evangelyn C. Alocilja http://www.mdpi.com/2079-6374/2/4/396 Angiotensin II is a key regulator of blood pressure and cardiovascular function in mammals. The conversion of angiotensin into its active form is carried out by Angiotensin I-Converting Enzyme (ACE). The measurement of ACE concentration in plasma or serum, its enzymatic activity, and the correlation between an insertion/deletion (I/D) genetic polymorphism of the ACE gene have been investigated as possible indicators of superior athletic performance in humans. In this context, other indicators of superior adaptation to exercise resulting in better athletic performance (such as ventricular hypertrophy, VO2 max, and competition results) were mostly used to study the association between ACE I/D polymorphism and improved performance. Despite the fact that the existing literature presents little consensus, there is sufficient scientific evidence to warrant further investigation on the usage of ACE activity and the I/D ACE gene polymorphism as biomarkers of superior athletic performance in humans of specific ethnicities or in athletes involved in certain sports. In this sense, a biomarker would be a substance or genetic component that could be measured to provide a degree of certainty, or an indication, of the presence of a certain trait or characteristic that would be beneficial to the athlete’s performance. Difficulties in interpreting and comparing the results of scientific research on the topic arise from dissimilar protocols and variation in study design. This review aims to investigate the current literature on the use of ACE I/D polymorphism as a biomarker of performance in humans through the comparison of scientific publications. Biosensors 2012-10-09 2 4 Review 10.3390/bios2040396 396 404 2079-6374 2012-10-09 doi: 10.3390/bios2040396 Maria Fernanda De Mello Costa Ron Slocombe http://www.mdpi.com/2079-6374/2/4/388 This work demonstrates the ability to electrospin reagents into water-soluble nanofibers resulting in a stable on-chip enzyme storage format. Polyvinylpyrrolidone (PVP) nanofibers were spun with incorporation of the enzyme horseradish peroxidase (HRP). Scanning electron microscopy (SEM) of the spun nanofibers was used to confirm the non-woven structure which had an average diameter of 155 ± 34 nm. The HRP containing fibers were tested for their change in activity following electrospinning and during storage. A colorimetric assay was used to characterize the activity of HRP by reaction with the nanofiber mats in a microtiter plate and monitoring the change in absorption over time. Immediately following electrospinning, the activity peak for the HRP decreased by approximately 20%. After a storage study over 280 days, 40% of the activity remained. In addition to activity, the fibers were observed to solubilize in the microfluidic chamber. The chromogenic 3,3′,5,5′-tetramethylbenzidine solution reacted immediately with the fibers as they passed through a microfluidic channel. The ability to store enzymes and other reagents on-chip in a rapidly dispersible format could reduce the assay steps required of an operator to perform. Biosensors 2012-10-08 2 4 Communication 10.3390/bios2040388 388 395 2079-6374 2012-10-08 doi: 10.3390/bios2040388 Minhui Dai Shengquan Jin Sam R. Nugen http://www.mdpi.com/2079-6374/2/4/377 Although still commonly used in clinical practice to screen and diagnose prostate cancer, there are numerous weaknesses of prostate-specific antigen (PSA) testing, including lack of specificity and the inability to distinguish between aggressive and indolent cancers. A promising prostate cancer biomarker, alpha-methylacyl-CoA racemase (AMACR), has been previously demonstrated to distinguish cancer from healthy and benign prostate cells with high sensitivity and specificity. However, no accurate clinically useful assay has been developed. This study reports the development of a single use, disposable biosensor for AMACR detection. Human blood samples were used to verify its validity, reproducibility and reliability. Plasma samples from 9 healthy males, 10 patients with high grade prostatic intraepithelial neoplasia (HGPIN), and 5 prostate cancer patients were measured for AMACR levels. The average AMACR levels in the prostate cancer patients was 10 fold higher (mean(SD) = 0.077 (0.10)) than either the controls (mean(SD) = 0.005 (0.001)) or HGPIN patients (mean(SD) = 0.004 (0.0005)). At a cutoff of between 0.08 and 0.9, we are able to achieve 100% accuracy in separating prostate cancer patients from controls. Our results provide strong evidence demonstrating that this biosensor can perform as a reliable assay for prostate cancer detection and diagnosis. Biosensors 2012-09-26 2 4 Article 10.3390/bios2040377 377 387 2079-6374 2012-09-26 doi: 10.3390/bios2040377 Po-Yuan Lin Kai-Lun Cheng James D. McGuffin-Cawley Fuh-Sheng Shieu Anna C. Samia Sanjay Gupta Matthew Cooney Cheryl L. Thompson Chung Chiun Liu http://www.mdpi.com/2079-6374/2/4/341 The great wealth of different surface sensitive techniques used in biosensing, most of which claim to measure adsorbed mass, can at first glance look unnecessary. However, with each technique relying on a different transducer principle there is something to be gained from a comparison. In this tutorial review, different optical and acoustic evanescent techniques are used to illustrate how an understanding of the transducer principle of each technique can be exploited for further interpretation of hydrated and extended polymer and biological films. Some of the most commonly used surface sensitive biosensor techniques (quartz crystal microbalance, optical waveguide spectroscopy and surface plasmon resonance) are briefly described and five case studies are presented to illustrate how different biosensing techniques can and often should be combined. The case studies deal with representative examples of adsorption of protein films, polymer brushes and lipid membranes, and describe e.g., how to deal with strongly vs. weakly hydrated films, large conformational changes and ordered layers of biomolecules. The presented systems and methods are compared to other representative examples from the increasing literature on the subject. Biosensors 2012-09-26 2 4 Review 10.3390/bios2040341 341 376 2079-6374 2012-09-26 doi: 10.3390/bios2040341 Rupert Konradi Marcus Textor Erik Reimhult http://www.mdpi.com/2079-6374/2/3/318 p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase) and pRL-CMV-luc (CMV promoter linked to Renilla luciferase) into marine flatfish flounder gill (FG) cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C) and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation), but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl) phthalate (DEHP), a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner. Biosensors 2012-09-10 2 3 Article 10.3390/bios2030318 318 340 2079-6374 2012-09-10 doi: 10.3390/bios2030318 Deyu Geng Zhixia Zhang Huarong Guo http://www.mdpi.com/2079-6374/2/3/305 We demonstrate here that thiol-ene chemistry can be used to provide side-chain functionalized monomers based on 3,4-propylenedioxythiophene (ProDOT) containing ionic, neutral, hydrophobic, and hydrophilic side chains. All reactions gave high yields and purification could generally be accomplished through precipitation. These monomers were polymerized either chemically or electro-chemically to give soluble materials or conductive films, respectively. This strategy provides for facile tuning of the solubility, film surface chemistry, and film morphology of this class of conducting polymers. Biosensors 2012-08-31 2 3 Article 10.3390/bios2030305 305 317 2079-6374 2012-08-31 doi: 10.3390/bios2030305 Kathleen E. Feldman David C. Martin http://www.mdpi.com/2079-6374/2/3/291 In this paper, label-free biosensing for antibody screening by periodic lattices of high-aspect ratio SU-8 nano-pillars (BICELLs) is presented. As a demonstration, the determination of anti-gestrinone antibodies from whole rabbit serum is carried out, and for the first time, the dissociation constant (KD = 6 nM) of antigen-antibody recognition process is calculated using this sensing system. After gestrinone antigen immobilization on the BICELLs, the immunorecognition was performed. The cells were interrogated vertically by using micron spot size Fourier transform visible and IR spectrometry (FT-VIS-IR), and the dip wavenumber shift was monitored. The biosensing assay exhibited good reproducibility and sensitivity (LOD = 0.75 ng/mL). Biosensors 2012-08-14 2 3 Article 10.3390/bios2030291 291 304 2079-6374 2012-08-14 doi: 10.3390/bios2030291 Francisco J. Ortega María-José Bañuls Francisco J. Sanza Rafael Casquel María Fe Laguna Miguel Holgado David López-Romero Carlos A. Barrios Ángel Maquieira Rosa Puchades http://www.mdpi.com/2079-6374/2/3/273 Profiling ligand function on G-protein coupled receptors (GPCRs) typically involves using transfected cells over-expressing a target of interest, a labelled ligand, and intracellular secondary messenger reporters. In contrast, label-free assays are sensitive enough to allow detection in native cells, which may provide a more physiologically relevant readout. Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR. The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells. Receptor activation was compared on label-free optical and impedance biosensors and contrasted with results from two traditional reporter assays. The rank order of potencies observed across label-free and pathway specific assays was similar. However, label-free read outs gave consistently lower potency values in both native and transfected cells. Relative to pathway-specific assays, these technologies measure whole-cell responses that may encompass multiple signalling events, including down-regulatory events, which may explain the potency discrepancies observed. These observations have important implications for screening compound libraries against GPCR targets and for selecting drug candidates for in vivo assays. Biosensors 2012-07-26 2 3 Article 10.3390/bios2030273 273 290 2079-6374 2012-07-26 doi: 10.3390/bios2030273 Reena Halai Daniel E. Croker Jacky Y. Suen David P. Fairlie Matthew A. Cooper http://www.mdpi.com/2079-6374/2/3/269 This editorial summarizes the general approaches of the electrochemical based biosensors described in the manuscripts published in this Special Issue. Electrochemical based biosensors are scientifically and economically important for the detection and early diagnosis of many diseases, and they will be increasing used and developed in the coming years. The importance of the selection of recognition processes, fabrication techniques and biosensor materials will be introduced. Biosensors 2012-07-24 2 3 Editorial 10.3390/bios2030269 269 272 2079-6374 2012-07-24 doi: 10.3390/bios2030269 Chung Chiun Liu http://www.mdpi.com/2079-6374/2/3/255 Polarization curves are of paramount importance for the detection of toxic components in microbial fuel cell (MFC) based biosensors. In this study, polarization curves were made under non-toxic conditions and under toxic conditions after the addition of various concentrations of nickel, bentazon, sodiumdodecyl sulfate and potassium ferricyanide. The experimental polarization curves show that toxic components have an effect on the electrochemically active bacteria in the cell. (Extended) Butler Volmer Monod (BVM) models were used to describe the polarization curves of the MFC under nontoxic and toxic conditions. It was possible to properly fit the (extended) BVM models using linear regression techniques to the polarization curves and to distinguish between different types of kinetic inhibitions. For each of the toxic components, the value of the kinetic inhibition constant Ki was also estimated from the experimental data. The value of Ki indicates the sensitivity of the sensor for a specific component and thus can be used for the selection of the biosensor for a toxic component. Biosensors 2012-07-11 2 3 Article 10.3390/bios2030255 255 268 2079-6374 2012-07-11 doi: 10.3390/bios2030255 Nienke E. Stein Hubertus V. M. Hamelers Gerrit van Straten Karel J. Keesman http://www.mdpi.com/2079-6374/2/3/245 Two quartz crystal microbalance oligonucleotide biosensors of a toxic microalga gene sequence (Alexandrium Minutum) have been designed. Grafting on a gold surface of 20-base thiol- or biotin-labeled probe, and selective hybridization with the complementary 20-base target, have been monitored in situ with a 27 MHz quartz crystal microbalance under controlled hydrodynamic conditions. The frequency of the set up is stable to within a few hertz, corresponding to the nanogram scale, for three hour experiments. DNA recognition by the two biosensors is efficient and selective. Hybridization kinetic curves indicate that the biosensor designed with the thiol-labeled probe is more sensitive, and that the biosensor designed with the biotin-labeled probe has a shorter time response and a higher hybridization efficiency. Biosensors 2012-07-04 2 3 Article 10.3390/bios2030245 245 254 2079-6374 2012-07-04 doi: 10.3390/bios2030245 Mathieu Lazerges Hubert Perrot Niriniony Rabehagasoa Chantal Compère http://www.mdpi.com/2079-6374/2/2/234 Aspartate aminotransaminase (AST) is a hepatocelluar enzyme released into the bloodstream when hepatic cells are damaged, resulting in elevated blood levels of AST. A single use, disposable biosensor prototype, composed of catalytic iridium nano-particles dispersed on carbon paste, was developed to detect enzymatically-produced H2O2 in AST-mediated reactions. This biosensor is capable of measuring AST levels in a phosphate buffer and undiluted human serum over the concentration range of 0 to 0.89 μg/mL AST concentration (corresponding to 0–250 UL−1 specific activity). The biosensor operates at relatively low oxidation potential (+0.3 volt (V) versus the printed Ag/AgCl), minimizing any potential chemical interference in human serum. The measurements of AST in human serum using the biosensor compared well with those measured by standard hospital spectrophotometric assays. This Ir-C biosensor may be useful for AST measurements in the clinical environment. Biosensors 2012-05-29 2 2 Article 10.3390/bios2020234 234 244 2079-6374 2012-05-29 doi: 10.3390/bios2020234 Chang-Jung Hsueh Joanne H. Wang Liming Dai Chung-Chiun Liu http://www.mdpi.com/2079-6374/2/2/221 The structural stability of pure SnO2 nanoparticles and highly sensitive SnO2-SiO2 nanocomposites (0–15 SiO2 wt%) has been investigated for conditions relevant to their utilization as chemoresistive gas sensors. Thermal stabilization by SiO2 co-synthesis has been investigated at up to 600 °C determining regimes of crystal size stability as a function of SiO2-content. For operation up to 400 °C, thermally stable crystal sizes of ca. 24 and 11 nm were identified for SnO2 nanoparticles and 1.4 wt% SnO2-SiO2 nanocomposites, respectively. The effect of crystal growth during operation (TO = 320 °C) on the sensor response to ethanol has been reported, revealing possible long-term destabilization mechanisms. In particular, crystal growth and sintering-neck formation were discussed with respect to their potential to change the sensor response and calibration. Furthermore, the effect of SiO2 cosynthesis on the cross-sensitivity to humidity of these noble metal-free SnO2-based gas sensors was assessed. Biosensors 2012-05-29 2 2 Article 10.3390/bios2020221 221 233 2079-6374 2012-05-29 doi: 10.3390/bios2020221 Antonio Tricoli http://www.mdpi.com/2079-6374/2/2/205 The detection of myoglobin (Myo), cardiac troponin I (cTnI), creatine kinase-MB (CK-MB), and b-type natriuretic peptide (BNP) plays a vital role in diagnosing cardiovascular diseases. Here we present single site-specific polyaniline (PANI) nanowire biosensors that can detect cardiac biomarkers such as Myo, cTnI, CK-MB, and BNP with ultra-high sensitivity and good specificity. Using single PANI nanowire-based biosensors integrated with microfluidic channels, very low concentrations of Myo (100 pg/mL), cTnI (250 fg/mL), CK-MB (150 fg/mL), and BNP (50 fg/mL) were detected. The single PANI nanowire-based biosensors displayed linear sensing profiles for concentrations ranging from hundreds (fg/mL) to tens (ng/mL). In addition, devices showed a fast (few minutes) response satisfying respective reference conditions for Myo, cTnI, CK-MB, and BNP diagnosis of heart failure and for determining the stage of the disease. This single PANI nanowire-based biosensor demonstrated superior biosensing reliability with the feasibility of label free detection and improved processing cost efficiency due to good biocompatibility of PANI to monoclonal antibodies (mAbs). Therefore, this development of single PANI nanowire-based biosensors can be applied to other biosensors for cancer or other diseases. Biosensors 2012-05-14 2 2 Article 10.3390/bios2020205 205 220 2079-6374 2012-05-14 doi: 10.3390/bios2020205 Innam Lee Xiliang Luo Jiyong Huang Xinyan Tracy Cui Minhee Yun http://www.mdpi.com/2079-6374/2/2/189 In view of the role ascribed to the peroxidatic activity of degradation products of cytochrome c (cyt c) in the processes of apoptosis, we investigate the catalytic potential of heme and of the cyt c derived heme peptide MP-11 to catalyse the cathodic reduction of hydrogen peroxide and to oxidize aromatic compounds. In order to check whether cyt c has an enzymatic activity in the native state where the protein matrix should suppress the inherent peroxidatic activity of its heme prosthetic group, we applied a biocompatible immobilization matrix and very low concentrations of the co-substrate H2O2. The biocatalysts were entrapped on the surface of a glassy carbon electrode in a biocompatible chitosan layer which contained gold nanoparticles. The electrochemical signal for the peroxide reduction is generated by the redox conversion of the heme group, whilst a reaction product of the substrate oxidation is cathodically reduced in the substrate indication. The catalytic efficiency of microperoxidase-11 is sufficient for sensors indicating HRP substrates, e.g., p-aminophenol, paracetamol and catechol, but also the hydroxylation of aniline and dehalogenation of 4-fluoroaniline. The lower limit of detection for p-aminophenol is comparable to previously published papers with different enzyme systems. The peroxidatic activity of cyt c immobilized in the chitosan layer for catechol was found to be below 1 per mill and for p-aminophenol about 3% as compared with that of heme or MP-11. Biosensors 2012-05-14 2 2 Article 10.3390/bios2020189 189 204 2079-6374 2012-05-14 doi: 10.3390/bios2020189 Aysu Yarman Bettina Neumann Maria Bosserdt Nenad Gajovic-Eichelmann Frieder W. Scheller http://www.mdpi.com/2079-6374/2/2/171 Infection control is a key aspect of wound management strategies. Infection results in chemical imbalances and inflammation in the wound and may lead to prolonged healing times and degradation of the wound surface. Frequent changing of wound dressings may result in damage to healing tissues and an increased risk of infection. This paper presents the first results from a monitoring system that is being developed to detect presence and growth of bacteria in real time. It is based on impedance sensors that could be placed at the wound-dressing interface and potentially monitor bacterial growth in real time. As wounds can produce large volumes of exudate, the initial system reported here was developed to test for the presence of bacteria in suspension. Impedance was measured using disposable silver-silver chloride electrodes. The bacteria Staphylococcus aureus were chosen for the study as a species commonly isolated from wounds. The growth of bacteria was confirmed by plate counting methods and the impedance data were analysed for discernible differences in the impedance profiles to distinguish the absence and/or presence of bacteria. The main findings were that the impedance profiles obtained by silver-silver chloride sensors in bacterial suspensions could detect the presence of high cell densities. However, the presence of the silver-silver chloride electrodes tended to inhibit the growth of bacteria. These results indicate that there is potential to create a real time infection monitor for wounds based upon impedance sensing. Biosensors 2012-05-09 2 2 Article 10.3390/bios2020171 171 188 2079-6374 2012-05-09 doi: 10.3390/bios2020171 Malcolm J. Farrow Iain S. Hunter Patricia Connolly http://www.mdpi.com/2079-6374/2/2/127 Cellular biochemical parameters can be used to reveal the physiological and functional information of various cells. Due to demonstrated high accuracy and non-invasiveness, electrochemical detection methods have been used for cell-based investigation. When combined with improved biosensor design and advanced measurement systems, the on-line biochemical analysis of living cells in vitro has been applied for biological mechanism study, drug screening and even environmental monitoring. In recent decades, new types of miniaturized electrochemical biosensor are emerging with the development of microfabrication technology. This review aims to give an overview of the microfabricated electrochemical cell-based biosensors, such as microelectrode arrays (MEA), the electric cell-substrate impedance sensing (ECIS) technique, and the light addressable potentiometric sensor (LAPS). The details in their working principles, measurement systems, and applications in cell monitoring are covered. Driven by the need for high throughput and multi-parameter detection proposed by biomedicine, the development trends of electrochemical cell-based biosensors are also introduced, including newly developed integrated biosensors, and the application of nanotechnology and microfluidic technology. Biosensors 2012-04-25 2 2 Review 10.3390/bios2020127 127 170 2079-6374 2012-04-25 doi: 10.3390/bios2020127 Jun Wang Chengxiong Wu Ning Hu Jie Zhou Liping Du Ping Wang http://www.mdpi.com/2079-6374/2/2/114 New silicon nitride coated optical gratings were tested by means of Optical Waveguide Lightmode Spectroscopy (OWLS). A thin layer of 10 nm of transparent silicon nitride was deposited on commercial optical gratings by means of sputtering. The quality of the layer was tested by x-ray photoelectron spectroscopy and atomic force microscopy. As a proof of concept, the sensors were successfully tested with OWLS by monitoring the concentration dependence on the detection of an antibody-protein pair. The potential of the Si3N4 as functional layer in a real-time biosensor opens new ways for the integration of optical waveguides with microelectronics. Biosensors 2012-04-10 2 2 Article 10.3390/bios2020114 114 126 2079-6374 2012-04-10 doi: 10.3390/bios2020114 Lorena Diéguez David Caballero Josep Calderer Mauricio Moreno Elena Martínez Josep Samitier http://www.mdpi.com/2079-6374/2/1/101 In this article we investigate the possibility of scaling down enzyme-gelatin modified electrodes by spin coating the enzyme-gelatin layer. Special attention is given to the electrochemical behavior of the selected enzymes inside the gelatin matrix. A glassy carbon electrode was used as a substrate to immobilize, in the first instance, horse heart cytochrome c (HHC) in a gelatin matrix. Both a drop dried and a spin coated layer was prepared. On scaling down, a transition from diffusion controlled reactions towards adsorption controlled reactions is observed. Compared to a drop dried electrode, a spin coated electrode showed a more stable electrochemical behavior. Next to HHC, we also incorporated catalase in a spin coated gelatin matrix immobilized on a glassy carbon electrode. By spincoating, highly uniform sub micrometer layers of biocompatible matrices can be constructed. A full electrochemical study and characterization of the modified surfaces has been carried out. It was clear that in the case of catalase, gluteraldehyde addition was needed to prevent leaking of the catalase from the gelatin matrix. Biosensors 2012-03-15 2 1 Article 10.3390/bios2010101 101 113 2079-6374 2012-03-15 doi: 10.3390/bios2010101 Karolien De Wael Stijn De Belder Sanaz Pilehvar Geert Van Steenberge Wouter Herrebout Hendrik A. Heering http://www.mdpi.com/2079-6374/2/1/83 The discovery over the past two decades of viable microbial communities within glaciers has promoted interest in the role of glaciers and ice sheets (the cryosphere) as contributors to subglacial erosion, global biodiversity, and in regulating global biogeochemical cycles. In situ or in-field detection and characterisation of microbial communities is becoming recognised as an important approach to improve our understanding of such communities. Within this context we demonstrate, for the first time, the ability to detect Gram-negative bacteria in glacial field-environments (including subglacial environments) via the detection of lipopolysaccharide (LPS); an important component of Gram-negative bacterial cell walls. In-field measurements were performed using the recently commercialised PyroGene® recombinant Factor C (rFC) endotoxin detection system and used in conjunction with a handheld fluorometer to measure the fluorescent endpoint of the assay. Twenty-seven glacial samples were collected from the surface, bed and terminus of a low-biomass Arctic valley glacier (Engabreen, Northern Norway), and were analysed in a field laboratory using the rFC assay. Sixteen of these samples returned positive LPS detection. This work demonstrates that LPS detection via rFC assay is a viable in-field method and is expected to be a useful proxy for microbial cell concentrations in low biomass environments. Biosensors 2012-03-09 2 1 Article 10.3390/bios2010083 83 100 2079-6374 2012-03-09 doi: 10.3390/bios2010083 Megan J. Barnett Jemma L. Wadham Miriam Jackson David C. Cullen http://www.mdpi.com/2079-6374/2/1/70 The focus of this paper is the development and investigation of properties of new nanostructured architecture for biosensors applications. Highly porous nanocomposite fibers were developed for use as active materials in biosensors. The nanocomposites comprised poly(lactic acid)(PLA)/multi-walled carbon nanotube (MWCNT) fibers obtained via solution-blow spinning onto indium tin oxide (ITO) electrodes. The electrocatalytic properties of nanocomposite-modified ITO electrodes were investigated toward hydrogen peroxide (H2O2) detection. We investigated the effect of carbon nanotube concentration and the time deposition of fibers on the sensors properties, viz., sensitivity and limit of detection. Cyclic voltammetry experiments revealed that the nanocomposite-modified electrodes displayed enhanced activity in the electrochemical reduction of H2O2, which offers a number of attractive features to be explored in development of an amperometric biosensor. Glucose oxidase (GOD) was further immobilized by drop coating on an optimized ITO electrode covered by poly(lactic acid)/carbon nanotube nanofibrous mats. The optimum biosensor response was linear up to 800 mM of glucose with a sensitivity of 358 nA·mM−1 and a Michaelis-Menten constant (KM) of 4.3 mM. These results demonstrate that the solution blow spun nanocomposite fibers have great potential for application as amperometric biosensors due to their high surface to volume ratio, high porosity and permeability of the substrate. The latter features may significantly enhance the field of glucose biosensors. Biosensors 2012-02-27 2 1 Article 10.3390/bios2010070 70 82 2079-6374 2012-02-27 doi: 10.3390/bios2010070 Juliano Elvis Oliveira Luiz Henrique Capparelli Mattoso Eliton Souto Medeiros Valtencir Zucolotto http://www.mdpi.com/2079-6374/2/1/57 This paper describes the functionalization of magnetoelastic (ME) materials with Parylene-C coating to improve the surface reactivity to cellular response. Previous study has demonstrated that vibrating ME materials were capable of modulating cellular adhesion when activated by an externally applied AC magnetic field. However, since ME materials are not inherently biocompatible, surface modifications are needed for their implementation in biological settings. Here, the long-term stability of the ME material in an aqueous and biological environment is achieved by chemical-vapor deposition of a conformal Parylene-C layer, and further functionalized by methods of oxygen plasma etching and protein adsorption. In vitro cytotoxicity measurement and characterization of the vibrational behavior of the ME materials showed that Parylene-C coatings of 10 µm or greater could prevent hydrolytic degradation without sacrificing the vibrational behavior of the ME material. This work allows for long-term durability and functionality of ME materials in an aqueous and biological environment and makes the potential use of this technology in monitoring and modulating cellular behavior at the surface of implantable devices feasible. Biosensors 2012-02-13 2 1 Article 10.3390/bios2010057 57 69 2079-6374 2012-02-13 doi: 10.3390/bios2010057 Hal R. Holmes Ee Lim Tan Keat Ghee Ong Rupak M. Rajachar http://www.mdpi.com/2079-6374/2/1/43 Single domain antibodies (sdAb) are the recombinantly expressed variable regions from the heavy-chain-only antibodies found in camelids and sharks. SdAb are able to bind antigens with high affinity, and most are capable of refolding after heat or chemical denaturation to bind antigen again. Starting with our previously isolated ricin binding sdAb determined to bind to four non-overlapping epitopes, we constructed a series of sdAb pairs, which were genetically linked through peptides of different length. We designed the series so that the sdAb are linked in both orientations with respect to the joining peptide. We confirmed that each of the sdAb in the constructs was able to bind to the ricin target, and have evidence that they are both binding ricin simultaneously. Through this work we determined that the order of genetically linked sdAb seems more important than the linker length. The genetically linked sdAb allowed for improved ricin detection with better limits of detection than the best anti-ricin monoclonal we evaluated, however they were not able to refold as well as unlinked component sdAb. Biosensors 2012-02-01 2 1 Article 10.3390/bios2010043 43 56 2079-6374 2012-02-01 doi: 10.3390/bios2010043 Richard H. Glaven George P. Anderson Dan Zabetakis Jinny L. Liu Nina C. Long Ellen R. Goldman http://www.mdpi.com/2079-6374/2/1/32 Rapid, sensitive detection methods are of utmost importance for the identification of pathogens related to health and safety. Herein we report the development of a nucleic acid sequence-based lateral flow assay which achieves a low limit of detection using chemiluminescence. On-membrane enzymatic signal amplification is used to reduce the limit of detection to the sub-femtomol level. To demonstrate this assay, we detected synthetic nucleic acid sequences representative of Trypanosoma mRNA, the causative agent for African sleeping sickness, with relevance in human and animal health in sub-Saharan Africa. The intensity of the chemiluminescent signal was evaluated by using a charge-coupled device as well as a microtiter plate reader. We demonstrated that our lateral flow chemiluminescent assay has a very low limit of detection and is easy to use. The limit of detection was determined to be 0.5 fmols of nucleic acid target. Biosensors 2012-01-18 2 1 Article 10.3390/bios2010032 32 42 2079-6374 2012-01-18 doi: 10.3390/bios2010032 Yuhong Wang Catherine Fill Sam R. Nugen http://www.mdpi.com/2079-6374/2/1/15 Biological defense and security applications demand rapid, sensitive detection of bacterial pathogens. This work presents a novel qualitative electrochemical detection technique which is applied to two representative bacterial pathogens, Bacillus cereus (as a surrogate for B. anthracis) and Escherichia coli O157:H7, resulting in detection limits of 40 CFU/mL and 6 CFU/mL, respectively, from pure culture. Cyclic voltammetry is combined with immunomagnetic separation in a rapid method requiring approximately 1 h for presumptive positive/negative results. An immunofunctionalized magnetic/polyaniline core/shell nano-particle (c/sNP) is employed to extract target cells from the sample solution and magnetically position them on a screen-printed carbon electrode (SPCE) sensor. The presence of target cells significantly inhibits current flow between the electrically active c/sNPs and SPCE. This method has the potential to be adapted for a wide variety of target organisms and sample matrices, and to become a fully portable system for routine monitoring or emergency detection of bacterial pathogens. Biosensors 2012-01-17 2 1 Article 10.3390/bios2010015 15 31 2079-6374 2012-01-17 doi: 10.3390/bios2010015 Emma B. Setterington Evangelyn C. Alocilja http://www.mdpi.com/2079-6374/2/1/1 Electrochemical aptasensors, which are based on the specificity of aptamer-target recognition, with electrochemical transduction for analytical purposes have received particular attention due to their high sensitivity and selectivity, simple instrumentation, as well as low production cost. Aptamers are functional nucleic acids with specific and high affinity to their targets, similar to antibodies. However, they are completely selected in vitro in contrast to antibodies. Due to their stability, easy chemical modifications and proneness to nanostructured device construction, aptamer-based sensors have been incorporated in a variety of applications including electrochemical sensing devices. In recent years, the performance of aptasensors has been augmented by incorporating novel nanomaterials in the preparation of better electrochemical sensors. In this review, we summarize the recent trends in the use of nanomaterials for developing electrochemical aptasensors. Biosensors 2012-01-13 2 1 Review 10.3390/bios2010001 1 14 2079-6374 2012-01-13 doi: 10.3390/bios2010001 Frank J. Hernandez Veli Cengiz Ozalp http://www.mdpi.com/2079-6374/1/4/164 Film bulk acoustic resonators (FBAR) are mass sensitive, label-free biosensors that allow monitoring of the interaction between biomolecules. In this paper we use the FBAR to measure the binding of calcium and the CaMKII peptide to calmodulin. Because the mass of the calcium is too small to be detected, the conformational change caused by the binding process is measured by monitoring the resonant frequency and the motional resistance of the FBAR. The resonant frequency is a measure for the amount of mass coupled to the sensor while the motional resistance is influenced by the viscoelastic properties of the adsorbent. The measured frequency shift during the calcium adsorptions was found to be strongly dependent on the surface concentration of the immobilized calmodulin, which indicates that the measured signal is significantly influenced by the amount of water inside the calmodulin layer. By plotting the measured motional resistance against the frequency shift, a mass adsorption can be distinguished from processes involving measurable conformational changes. With this method three serial processes were identified during the peptide binding. The results show that the FBAR is a promising technology for the label-free measurement of conformational changes. Biosensors 2011-12-14 1 4 Article 10.3390/bios1040164 164 176 2079-6374 2011-12-14 doi: 10.3390/bios1040164 Martin Nirschl Johannes Ottl Janos Vörös http://www.mdpi.com/2079-6374/1/4/153 In this study, we have successfully demonstrated the fabrication of a biosensor based on well aligned single-crystal zinc oxide (ZnO) nanorods which were grown on gold coated glass substrate using a low temperature aqueous chemical growth (ACG) method. The ZnO nanorods were immobilized with penicillinase enzyme using the physical adsorption approach in combination with N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) as cross linking molecules. The potentiometric response of the sensor configuration revealed good linearity over a large logarithmic concentration range from 100 µM to 100 mM. During the investigations, the proposed sensor showed a good stability with high sensitivity of ~121 mV/decade for sensing of penicillin. A quick electrochemical response of less than 5 s with a good selectivity, repeatability, reproducibility and a negligible response to common interferents such as Na1+, K1+, d-glucose, l-glucose, ascorbic acid, uric acid, urea, sucrose, lactose, glycine, penicilloic acid and cephalosporins, was observed. Biosensors 2011-10-27 1 4 Article 10.3390/bios1040153 153 163 2079-6374 2011-10-27 doi: 10.3390/bios1040153 Zafar Hussain Ibupoto Syed Muhammad Usman Ali Kimleang Khun Chan Oeurn Chey Omer Nur Magnus Willander http://www.mdpi.com/2079-6374/1/4/134 A passive and wireless sensor was developed for monitoring pressure in vivo. Structurally, the pressure sensor, referred to as the magneto-harmonic pressure sensor, is an airtight chamber sealed with an elastic pressure membrane. A strip of magnetically-soft material is attached to the bottom of the chamber and a permanent magnet strip is embedded inside the membrane. Under the excitation of an externally applied AC magnetic field, the magnetically-soft strip produces a higher-order magnetic signature that can be remotely detected with an external receiving coil. As ambient pressure varies, the pressure membrane deflects, altering the separation distance between the magnetically-soft strip and the permanent magnet. This shifts the higher-order harmonic signal, allowing for detection of pressure change as a function of harmonic shifting. The wireless, passive nature of this sensor technology allows for continuous long-term pressure monitoring, particularly useful for biomedical applications such as monitoring pressure in aneurysm sac and sphincter of Oddi. In addition to demonstrating its pressure sensing capability, an animal model was used to investigate the efficacy and feasibility of the pressure sensor in a biological environment. Biosensors 2011-10-17 1 4 Article 10.3390/bios1040134 134 152 2079-6374 2011-10-17 doi: 10.3390/bios1040134 Ee Lim Tan Andrew J. DeRouin Brandon D. Pereles Keat Ghee Ong http://www.mdpi.com/2079-6374/1/3/118 Diamond is a material of interest due to its unique combination of properties, including its chemical inertness and biocompatibility. Polycrystalline diamond (poly-C) has been used in experimental biosensors that utilize electrochemical methods and antigen-antibody binding for the detection of biological molecules. Boron-doped poly-C electrodes have been found to be very advantageous for electrochemical applications due to their large potential window, low background current and noise, and low detection limits (as low as 500 fM). The biocompatibility of poly-C is found to be comparable, or superior to, other materials commonly used for implants, such as titanium and 316 stainless steel. We have developed a diamond-based, neural microelectrode-array (MEA), due to the desirability of poly-C as a biosensor. These diamond probes have been used for in vivo electrical recording and in vitro electrochemical detection. Poly-C electrodes have been used for electrical recording of neural activity. In vitro studies indicate that the diamond probe can detect norepinephrine at a 5 nM level. We propose a combination of diamond micro-machining and surface functionalization for manufacturing diamond pathogen-microsensors. Biosensors 2011-08-15 1 3 Article 10.3390/bios1030118 118 133 2079-6374 2011-08-15 doi: 10.3390/bios1030118 Michael W. Varney Dean M. Aslam Abed Janoudi Ho-Yin Chan Donna H. Wang http://www.mdpi.com/2079-6374/1/3/107 Alanine aminotransaminase (ALT), is an enzyme that normally resides in serum and body tissues, especially in the liver. It is released into the serum as a result of tissue injury; hence the concentration of ALT in the serum may be increased with acute damage to hepatic cells. A single use, disposable biosensor, comprising iridium nano-particle as catalyst dispersed on carbon paste, has been developed for the determination of ALT concentration. The biosensor is based on quantifying H2O2 concentration produced by a serial of ALT enzymatic reactions. It operates well at room temperature in different physiological fluids: phosphate buffer, calf serum and human serum for ALT concentration of 0–544 ng/mL. Experimental results in human serum are compared to those obtained by spectrophotometric assays with excellent agreement. Therefore, the Ir/C biosensor shows good relationship on the dilution of concentrated ALT clinical applications. Biosensors 2011-07-12 1 3 Article 10.3390/bios1030107 107 117 2079-6374 2011-07-12 doi: 10.3390/bios1030107 Chang-Jung Hsueh Joanne H. Wang Liming Dai Chung-Chiun Liu http://www.mdpi.com/2079-6374/1/3/93 Triacetone triperoxide (TATP) is a primary explosive, which was used in various terrorist attacks in the past. For the development of biosensors, immunochemical µ-TAS, electronic noses, immunological test kits, or test strips, the availability of antibodies of high quality is crucial. Recently, we presented the successful immunization of mice, based on the design, synthesis, and conjugation of a novel TATP derivative. Here, the long-term immunization of rabbits is shown, which resulted in antibodies of extreme selectivity and more than 1,000 times better affinity in relation to the antibodies from mice. Detection limits below 10 ng L−1 (water) were achieved. The working range covers more than four decades, calculated from a precision profile. The cross-reactivity tests revealed an extraordinary selectivity of the antibodies—not a single compound could be identified as a relevant cross-reactant. The presented immunoreagent might be a major step for the development of highly sensitive and selective TATP detectors particularly for security applications. Biosensors 2011-07-07 1 3 Article 10.3390/bios1030093 93 106 2079-6374 2011-07-07 doi: 10.3390/bios1030093 Maria Astrid Walter Ulrich Panne Michael G. Weller http://www.mdpi.com/2079-6374/1/3/70 Label-free biomolecular interaction analysis is an important technique to study the chemical binding between e.g., protein and protein or protein and small molecule in real-time. The parameters obtained with this technique, such as the affinity, are important for drug development. While the surface plasmon resonance (SPR) instruments are most widely used, new types of sensors are emerging. These developments are generally driven by the need for higher throughput, lower sample consumption or by the need of complimentary information to the SPR data. This review aims to give an overview about a wide range of sensor transducers, the working principles and the peculiarities of each technology, e.g., concerning the set-up, sensitivity, sensor size or required sample volume. Starting from optical technologies like the SPR and waveguide based sensors, acoustic sensors like the quartz crystal microbalance (QCM) and the film bulk acoustic resonator (FBAR), calorimetric and electrochemical sensors are covered. Technologies long established in the market are presented together with those newly commercially available and with technologies in the early development stage. Finally, the commercially available instruments are summarized together with their sensitivity and the number of sensors usable in parallel and an outlook for potential future developments is given. Biosensors 2011-07-01 1 3 Review 10.3390/bios1030070 70 92 2079-6374 2011-07-01 doi: 10.3390/bios1030070 Martin Nirschl Florian Reuter Janos Vörös http://www.mdpi.com/2079-6374/1/2/58 In this article, we describe the kinetic ELISA of Blue Tongue and Epizootic Hemorrhagic Disease viral antibodies in microfluidic channels by monitoring the rate of generation of the enzyme reaction product under static conditions. It has been shown that this format of the immunoassay allows very reliable quantitation of the target species using inexpensive glass microchips and a standard epifluorescence microscope system coupled to a CCD camera. For the viral antibodies assayed here, the limit of detection (LOD) for the analyte concentration in our microchips was established to be 3–5 times lower than that obtained on commercial microwell plates using a fiftieth of the sample volume and less than a third of the incubation time. Our analyses further show that when compared to the end-point ELISA format, the kinetic mode of this assay yields an improvement in the LOD by over an order of magnitude in microfluidic devices. This benefit is primarily realized as the observed variation in the background fluorescence (signal at the start of the enzyme reaction period) was significantly larger than that in the rate of signal generation upon repeating these assays in different microchannels/microchips. Because the kinetic ELISA results depend only on the latter quantity, the noise level in them was substantially lower compared to that in its end-point counterpart in which the absolute fluorescence measurements are of greater significance. While a similar benefit was also recorded through implementation of kinetic ELISAs on the microwell platform, the improvement in LOD registered in that system was not as significant as was observed in the case of microfluidic assays. Biosensors 2011-06-17 1 2 Article 10.3390/bios1020058 58 69 2079-6374 2011-06-17 doi: 10.3390/bios1020058 Naoki Yanagisawa Debashis Dutta http://www.mdpi.com/2079-6374/1/2/46 By simultaneously measuring the bulk media and electrode interface voltages of a yeast (Saccharomyces cerevisiae) suspension subjected to an AC voltage, a yeast-dependent nonlinear response was found only near the current injection electrodes. Computer simulation of yeast near a current injection electrode found an enhanced voltage drop across the yeast near the electrode due to slowed charging of the electrode interfacial capacitance. This voltage drop is sufficient to induce conformation change in membrane proteins. Disruption of the mitochondrial electron transport chain is found to significantly change the measured nonlinear current response, suggesting nonlinear impedance can be used as a non-invasive probe of cellular metabolic activity. Biosensors 2011-04-11 1 2 Article 10.3390/bios1020046 46 57 2079-6374 2011-04-11 doi: 10.3390/bios1020046 Akilan Palanisami George T. Mercier Jie Fang John H. Miller Jr. http://www.mdpi.com/2079-6374/1/2/36 This study aims to measure the effect of toxic aqueous solutions of metals on the mobility of Artemia salina nauplii by using digital image processing. The instrument consists of a camera with a macro lens, a dark chamber, a light source and a laptop computer. Four nauplii were inserted into a macro cuvette, which contained copper, cadmium, iron and zinc ions at various concentrations. The nauplii were then filmed inside the dark chamber for two minutes and the video sequence was processed by a motion tracking algorithm that estimated their mobility. The results obtained by this system were compared to the mortality assay of the Artemia salina nauplii. Despite the small number of tested organisms, this system demonstrates great sensitivity in quantifying the mobility of the nauplii, which leads to significantly lower EC50 values than those of the mortality assay. Furthermore, concentrations of parts per trillion of toxic compounds could be detected for some of the metals. The main novelty of this instrument relies in the sub-pixel accuracy of the tracking algorithm that enables robust measurement of the deterioration of the mobility of Artemia salina even at very low concentrations of toxic metals. Biosensors 2011-03-28 1 2 Article 10.3390/bios1020036 36 45 2079-6374 2011-03-28 doi: 10.3390/bios1020036 Varvara Kokkali Ioannis Katramados Jeffrey D. Newman http://www.mdpi.com/2079-6374/1/1/23 Here we report the fabrication and characterization of pH sensors using aligned single-walled carbon nanotubes (SWNTs). The SWNTs are dispersed in deionized (DI) water after chemical functionalization and filtration. They are deposited and organized on silicon substrates with the dielectrophoresis process. Electrodes with “teeth”-like patterns—fabricated with photolithography and wet etching—are used to generate concentrated electric fields and strong dielectrophoretic forces for the SWNTs to deposit and align in desired locations. The device fabrication is inexpensive, solution-based, and conducted at room temperature. The devices are used as pH sensors with the electrodes as the testing pads and the dielectrophoretically captured SWNTs as the sensing elements. When exposed to aqueous solutions with various pH values, the SWNTs change their resistance accordingly. The SWNT-based sensors demonstrate a linear relationship between the sensor resistance and the pH values in the range of 5–9. The characterization of multiple sensors proves that their pH sensitivity is highly repeatable. The real-time data acquisition shows that the sensor response time depends on the pH value, ranging from 2.26 s for the pH-5 solution to 23.82 s for the pH-9 solution. The long-term stability tests illustrate that the sensors can maintain their original sensitivity for a long period of time. The simple fabrication process, high sensitivity, and fast response of the SWNT-based sensors facilitate their applications in a wide range of areas. Biosensors 2011-01-31 1 1 Article 10.3390/bios1010023 23 35 2079-6374 2011-01-31 doi: 10.3390/bios1010023 Pengfei Li Caleb M. Martin Kan Kan Yeung Wei Xue http://www.mdpi.com/2079-6374/1/1/13 Nonlinear dielectric spectroscopy (NDS) is a non-invasive probe of cellular metabolic activity with potential application in the development of whole-cell biosensors. However, the mechanism of NDS interaction with metabolic membrane proteins is poorly understood, partly due to the inherent complexity of single cell organisms. Here we use the light-activated electron transport chain of spinach thylakoid membrane as a model system to study how NDS interacts with metabolic activity. We find protein modification, as opposed to membrane pump activity, to be the dominant source of NDS signal change in this system. Potential mechanisms for such protein modifications include reactive oxygen species generation and light-activated phosphorylation. Biosensors 2011-01-31 1 1 Article 10.3390/bios1010013 13 22 2079-6374 2011-01-31 doi: 10.3390/bios1010013 Jie Fang Akilan Palanisami Kimal Rajapakshe William R. Widger John H. Miller http://www.mdpi.com/2079-6374/1/1/4 With the rapid growth of Biological/Biomedical MicroElectroMechanical Systems (BioMEMS) and microfluidic-based lab-on-a-chip (LOC) technology to biological and biomedical research and applications, demands for educated and trained researchers and technicians in these fields are rapidly expanding. Universities are expected to develop educational plans to address these specialized needs in BioMEMS, microfluidic and LOC science and technology. A course entitled BioMEMS and Lab-on-a-Chip was taught recently at the senior undergraduate and graduate levels in the Department of Computer Science and Electrical Engineering at West Virginia University (WVU). The course focused on the basic principles and applications of BioMEMS and LOC technology to the areas of biomedicine, biology, and biotechnology. The course was well received and the enrolled students had diverse backgrounds in electrical engineering, material science, biology, mechanical engineering, and chemistry. Student feedback and a review of the course evaluations indicated that the course was effective in achieving its objectives. Student presentations at the end of the course were a highlight and a valuable experience for all involved. The course proved successful and will continue to be offered regularly. This paper provides an overview of the course as well as some development and future improvements. Biosensors 2011-01-20 1 1 Article 10.3390/bios1010004 4 12 2079-6374 2011-01-20 doi: 10.3390/bios1010004 Yuxin Liu http://www.mdpi.com/2079-6374/1/1/1 The journal Biosensors has been started as a peer-reviewed, open access journal. As editors, we believe that it will fulfill an important role in the community of researchers and developers in the field of biosensors. The addition of a “free access” journal to the existing, high quality publications in this field is something that we believe is very important in a field which is now so entwined with commercial activity and where researchers aim, not only at academic research, but on the development of products at a potentially massive scale. For these researchers, it is important that they can publish their results in a journal that guarantees quality that comes from peer-review, but that at the same time breaks the traditional boundaries of academic journals which need a subscription or a pay-per-view option to access the published data. Biosensors 2011-01-19 1 1 Editorial 10.3390/bios1010001 1 3 2079-6374 2011-01-19 doi: 10.3390/bios1010001 Jeffrey D. Newman