Biosensors and Bioelectronics (v.24, #7)

The immobilization of nucleic acids on solid supports has been widely used in the detection of DNA and other biomolecules in sensor technology. Because three dimensional (3-D) hydrogel matrixes offer significant advantages for capturing probes over more conventional two dimensional (2-D) rigid substrates and the ability to provide a solution-mimicking environment, they are becoming increasingly attractive as desired supports for bio-analysis. Acrylamide-modified nucleic acids and acrylamide monomers being polymerized directly to immobilize nucleic acids is only one-step chemical process which is not interfered by exterior surroundings, and the 3-D polyacrylamide gel fabricated by this method is not required to be activated by some labile chemical treatments. Moreover, the attachment is extremely stable to withstand the cycling process involved in the polymerase chain reaction (PCR). In this paper, the development of polymerizing immobilization of acrylamide-modified nucleic acids is reviewed, and its applications in DNA sequence high-throughput analysis including mutation analysis and the whole genome sequencing are summarized.
Keywords: Acrylamide-modified nucleic acids; 3-D microarray; Polymerizing immobilization; Application; Mutation detection;

A doubly amplified electrochemical immunoassay for carcinoembryonic antigen by Zhiqiang Gao; Jie Zhang; Boon Ping Ting (1825-1830).
An ultrasensitive electrochemical immunoassay (EIA) for the detection of carcinoembryonic antigen (CEA) is described in this report. The assay involves utilizing enzyme-catalyzed deposition of a redox polymer and electrocatalytic oxidation of ascorbic acid (AA) by the deposited redox polymer, a dual-amplification scheme to enhance analytical signals. Briefly, CEA capturing antibody and redox polymer anchoring agent were covalently immobilized on a gold electrode. After incubating with CEA, the electrode was treated in detection antibody–glucose oxidase conjugate solution. Thereafter, it was dipped into the redox polymer solution. Upon the addition of glucose, the redox polymer was enzymatically reduced and deposited on the electrode surface. The deposited redox polymer exhibits excellent electrocatalytic activity towards the oxidation of AA. Consequently, CEA could be quantified amperometrically. This electrochemical immunoassay combines the specificity of the immunological reaction with the sensitivity of the doubly amplified electrochemical detection.
Keywords: Immunoassay; Carcinoembryonic antigen; Amperometry; Redox polymer; Glucose oxidase;

Ligand–receptor interactions in complex media: A new type of biosensors for the detection of coagulation factor VIII by A. Goldzstein; A. Aamouche; F. Homblé; M. Voué; J. Conti; J. De Coninck; S. Devouge; J. Marchand-Brynaert; E. Goormaghtigh (1831-1836).
Detection of receptor–ligand interaction in complex media remains a challenging issue. We report experimental results demonstrating the specific detection of the coagulation factor VIII in the presence of a large excess of other proteins using the new BIA-ATR technology based on attenuated total reflection Fourier transform infrared spectroscopy. The principle of the detection is related to the ability of factor VIII molecules to bind to lipid membranes containing at least 8% phosphatidylserine. Several therapeutic concentrates of factor VIII were analyzed and the binding of the coagulation factor was monitored as a function of time. We show that a non-specific adsorption of stabilizing agents (typically, von Willebrand factor and human serum albumin) may be avoided by controlling the geometry of the ATR element. A linear response of the sensors as a function of the factor VIII concentration is described for different lipid membrane compositions.
Keywords: Ligand–receptor interaction; FTIR-ATR; Alkylsilanes; Haemophilia; Factor VIII; Molecular diagnosis;

BioCapacitor—A novel category of biosensor by Takuya Hanashi; Tomohiko Yamazaki; Wakako Tsugawa; Stefano Ferri; Daisuke Nakayama; Masamitsu Tomiyama; Kazunori Ikebukuro; Koji Sode (1837-1842).
This research reports on the development of an innovative biosensor, known as BioCapacitor, in which biological recognition elements are combined with a capacitor functioning as the transducer. The analytical procedure of the BioCapacitor is based on the following principle: a biocatalyst, acting as a biological recognition element, oxidizes or reduces the analyte to generate electric power, which is then charged into a capacitor via a charge pump circuit (switched capacitor regulator) until the capacitors attains full capacity. Since the charging rate of the capacitor depends on the biocatalytic reaction of the analyte, the analyte concentration can be determined by monitoring the time/frequency required for the charge/discharge cycle of the BioCapacitor via a charge pump circuit. As a representative model, we constructed a BioCapacitor composed of FAD-dependent glucose dehydrogenase (FADGDH) as the anodic catalyst, and attempted a glucose measurement. Charge/discharge frequency of the BioCapacitor increased with the increasing glucose concentration, exhibiting good correlation with glucose concentration. We have also constructed a wireless sensing system using the BioCapacitor combined with an infrared light emitting diode (IRLED), an IR phototransistor system. In the presence of glucose, the IRLED signal was observed due to the discharge of the BioCapacitor and detected by an IR phototransistor in a wireless receiver. Therefore, a BioCapacitor employing FADGDH as its anodic catalyst can be operated as a self-powered enzyme sensor.
Keywords: Biofuel cells; Glucose dehydrogenase; Continuous glucose monitoring; Glucose sensor; Capacitor; Wireless sensing system;

Bilayer lipid membrane (BLM) based ion selective electrodes at the meso-, micro-, and nano-scales by Bingwen Liu; Daniel Rieck; Bernard J. Van Wie; Gary J. Cheng; David F. Moffett; David A. Kidwell (1843-1849).
This paper presents a novel method for making micron-sized apertures with tapered sidewalls and nano-sized apertures. Their use in bilayer lipid membrane-based ion selective electrode design is demonstrated and compared to mesoscale bilayers and traditional PVC ion selective electrodes. Micron-sized apertures are fabricated in SU-8 photoresist films and vary in diameter from 10 to 40 μm. The tapered edges in SU-8 films are desired to enhance bilayer lipid membrane (BLM) formation and are fabricated by UV-light overexposure. Nano-apertures are made in boron diffused silicon film. The membranes are used as septa to separate two potassium chloride solutions of different concentrations. Lecithin BLMs are assembled on the apertures by ejecting lipid solution. Potassium ionophore, dibenzo-18-crown-6, is incorporated into BLMs by dissolving it in the lipid solution before membrane assembly. Voltage changes with increasing potassium ion concentrations are recorded with an A/D converter. Various ionophore concentrations in BLMs are investigated. At least a 1% concentration is needed for consistent slopes. Electrode response curves are linear over the 10−6 to 0.1 M range with a sub-Nernstian slope of 20 mV per Log concentration change. This system shows high selectivity to potassium ions over potential interfering sodium ions. BLMs on the three different aperture sizes at the meso-, micro-, and nano-scales all show similar linear ranges and limits of detection (LODs) as PVC ion selective membranes.
Keywords: Ion selective electrodes; Bilayer lipid membranes; Micro-fabrication; Dibenzo-18-crown-6;

Microbeads on microposts: An inverted architecture for bead microarrays by Luisa Filipponi; Prashant D. Sawant; Florin Fulga; Dan V. Nicolau (1850-1857).
The rapid development of genomics and proteomics requires accelerated improvement of the microarrays density, multiplexing, readout capabilities and cost-effectiveness. The bead arrays are increasingly attractive because of their self-assembly-based fabrication, which alleviates many problems of top-down microfabrication. Here we present a simple, reliable, robust and modular technique for the fabrication of bead microarrays, which combines the directed assembling of beads in microstructures and PDMS-based replica molding. The beads are first self-assembled in pyramidal microwells fabricated by anisotropic etching of silicon substrates, then transferred on the apex of PDMS pyramids that replicate the silicon microstructures. The arrays are chemically and biochemically robust; they are spatially addressable and have the potential for being informationally addressable; and they appear to offer better readout capabilities than the classical microarrays.
Keywords: Microarray; Bead arrays; Self-assembly; Replica molding; PDMS; Spatially addressable;

Highly sensitive amperometric biosensors for phenols based on polyaniline–ionic liquid–carbon nanofiber composite by Jing Zhang; Jianping Lei; Yiyin Liu; Jianwei Zhao; Huangxian Ju (1858-1863).
A novel polyaniline–ionic liquid–carbon nanofiber (PANI–IL–CNF) composite was greenly prepared by in situ one-step electropolymerization of aniline in the presence of IL and CNF for fabrication of amperometric biosensors. The scanning electron micrographs confirmed that the PANI uniformly grew along with the structure of CNF and the PANI–IL–CNF composite film showed a fibrillar morphology with the diameter of around 95 nm. A phenol biosensor was constructed by immobilizing tyrosinase on the surface of the composite modified glassy carbon electrode via the cross-linking step with glutaraldehyde. The biosensor exhibited a wide linear response to catechol ranging from 4.0 × 10−10 to 2.1 × 10−6  M with a high sensitivity of 296 ± 4 A M−1  cm−2, a limit of detection down to 0.1 nM at the signal to noise ratio of 3 and applied potential of −0.05 V. According to the Arrhenius equation, the activation energy for enzymatic reaction was calculated to be 38.8 kJ mol−1 using catechol as the substrate. The apparent Michaelis–Menten constants of the enzyme electrode were estimated to be 1.44, 1.33, 1.16, 0.65 μM for catechol, p-cresol, phenol, m-cresol, respectively. The functionalization of CNF with PANI in IL provided good biocompatible platform for biosensing and biocatalysis.
Keywords: Nanocomposite; Biosensors; Phenol; Carbon nanofiber; Polyaniline; Ionic liquid;

Rhodamine B isothiocyanate (RhBITC) is a prototype dye molecule that is widely used as a fluorescent tag in a variety of biological applications. We report in this work that once RhBITC is adsorbed onto Ag on silica or polystyrene beads, it exhibits not only a strong surface-enhanced Raman scattering (SERS) signal but also a measurable amount of fluorescence. The RhBITC-modified Ag-deposited silica or polystyrene beads disperse well in ethanol, and they are also readily coated in water with polyelectrolytes for their further derivatization with biological molecules of interest that can bind to target molecules. The application prospects of these materials are thus expected to be very high especially in the areas of biological sensing and recognition that rely heavily on optical and spectroscopic properties. For instance, on the basis of the nature of the SERS peaks of RhBITC, those Ag-deposited silica or polystyrene beads were confirmed, after attaching biotin groups over RhBITC, to selectively recognize streptavidin molecules down to concentrations of 10−13  M based on a signal-to-noise ratio of 3. The biotin–streptavidin interaction was also confirmed from the photoluminescence of RhBITC.
Keywords: Surface-enhanced Raman scattering; Fluorescence; Silica bead; Polystyrene bead; Rhodamine B isothiocyanate; Biotin–streptavidin interaction;

Magnetophoretic position detection for multiplexed immunoassay using colored microspheres in a microchannel by Young Ki Hahn; Jae-Byum Chang; Zongwen Jin; Hak-Sung Kim; Je-Kyun Park (1870-1876).
This paper demonstrates a new magnetophoretic position detection method for multiplexed immunoassay using colored microspheres as an encoding tool in a microchannel. Colored microspheres conjugated with respective capture molecules are incubated with a mixture of target analytes, followed by reaction with the probe molecules which had been conjugated with superparamagnetic nanoparticles (SMNPs). Under the magnetic field gradient, the resulting microspheres are deflected from their focused streamlines in a microchannel, and respective colored microspheres are detected using color charge-coupled device (CCD) in a specific detection region of the microchannel. The color and position of respective colored microspheres are automatically decoded and analyzed by MATLAB program, and the position was correlated with the concentration of corresponding target analytes. As a proof-of-concept, we attempted to assay simultaneously three types of biotinylated immunoglobuline Gs (IgGs), such as goat, rabbit and mouse IgGs, using colored microspheres (red, yellow and blue, respectively). As the capture molecules, corresponding anti-IgGs were employed and target analytes were probed using streptavidin-modified superparamagnetic nanoparticles. As a result, three analytes were simultaneously assayed using colored microspheres with high accuracy, and detection limits of goat IgG, rabbit IgG and mouse IgG were estimated to be 10.9, 30.6 and 12.1 fM, respectively. In addition, with adjustment of the flow rate and detection zone, the dynamic range could be controlled by more than one order of magnitude.
Keywords: Magnetophoresis; Multiplexed immunoassay; Microchannel; Colored microspheres; Superparamagnetic nanoparticles;

Fabrication of a highly sensitive penicillin sensor based on charge transfer techniques by Seung-Ro Lee; M.M. Rahman; Kazuaki Sawada; Makoto Ishida (1877-1882).
A highly sensitive penicillin biosensor based on a charge-transfer technique (CTTPS) has been fabricated and demonstrated in this paper. CTTPS comprised a charge accumulation technique for penicilloic acid and H+ ions perception system. With the proposed CTTPS, it is possible to amplify the sensing signals without external amplifier by using the charge accumulation cycles. The fabricated CTTPS exhibits excellent performance for penicillin detection and exhibit a high-sensitivity (47.852 mV/mM), high signal-to-noise ratio (SNR), large span (1445 mV), wide linear range (0–25 mM), fast response time (<3 s), and very good reproducibility. A very lower detection limit of about 0.01 mM was observed from the proposed sensor. Under optimum conditions, the proposed CTTPS outstripped the performance of the widely used ISFET penicillin sensor and exhibited almost eight times greater sensitivity as compared to ISFET (6.56 mV/mM). The sensor system is implemented for the measurement of the penicillin concentration in penicillin fermentation broth.
Keywords: Penicillin sensor; CTTPS; ISFET; ENFET; Cyclic voltammetry;

Robust and real-time monitoring of nerve regeneration using implantable flexible microelectrode array by Yong-Ho Kim; Chungkeun Lee; Kang-Min Ahn; Myoungho Lee; Yong-Jun Kim (1883-1887).
This paper presents a robust and quasi-real-time monitoring method for examining the degree of regeneration of peripheral nerves using a polymer-based implantable microelectrode array (IMA). The IMA was implanted in the sciatic nerves of rats using two different approaches: (1) direct implantation which was achieved by directly suturing to nerve stumps for short-term regeneration and (2) assembly implantation which was achieved by implanting the IMA assembled with a resorbable conduit that cross a 4-mm nerve defect. The regeneration of the injury was evaluated by measuring the nerve signals following foot pricking and impulse stimulating to the proximal stump. The direct implantation produced a peak voltage of 3.43 mV before amplification and a conduction velocity of 19.8 m/s. The assembly implantation produced a peak voltage of 1.51 mV and a conduction velocity of 18 m/s. The degree of regeneration was also examined using gait and morphological analyses. The sciatic function index was −38.5 ± 12.5 for direct implantation and −64.9 ± 7.5 for assembly implantation. The morphologies of the regenerated nerves were examined using toluidine blue, transmission electron microscopy images, and pilot tests. Te regeneration ratios after the direct and assembly implantation were 45% and 21%, respectively.
Keywords: Monitoring tool; Degree of regeneration; Peripheral nerve; Microelectrode;

Label-free impedimetric immunosensor for sensitive detection of ochratoxin A by Abd-Elgawad Radi; Xavier Muñoz-Berbel; Vasilica Lates; Jean-Louis Marty (1888-1892).
A novel label-free electrochemical impedimetric immunosensor for sensitive detection of ochratoxin A (OTA) was reported. A two-step reaction protocol was elaborated to modify the gold electrode. The electrode was first derivatized by electrochemical reduction of in situ generated 4-carboxyphenyl diazonium salt (4-CPDS) in acidic aqueous solution yielded stable 4-carboxyphenyl (4-CP) monolayer. The ochratoxin A antibody was then immobilized making use of the carbodiimide chemistry. The steps of the immunosensor elaboration and the immunochemical reaction between ochratoxin A and the surface-bound antibody were interrogated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect ochratoxin A. The increase in electron-transfer resistance (ΔR et) values was linearly proportional to the concentration of OTA in the range of 1–20 ng mL−1, with a detection limit of 0.5 ng mL−1.
Keywords: Immunosensor; Ochratoxin A; Diazonium salt; Electrochemical impedance spectroscopy;

Rapid DNA multi-analyte immunoassay on a magneto-resistance biosensor by M. Koets; T. van der Wijk; J.T.W.M. van Eemeren; A. van Amerongen; M.W.J. Prins (1893-1898).
We present the rapid and sensitive detection of amplified DNA on a giant magneto-resistance sensor using superparamagnetic particles as a detection label. The one-step assay is performed on an integrated and miniaturized detection platform suitable for application into point-of-care devices. A double-tagged PCR amplification product of the LamB gene of the Escherichia coli bacterium was used to investigate binding kinetics of the assay. We applied magnetic actuation to concentrate the target–particle complexes at the sensor surface and to remove unbound particles from the sensor surface. We achieved biological dose-response curves detecting 4–250 pM amplicon concentrations in a one-step format in total assay times of less than 3 min. Using various tag-antibody combinations specific for one of the individual genes, multi-analyte detection is shown of several antibiotic resistance genes of the food pathogen Salmonella.
Keywords: GMR; DNA; Magnetic particle; Biosensor; Rapid assay; Magnetic actuation;

This study presents an ultra-sensitive technique for the electrochemical detection of the mutated BRAF gene associated with papillary thyroid carcinomas (PTC). In the proposed approach, a biotinylated 30-nucleotides probe DNA was immobilized in a streptavidin-modified 96-well microtiter plate and the free active sites of the streptavidin were blocked using biotinylated bovine serum albumin (BSA). The biotinylated target DNA was then added and allowed to hybridize with the immobilized probe DNA for 30 min. Subsequently, streptavidin-labeled gold nanoparticles were added, and a nanoparticle enlargement process was performed using gold ion solution and formaldehyde reductant. The gold particles were then dissolved in bromide and DNA hybridization detection process was performed using a square wave stripping voltammetry (SWSV) technique. The results indicated a stable SWSV response in differential detection between blank solution and target DNA solution with a concentration of 130 aM. Moreover, the coefficient of determination (R 2) of the semi-log plot of the SWSV response current against the target DNA concentration (0.52–1300 aM) was found to be 0.9982. The detection limit was estimated to be 0.35 aM (based on a signal-to-noise ratio of 3:1). This value was approximately three orders of magnitude lower than that obtained using the same method but without gold amplification process. Finally, the proposed approach is successful in differentiating between the mutant and wildtype BRAF sequences that are present in genuine 224-nucleotides DNA.
Keywords: Gold nanoparticle biomarkers; Autocatalytic deposition; Square wave stripping voltammetry (SWSV); BRAF gene mutation; Papillary thyroid carcinoma (PTC);

Innovative antimicrobial susceptibility testing method using surface plasmon resonance by Ya-Ling Chiang; Chi-Hung Lin; Muh-Yong Yen; Yuan-Deng Su; Shean-Jen Chen; How-foo Chen (1905-1910).
Utilizing the ultra sensitivity of surface plasmon resonance (SPR) biosensor to examine drug resistance of bacteria was studied in this research. Susceptible and resistant strains of Escherichia coli JM109 to ampicillin and those of Staphylococcus epidermidis to tetracycline, served as a blind test, were examined. The bacteria adhered on the Au thin film was treated by the injection of antibiotic flow. The optical property change of the bacteria responded to antibiotics were recorded through SPR mechanism. As a result, the susceptible strain of E. coli generally revealed more than three times of SPR angle shift when compared to the resistant one; the susceptible strain of S. epidermidis revealed irregular SPR angle shift while the resistant strain kept the SPR angle almost unchanged. The new SPR method took less than 2 h of antibiotic treatment time to complete the antimicrobial susceptibility test. Different from conventional applications of SPR, specific antibodies is not required in this method. As compared to the conventional assays, Kirby-Bauer disk diffusion and variations of broth microdilution usually take 1 day to weeks to issue the report. Using this SPR assay can greatly reduce the waiting period for laboratory tests, and can therefore benefit patients who need proper antibiotic treatments to control bacterial infections. The sensitivity of the SPR biosensor built for the application is around 1.4 × 10−4 on the refractive index.
Keywords: Surface plasmon resonance; Optical biosensor; Antimicrobial susceptibility test; Escherichia coli; Staphylococcus epidermidis; Ampicillin; Tetracycline;

Micro-multi-probe electrode array to measure neural signals by Chang-Hsiao Chen; Da-Jeng Yao; Sin-Hua Tseng; Shao-Wei Lu; Chuan-Chin Chiao; Shih-Rung Yeh (1911-1917).
A multi-electrode array (MEA) with 16 channels was designed to record simultaneously the velocity of conduction of neurons in a measurement system for bio-medical applications. MEA were fabricated with MEMS technology on a silicon-on-insulator (SOI) wafer, which controls the thickness of the probe effectively. All used probes have length 3 mm and width 100 μm. The thickness of the probe, 25 μm, was defined by the thickness of the device layer on the SOI wafer. The multiple probes with a 16-site recording electrode array have been manufactured; their strength was tested with a force gauge and their electrical performance was tested with an impedance measurement system. The readout circuitry comprises an array of 16-site preamplifiers fully integrated on a chip that is capable of signal processing to improve the signal-noise-ratio (SNR). To demonstrate the capability, multiple neural signals were recorded simultaneously with all electrodes from each separate probe. To verify its capability of measuring neural signals, the MEA was used to measure these signals from the electrophysiology system of crayfish. The velocity of neural conduction recorded with a fabricated MEA is shown, and is comparable with a measurement with a traditional glass pipette. The MEA for recording neural signals would be improved in further development.
Keywords: Multi-electrode array; Extracellular record; Conduction Velocity; MEMS;

This paper reports on the synthesis and characterization of a novel electrochemical label for sensitive electrochemical stripping metalloimmunoassays based on silver dendrimer-encapsulated nanoparticles (NPs). Silver dendrimer nanocomposites (Ag-DNCs) were synthesized from a generation 5–7 (G5–7) hydroxyl-terminated ethylenediamine-core-type (2-carbon core) PAMAM dendrimer. Several fixed ratios of Ag+/dendrimer were prepared with the aim to obtain stable nanocomposites with maximal silver loading in the interior of a polymeric shell. Synthesized Ag-DNCs were characterized by UV–vis spectrophotometry, atomic force microscopy (AFM), transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). The G7 Ag-DNC synthesized in 2000 molar excess of silver (1/4 ratio of tertiary amine/Ag+) turned out a more suitable candidate for the label development. By combination of the differential pulse voltammetry (DPV) and the anodic stripping analysis (ASV) on a carbon electrode, down to 1.35 × 10+10 of individual Ag-DNCs (LOD = 0.9 pM, 25 ml volume) was detected after the dissolution of silver nanoparticles in a diluted nitric acid. The potential advantages of proposed electrochemical label are discussed.
Keywords: Dendrimer nanocomposite; Silver enhancement; Stripping analysis; Immunosensors; Metalloimmunoassay;

Functionalizable surface platform with reduced nonspecific protein adsorption from full blood plasma—Material selection and protein immobilization optimization by Hana Vaisocherová; Zheng Zhang; Wei Yang; Zhiqiang Cao; Gang Cheng; Allen D. Taylor; Marek Piliarik; Jiří Homola; Shaoyi Jiang (1924-1930).
In this work, zwitterionic polymers are investigated as ultra-low fouling and functionalizable coatings for biosensors, nanoparticle-based diagnostics, and microarrays to enable detections in real-world complex media. The effect of the spacer length between the two charged groups on the nonfouling properties of zwitterionic poly(carboxybetaine acrylamide) (polyCBAA) was studied in blood plasma and serum. The polyCBAA polymer with an ethylene spacer was selected for protein immobilization studies. A polyCBAA-coated surface was functionalized with antibodies using a simple and fast amino coupling chemistry for direct protein immobilization in two simple steps: surface activation and protein immobilization/background deactivation. The effect of pH was found to be very important for both steps and it was optimized. The functionalized polyCBAA surface exhibited very low fouling properties even when exposed to undiluted blood plasma for more than 6 h with <7 ng/cm2 of adsorbed proteins. The biological activity of the immobilized proteins was demonstrated with the detection of a model protein in undiluted blood plasma. A recently developed highly sensitive four-channel surface plasmon resonance (SPR) sensor was used for the evaluation of specific and nonspecific protein adsorption to these surfaces.
Keywords: Zwitterionic poly(carboxybetaine); Functionalization; Ultra-low fouling; Blood;

Factors affecting the performance of microbial fuel cells for sulfur pollutants removal by Feng Zhao; Nelli Rahunen; John R. Varcoe; Alexander J. Roberts; Claudio Avignone-Rossa; Alfred E. Thumser; Robert C.T. Slade (1931-1936).
A microbial fuel cell (MFC) has been developed for removal of sulfur-based pollutants and can be used for simultaneous wastewater treatment and electricity generation. This fuel cell uses an activated carbon cloth + carbon fibre veil composite anode, air-breathing dual cathodes and the sulfate-reducing species Desulfovibrio desulfuricans. 1.16 g dm−3 sulfite and 0.97 g dm−3 thiosulfate were removed from the wastewater at 22 °C, representing sulfite and thiosulfate removal conversions of 91% and 86%, respectively. The anode potential was controlled by the concentration of sulfide in the compartment. The performance of the cathode assembly was affected by the concentration of protons in the cation-exchanging ionomer with which the electrocatalyst is co-bound at the three-phase (air, catalyst and support) boundary.
Keywords: Microbial fuel cell; Anode; Cathode; Sulfur pollutants; Bacteria;

Controlled torque on superparamagnetic beads for functional biosensors by X.J.A. Janssen; A.J. Schellekens; K. van Ommering; L.J. van IJzendoorn; M.W.J. Prins (1937-1941).
We demonstrate that a rotating magnetic field can be used to apply a controlled torque on superparamagnetic beads which leads to a tunable bead rotation frequency in fluid. Smooth rotation is obtained for field rotation frequencies many orders of magnitude higher than the bead rotation frequency. A quantitative model is developed, based on results from a comprehensive set of experiments at different field strengths and frequencies. At low frequencies (<10 Hz), rotation is due to a small permanent magnetic moment in the bead. At high frequencies (kHz–MHz), the torque results from a phase lag between the applied field and the induced magnetic moment, caused by the non-zero relaxation time of magnetic nanoparticles in the bead. The control of torque and rotation will enable novel functional assays in bead-based biosensors.
Keywords: Superparamagnetic beads; Controlled torque; Rotation; Functional biosensor; Magnetic moment; Nanoparticle size distribution;

Easily made single-walled carbon nanotube surface microelectrodes for neuronal applications by Gemma Gabriel; Rodrigo Gómez; Markus Bongard; Nuria Benito; Eduardo Fernández; Rosa Villa (1942-1948).
The present work examines the feasibility of a simple method for using single-walled carbon nanotubes (SWNT) to fabricate multielectrode arrays (MEA) for electrophysiological recordings. A suspension of purified SWNTs produced by arc discharged was directly deposited onto standard platinum electrodes. The in vitro impedance and electrochemical characterizations demonstrated the enhanced electrical properties of the SWNT microelectrode array. To test its functionality we performed extracellular ganglion cell recordings in isolated superfused rabbit retinas. Our results showed that SWNT based electrode arrays have potential advantages over metal electrodes and can be successfully used to record the single and multi-unit activity of ganglion cell populations.
Keywords: Carbon nanotubes; Impedance spectroscopy; Multielectrode recordings; Retina;

Rapid detection of anti-chromatin autoantibodies in human serum using a portable electrochemical biosensor by Konstantin N. Konstantinov; Ravil A. Sitdikov; Gabriel P. Lopez; Plamen Atanassov; Robert L. Rubin (1949-1954).
The current system employed electrochemical sensor technology for the measurement of autoantibodies in human sera. Anti-chromatin antibodies are an early and sensitive indicator of systemic lupus erythematosus (SLE) and are typically quantified using enzyme-linked immunosorbent assays (ELISA). Electrochemical detection compared favorably with ELISA using a sample of 30 SLE sera (r  = 0.9), and non-specific binding by normal serum immunoglobulin was undetectable. The electrochemical sensor assay required <20 min processing time and utilized a hand-held apparatus with a disposable electrode. These results demonstrate the applicability of this technology to the rapid measurement of a clinically relevant analyte with an apparatus of potential simplicity and low cost.
Keywords: Autoantibody immunoassay; Electrochemical sensor; Point-of-care biosensor;

Electrochemical scanning of DNA point mutations via MutS protein-mediated mismatch recognition by Huan Chen; Xiang-Jun Liu; Ya-Li Liu; Jian-Hui Jiang; Guo-Li Shen; Ru-Qin Yu (1955-1961).
MutS protein is an important part of the DNA repair system which can specifically recognize and bind all possible single-base mismatches as well as 1–4 base insertion or deletion loops with varying affinities independent of other proteins or cofactors. In this paper, a new approach for electrochemical gene mutation detection based on the utilization of MutS protein for the mutation recognition and spontaneously intercalated methylene blue (MB) markers for electrochemical signal generation is described. This method involves the immobilization of MutS protein onto the gold electrode, the hybridization of target DNA to form homoduplex or heteroduplex DNA, the application of MutS protein for the mutation recognition, and finally the intercalation of MB. The background is very low because MutS protein binds DNA containing mispaired and unpaired bases but does not bind equally well to DNA without mismatches or single-stranded DNA. The proposed approach has been successfully implemented for the identification of single-base mutation in −28 site of the β-thalassemia gene with a detection limit of 5.6 × 10−13  M, demonstrating that this method provides a highly specific and cost-efficient approach for point mutation detection.
Keywords: Mediated mismatch recognition; MutS protein; Gene mutation; Methylene blue; Single nucleotide polymorphisms;

Development of a bio-electrochemical assay for AFB1 detection in olive oil by Ines Ben Rejeb; Fabiana Arduini; Adina Arvinte; Aziz Amine; Mohamed Gargouri; Laura Micheli; Camelia Bala; Danila Moscone; Giuseppe Palleschi (1962-1968).
A novel biosensor assay format for aflatoxin based on acetylcholinesterase (AChE) inhibition by aflatoxin B1 (AFB1) is proposed. The AChE was present in solution and an amperometric choline oxidase biosensor was used for monitoring its residual activity. To create the biosensor, the choline oxidase was immobilized by cross-linking onto screen-printed electrodes modified with Prussian Blue (PB) and these were used to detect the H2O2 at low potential (−0.05 V versus a screen-printed internal silver pseudoreference electrode).For the development of the AFB1 assay, several parameters such as AChE and substrate concentration, the methanol effect, and pH were evaluated and optimized. The linear working range was assessed to be 10–60 ppb. Concentrations as low as 2 ppb, which correspond to the legal limit of AFB1 in food for humans, were detected after a pre-concentration step. The suitability of the method was evaluated using commercial olive oil samples. A recovery equal to 78 ± 9% for 10 ppb of AFB1 in olive oil samples was obtained.
Keywords: Acetylcholinesterase; Aflatoxin B1; Inhibition; Prussian Blue; Screen-printed electrode; Olive oil;

Optical modeling of bioluminescence in whole cell biosensors by Hadar Ben-Yoav; Tal Elad; Omer Shlomovits; Shimshon Belkin; Yosi Shacham-Diamand (1969-1973).
Bioluminescence-based whole cell biosensors are devices that can be very useful for environmental monitoring applications. The advantages of these devices are that they can be produced as a single-chip, low-power, rugged, inexpensive component, and can be deployed in a variety of non-laboratory settings. However, such biosensors encounter inherent problems in overall system light collection efficiency. The light emitted from the bioluminescent microbial cells is isotropic and passes through various media before it reaches the photon detectors. We studied the bioluminescence distribution and propagation in microbial whole cell biochips. Optical emission and detection were modeled and simulated using an optical ray tracing method. Light emission, transfer and detection were simulated and optimized with respect to two fundamental system parameters: system geometry and bacterial concentration. Optimization elucidated some of the optical aspects of the biochip, e.g. detector radius values between 300 and 750 μm, and bacterial fixation radius values between 800 and 1200 μm. Understanding theses aspects may establish a basis for future optimization of similar chips.
Keywords: Whole cell biosensor; Bioluminescence; Stray light analysis; Water toxicity; Optical modeling; Biochip;

Design and validation of a low cost surface plasmon resonance bioanalyzer using microprocessors and a touch-screen monitor by Jiandong Hu; Jingfang Hu; Fukun Luo; Wei Li; Guoliang Jiang; Zhengfeng Li; Runna Zhang (1974-1978).
An economical and high-performance bioanalyzer, with no use of laptop computer, based on the use of TSPR1k23 biosensors was systematically designed, and validated experimentally for its high performance. The analyzer is composed of a micro-flow cell, a thermoelectric cooler (TEC), a clamp, a touch-screen monitor, and an electronic control unit (ECU) incorporated with photoelectric conversion device. The micro-flow cell is made of stainless steel with high thermal conductivity, and the micro-flow system is based on PID temperature-controlled algorithm to keep the constant temperature (25 °C) of the liquid sample via thermal exchange with the clamp. With a peristaltic pump implemented by an injection loop flow system, the bioanalyzer allows the core sensor to be completely exposed to samples. The touch-screen monitor displays the normalized response signal values updated every 0.25 s, with a typical noise level less than 5 RU (response unit) within 2 h. The bioanalyzer was validated using hepatitis B surface antigen (HBsAg) as an example. Anti-HBsAg monoclonal antibody is adhered to the surface of the sensor chip by a bifunctional cross-linker with the technology of self-assembly. The duration of the HBsAg measurement only lasts 5 min with a dilution factor ranging from 200 to 1200, optimized with a R-squared value 0.998. The results suggested that the bioanalyzer has higher selectivity, lower cost, expanded detection limit, and shorter measuring time as compared with the HBsAg ELISA kit, especially for low concentrations of analyte.
Keywords: Bioanalyzer; Surface plasmon resonance; Micro-flow cell; Touch-screen monitor;

We report a sensitively amplified electrochemical aptasensor using adenosine triphosphate (ATP) as a model. ATP is a multifunctional nucleotide that is most important as a “molecular currency” of intracellular energy transfer. In the sensing process, duplexes consisting of partly complementary strand (PCS1), ATP aptamer (ABA) and another partly complementary strand (PCS2) were immobilized onto Au electrode through the 5′-HS on the PCS1. Meanwhile, PCS2 was grafted with the Au nanoparticles (AuNPs) to amplify the detection signals. In the absence of ATP, probe methylene blue (MB) bound to the DNA duplexes and also bound to guanine bases specifically to produce a strong differential pulse voltammetry (DPV) signal. But when ATP exists, the ABA-PCS2 or ABA-PCS1 part duplexes might be destroyed, which decreased the amount of MB on the electrode and led to obviously decreased DPV signal. This phenomenon can be used to detect ATP and get a very sensitive detection limit low to 0.1 nM, and the detection range could extend up to 10−7  M. Compared to the sensing platform without PCS2 grafted AuNPs, amplified function of this sensing system was also evidently proved. Therefore, such PCS1-ABA-PCS2/AuNPs sensing system could provide a promising signal-amplified model for aptamer-based small-molecules detection.
Keywords: Electrochemical aptasensor; Au nanoparticles; ATP detection; Small molecule; Methylene blue;

Disposable electrochemical immunosensor for carcinoembryonic antigen using ferrocene liposomes and MWCNT screen-printed electrode by Subramanian Viswanathan; Chinnakkaruppanan Rani; Annadurai Vijay Anand; Ja-an Annie Ho (1984-1989).
Disposable electrochemical immunosensor for the detection of carcinoembryonic antigen (CEA) in saliva and serum was developed. Monoclonal anti-CEA antibodies (αCEA) were covalently immobilized on polyethyleneimine wrapped multiwalled carbon nanotubes screen-printed electrode. A sandwich immunoassay was performed with CEA and αCEA tagged ferrocene carboxylic acid encapsulated liposomes (αCEA-FCL). The square wave voltammetry (SWV) was employed to analyze faradic redox responses of the released ferrocene carboxylic acid from the immunoconjugated liposomes on the electrode surface. The magnitude of the SWV peak current was directly related to the concentration of CEA. The calibration curve for CEA concentration was in the range of 5 × 10−12 to 5 × 10−7  g mL−1 with a detection limit of 1 × 10−12  g mL−1 (S/N = 3). This method provides a high precise and sensitive determination of CEA in human blood serum and saliva samples.
Keywords: Immunosensors; Carcinoembryonic antigen; Liposome; Carbon nanotube; Screen-printed electrode;

Mycoplasma genitalium (Mg) is the smallest and simplest self-replicating bacteria lacking of cell wall and is a human pathogen causing various diseases. This paper describes the real-time, long-term and in situ monitoring of the growth of Mg and evaluation of the effect of the antibiotics tetracycline and levofloxacin on the growth using a wireless magnetoelastic sensor. The sensor is fabricated by coating a magnetoelastic strip with a polyurethane protecting film. In response to a time-varying magnetic field, the sensor longitudinally vibrates at a resonance frequency, emitting magnetic flux that can be remotely detected by a pick-up coil. No physical connections between the sensor and the detection system are required. The wireless property facilitates aseptic operation. The adhesion of Mg on the sensor surface results in a decrease in the resonance frequency, which is proportional to the concentration of Mg. The shift of the resonance frequency–time curves shows that under routine culture condition the growth curve of Mg is composed of three phases those are lag, logarithmic and stationary phase, respectively. In the presence of the antibiotics, the lag phase in the growth inhibition curves is prolonged obviously and the stationary phase is substituted by a decline phase. The growth inhibition of Mg is related to the concentration of the antibiotics. The MIC50 (minimal inhibitory concentration) of Mg incubated in the presence of the antibiotics for 120 h is calculated to be 1.5 and 0.5 μg/mL for tetracycline and levofloxacin, respectively.
Keywords: Magnetoelastic sensor; Mycoplasma genitalium; Wireless; Tetracycline; Levofloxacin;

Optical biosensing based on fluorescence detection has arguably become the standard technique for quantifying extents of hybridization between surface-immobilized probes and fluorophore-labeled analyte targets in DNA microarrays. However, electrochemical detection techniques are emerging which could eliminate the need for physically bulky optical instrumentation, enabling the design of portable devices for point-of-care applications. Unlike fluorescence detection, which can function well using a passive substrate (one without integrated electronics), multiplexed electrochemical detection requires an electronically active substrate to analyze each array site and benefits from the addition of integrated electronic instrumentation to further reduce platform size and eliminate the electromagnetic interference that can result from bringing non-amplified signals off chip. We report on an active electrochemical biosensor array, constructed with a standard complementary metal-oxide-semiconductor (CMOS) technology, to perform quantitative DNA hybridization detection on chip using targets conjugated with ferrocene redox labels. A 4 × 4 array of gold working electrodes and integrated potentiostat electronics, consisting of control amplifiers and current-input analog-to-digital converters, on a custom-designed 5 mm × 3 mm CMOS chip drive redox reactions using cyclic voltammetry, sense DNA binding, and transmit digital data off chip for analysis. We demonstrate multiplexed and specific detection of DNA targets as well as real-time monitoring of hybridization, a task that is difficult, if not impossible, with traditional fluorescence-based microarrays.
Keywords: DNA detection; CMOS; Electrochemical biosensor array;

Based on the newly discovered artificial enzyme formed by mixing hemin with supramolecular hydrogels via the self-assembly of amphiphilic oligopeptides, we prepared a novel organic phase artificial enzyme electrode by coating the artificial enzyme on an electrode which was then covered with sodium alginate for protection. Scanning electron micrograph showed that the supramolecular hydrogel kept its nanofibers structure on the electrode surface. Hemin dispersed in the supermolecular hydrogel as monomer greatly promotes its direct electrochemistry behavior in organic solvents. At the same time, this electrode exhibited higher electrocatalytic ability to tert-butyl hydroperoxide (TBHP) than free hemin modified electrode (free hemin mainly present as dimer). As low as 27 μM TBHP could be detected with a linear range from 6.6 × 10−5 to 1.27 × 10−2  M via amperometric method. The biosensor can reach 95% of the steady-state current in about 10 ± 2 s. More importantly, it can be applied in both hydrophilic and hydrophobic solvents without adding extra buffer or mediators to them that cannot be received by most traditional organic phase enzyme electrodes. This unique property greatly promotes the development of the organic phase enzyme electrodes by facilitating the detection of different kinds of substrates of the hemin-based artificial enzyme soluble in hydrophilic and hydrophobic solvents. The artificial enzyme electrode was successfully used to determine organic peroxides in body lotion samples.
Keywords: Artificial enzyme electrode; Hemin; tert-Butyl hydroperoxide; Nonaqueous solvents; Supermolecular hydrogel;

Hollow spherical nanostructured polydiphenylamine for direct electrochemistry and glucose biosensor by P. Santhosh; K.M. Manesh; S. Uthayakumar; A.I. Gopalan; K.-P. Lee (2008-2014).
Nanostructured, hollow spheres of polydiphenylamine (HS-PDPA) are prepared through a “soft template assisted self-assembly” approach. An enzymatic glucose biosensor is fabricated through immobilizing glucose oxidase (GOx) into HS-PDPA matrix. The HS-PDPA–GOx electrode exhibits a pair of well-defined reversible redox peaks with a fast heterogeneous electron transfer rate. At an applied potential of +0.65 V, HS-PDPA–GOx electrode possesses high sensitivity (1.77 μA mM−1  cm−2), stability and reproducibility towards glucose. The amperometric current response of HS-PDPA–GOx to glucose is linear in the concentration range between 1 and 28 mM with a detection limit of 0.05 mM (S/N = 3). Also, HS-PDPA–GOx electrode shows high selectivity towards glucose in the presence of ascorbic acid, uric acid and acetaminophen at their maximum physiological concentrations.
Keywords: Nanostructured conducting polymer; Polydiphenylamine; Glucose oxidase; Glucose; Amperometric biosensor;

Here we report a new glucose sensing electrode based on a poly(3,4-ethylenedioxythiophene) (PEDOT)/Prussian blue (PB) bilayer and multi-walled carbon nanotubes (CNT). The bilayer was prepared on a flexible screen-printed carbon electrode (SPCE) by sequential electrodeposition. The inner PB layer was electrodeposited first for detecting H2O2 from glucose oxidation; the outer PEDOT layer was electropolymerized on a baked or an unbaked PB film to entrap glucose oxidase (GOD). It was observed that the stability of PB in phosphate buffered saline (pH 7.4) was attained by post-deposition bake at 100 °C and the outer PEDOT layer both. In addition, a baked PB film enhanced the subsequent PEDOT growth and the corresponding GOD entrapment. As a result, the bilayer enzyme electrode showed highly resolved and reproducible signals (R.S.D. = 2.54%) to glucose samples from 100 μM to 1 M during a flow-injection analysis (FIA) at −0.1 V vs. Ag/AgCl. The sensitivity of the linear range (1–10 mM) was 2.67 μA cm−2  mM−1. Moreover, the electrode retained ca. 82% of the original response after 1-month storage in PBS, pH 6.0 at 4 °C and could determine the glucose level in human serum precisely. Besides, it was found that CNT incorporation could further improve the sensitivity and could achieve μM-range glucose detection.
Keywords: Glucose; Flexible biosensor; Flow-injection assay (FIA); Multi-walled carbon nanotubes (MWCNTs); Prussian blue (PB); Poly(3,4-etheylenedioxythiophene) (PEDOT); Screen printing;

Function of microbial fuel cell (MFC) as bio-electrochemical treatment system in concurrence with power generation was evaluated with composite chemical wastewater at high loading conditions (18.6 g COD/l; 56.8 g TDS/l). Two dual chambered MFCs [non-catalyzed graphite electrodes; mediatorless anode] were studied separately with aerated and potassium ferricyanide catholytes under similar anodic operating conditions [mixed consortia; pH 6]. Marked improvement in power output was observed at applied higher substrate loading rate for extended period of time without any process inhibition. Catholyte nature showed significant influence on power generation [ferricyanide—651 mV; 18.22 mA; 6230 mW/kg CODR (500 Ω); 2321.69 mA/m2 (100 Ω); 11.80 mW/m3 and aerated—578 mV; 10.23 mA; 2450 mW/kg CODR (400 Ω); 1220.68 mA/m2 (100 Ω); 5.64 mW/m3] but not on wastewater treatment efficiency. Along with enhanced substrate degradation, relatively good removal of color (31%) and TDS (51%) was also observed during MFC operation, which might be attributed to the diverse bio-electrochemical processes triggered due to substrate metabolism and subsequent in situ bio-potential (voltage) generation. Apart from power generation, various unit operations pertaining to wastewater treatment viz., biological (anaerobic) process, electrochemical decomposition and electrochemical oxidation were found to occur symbiotically in the anode chamber. Among them anaerobic metabolism is considered to be a crucial and important rate limiting step. In view of inherent advantages, function of MFC as integrated bio-electrochemical treatment system in the direction of various wastewater treatment operations can be exploited.
Keywords: Bio-potential; Electrochemical oxidation; Color; Mixed consortia; Chemical wastewater treatment; Total dissolved solids (TDS);

Novel enzyme biosensor for hydrogen peroxide via supramolecular associations by Conrado Camacho; Belkis Chico; Roberto Cao; Juan C. Matías; Javier Hernández; Ilaria Palchetti; Benjamin K. Simpson; Marco Mascini; Reynaldo Villalonga (2028-2033).
A polythiolated-β-cyclodextrin polymer was synthesized and used as a coating material for gold electrodes. The functionalized electrodes were employed for immobilizing adamantane-modified horseradish peroxidase via supramolecular associations. The enzyme-containing electrode was used as an amperometric biosensor device with 1 mM hydroquinone as electrochemical mediator. The biosensor exhibited a fast amperometric response (10 s), a good linear response toward H2O2 concentrations between 28 μM and 5.5 mM, and a low detection limit of 7 μM. The biosensor showed a sensitivity of 109 μA/M cm2 and retained 98% of its initial electrocatalytic activity after 40 days of storage at 4 °C in 50 mM sodium phosphate buffer pH 7.0. The host–guest supramolecular nature of the immobilization method was confirmed by cyclic voltammetry.
Keywords: Biosensor; β-Cyclodextrin; Enzyme immobilization; Horseradish peroxidase; Supramolecular complex;

An optical sensor for detection of glucose is implemented by incorporating a carbohydrate sensitive hydrogel as a Fabry-Perot cavity at the end of optical fiber for high sensitivity readout of the gel length. The glucose sensing functionality was achieved by incorporating boronic acid moieties into an acrylamide-based hydrogel. The interaction between glucose and boronic acid changes the driving forces for gel swelling thus inducing a glucose sensitive hydrogel swelling. The effects on the carbohydrate swelling response, with respect to sensitivity and selectivity, by incorporation of a cationic monomer, dimethyl-aminopropyl acrylamide, into the boronic acid functionalized responsive gels were determined. The linear gel swelling response in aqueous solutions at aqueous 2.5 mM carbohydrates were determined to −1760 nm/mM for glucose whereas mannose, sucrose, fructose and galactose displayed a response of about 10% of the glucose response for the hydrogels containing 10 mol% dimethylaminopropyl acrylamide. This gel composition with 10 mol% dimethylaminopropyl acrylamide is the most promising for detection of glucose at physiological pH and ionic strength. A mechanism where carbohydrate specific stabilisation of the boronic acid group and possible carbohydrate mediated additional crosslinking of the elastically active polymer chains is suggested.
Keywords: Hydrogel; Swelling; Glucose sensor; Nanometer resolution; Fiber-optic;

Nanoporous cerium oxide thin film for glucose biosensor by Shibu Saha; Sunil K. Arya; S.P. Singh; K. Sreenivas; B.D. Malhotra; Vinay Gupta (2040-2045).
Nanoporous cerium oxide (CeO2) thin film deposited onto platinum (Pt) coated glass plate using pulsed laser deposition (PLD) has been utilized for immobilization of glucose oxidase (GOx). Atomic force microscopy studies reveal the formation of nanoporous surface morphology of CeO2 thin film. Response studies carried out using differential pulsed voltammetry (DPV) and optical measurements show that the GOx/CeO2/Pt bio-electrode shows linearity in the range of 25–300 mg/dl of glucose concentration. The low value of Michaelis-Menten constant (1.01 mM) indicates enhanced enzyme affinity of GOx to glucose. The observed results show promising application of the nanoporous CeO2 thin film for glucose sensing application without any surface functionalization or mediator.
Keywords: Glucose; Cerium oxide; Biosensor;

A diamond-based biosensor for the recording of neuronal activity by Paolo Ariano; Alessandro Lo Giudice; Andrea Marcantoni; Ettore Vittone; Emilio Carbone; Davide Lovisolo (2046-2050).
We have developed a device for recording the extracellular electrical activity of cultured neuronal networks based on a hydrogen terminated (H-terminated) conductive diamond. GT1-7 cells, a neuronal cell line showing spontaneous action potentials firing, could maintain their functional properties for days in culture when plated on the H-terminated diamond surface. The recorded extracellular electrical activity appeared in the form of well-resolved bursts of fast and slow biphasic signals with a mean duration of about 8 ms for the fast and 60 ms for the slow events. The time courses of these signals were in good agreement with those recorded by means of conventional microelectrode array (MEAs) and with the negative derivative of the action potentials intracellularly recorded with the patch clamp technique from single cells. Thus, although hydrophobic in nature, the conductive H-terminated diamond surface is able to reveal the spontaneous electrical activity of neurons mainly by capacitative coupling to the cell membrane. Having previously shown that the optical properties of H-terminated diamond allow to record cellular activity by means of fluorescent probes (Ariano, P., Baldelli, P., Carbone, E., Giardino, A., Lo Giudice, A., Lovisolo, D., Manfredotti, C., Novara, M., Sternschulte, H., Vittone, E., 2005. Diam. Relat. Mater. 14, 669–674), we now provide evidence for the feasibility of using diamond-based cellular biosensors for multiparametrical recordings of electrical activity from living cells.
Keywords: Diamond; Hydrogen terminated diamond; Neuronal cells; Electrical activity; Extracellular recording;

Ultrasensitive immunoassay of 7-aminoclonazepam in human urine based on CdTe nanoparticle bioconjugations by fabricated microfluidic chip by Wei Chen; Chifang Peng; Zhengyue Jin; Ruirui Qiao; Wuyang Wang; Shuifang Zhu; Libing Wang; Qinhui Jin; Chuanlai Xu (2051-2056).
The present paper described a rapid and ultrasensitive detection method using a microfluidic chip for analyzing 7-aminoclonazepam (7-ACZP) residues in human urine. A microfluidic chip-based immunoassay with laser-induced fluorescence (LIF) detection based on the water-soluble denatured bovine serum albumin (dBSA)-coated CdTe quantum dots (QDs) was prepared for the ultrasensitive detection of 7-ACZP. The whole procedure including the chip and the control software was designed and constructed in our own laboratory. The detection of 7-ACZP could be completed within 5 min. The results demonstrated that under the optima conditions, 7-ACZP residues could be detected with a precision of 5% relative standard deviation (RSD), and the linear range and the limit of detection (LOD) for 7-ACZP were 1.1–60.1 and 0.021 ng mL−1, respectively. This method was compared with ELISA and showed a good correlation. This microfluidic chip with LIF detection was applied to the determination of 7-ACZP residues in positive human urine samples, and the results were confirmed by high-performance liquid chromatography and tandem mass spectrometry (LC/MS/MS). This ultrasensitive detection technique was proved to be practical for clinical use.
Keywords: Ultrasensitive; Immunoassay; 7-Aminoclonazepam; CdTe nanoparticle; Microfluidic Chip; LC/MS/MS;

Immunoassay for folic acid detection in vitamin-fortified milk based on electrochemical magneto sensors by A. Lermo; S. Fabiano; S. Hernández; R. Galve; M.-P. Marco; S. Alegret; M.I. Pividori (2057-2063).
An immunoassay-based strategy for folic acid in vitamin-fortified milk with electrochemical detection using magneto sensors is described for the first time. Among direct and indirect competitive formats, best performance was achieved with an indirect competitive immunoassay. The immunological reaction for folic acid (FA) detection was performed, for the first time on the magnetic bead as solid support by the covalent immobilization of a protein conjugate BSA-FA on tosyl-activated magnetic bead. Further competition for the specific antibody between FA in the food sample and FA immobilized on the magnetic bead was achieved, followed by the reaction with a secondary antibody conjugated with HRP (AntiIgG-HRP). Then, the modified magnetic beads were easily captured by a magneto sensor made of graphite–epoxy composite (m-GEC) which was also used as the transducer for the electrochemical detection. The performance of the immunoassay-based strategy with electrochemical detection using magneto sensors was successfully evaluated using spiked-milk samples and compared with a novel magneto-ELISA based on optical detection. The detection limit was found to be of the order of μg l−1 (13.1 nmol l−1, 5.8 μg l−1) for skimmed milk. Commercial vitamin-fortified milk samples were also evaluated obtaining good accuracy in the results. This novel strategy offers great promise for rapid, simple, cost-effective and on-site analysis of biological and food samples.
Keywords: Folic acid; Electrochemical immunosensor; Indirect competitive immunoassay; Magnetic beads; Fortified milk;

A new strategy for the electrochemical detection and signal amplification of DNA at gold electrodes is described. Current methodologies for DNA biosensing based on the electrochemical detection of electroactive base-specific labels such as methylene blue (MB) suffer from lengthy incubation and washing steps. Addressing these limitations, we report a novel approach for the electrochemical quantification of surface hybrid, using the control gene LTA, 107 bases long, as a model target. An array of 15 gold electrodes was used to detect the formation of hybridised duplex following interaction of non-hybridised guanine bases with MB present in solution. Upon hybridisation the number of free guanines present at the electrode surface increased from 8 to 25 due to guanine bases present in the target sequence which did not participate in hybridisation and remained free to interact directly with methylene blue. This increase in free guanines consequently concentrated MB directly at the electrode surface. We found that the MB signal recorded for 100 nM of the complementary LTA was typically 2.14 times higher than that of the non-hybridised state. Very low cross-reactivity (<7%) with a non-complementary probe was recorded. The assay was optimised with regards to methylene blue concentration, hybridisation time and regeneration. The assay was quantitative and linear in the range of 6.25–50 nM target DNA exhibiting an LOD of 17.5 nM. The assay was rapid and easy to perform, with no need for lengthy incubations with the methylene blue label or requirement for washing steps. Ongoing work addresses the impact of guanine location on the signal in order to tailor design specific signalling domains of PCR products.
Keywords: Electrode array; Methylene blue; DNA detection; Clinical diagnostics;

Design rule for optimization of microelectrodes used in electric cell-substrate impedance sensing (ECIS) by Dorielle T. Price; Abdur Rub Abdur Rahman; Shekhar Bhansali (2071-2076).
This paper presents an experimentally derived design rule for optimization of microelectrodes used in electric cell-substrate impedance sensing (ECIS) up to 10 MHz. The effect of change in electrode design (through electrode sensor area, lead trace widths, and passivation coating thickness) on electrode characteristics was experimentally evaluated using electrochemical impedance spectroscopy (EIS) measurements and analyzed using equivalent circuit models. A parasitic passivation coating impedance was successfully minimized by designing electrodes with either a thicker passivation layer or a smaller lead trace area. It was observed that the passivated lead trace area to passivation coating thickness ratio has a critical value of 5.5, under which the impedance contribution of the coating is minimized. The optimized design of ECIS-based microelectrode devices reported in this work will make it possible to probe the entire beta dispersion region of adherent biological cell layers by reducing measurement artifacts and improving the quality of data across the beta-dispersion region. The new design will enable the use of the commonly used ECIS technique to measure real-time cellular properties in high frequency ranges (beta dispersion) that was not possible thus far.
Keywords: ECIS optimization; Passivation coating capacitance; Microelectrode;

Screen printing as cost-efficient fabrication method for DNA-chips with electrical readout for detection of viral DNA by Thomas Schüler; Tim Asmus; Wolfgang Fritzsche; Robert Möller (2077-2084).
The fast development in the field of DNA analytics is driven by the need for cost-effective and high-throughput methods for the detection of biomolecules. The detection of DNA using metal nanoparticles as labels is an interesting alternative to the standard fluorescence technique. Fluorescence is highly sensitive and broadly established, but shows limitations, for example instability of the signal and the requirement for sophisticated and high-cost equipment. A recently developed approach realizes a method for the electrical detection of DNA, based on the induction of silver nanoparticles growth in microelectrode gaps on the surface of a DNA-chip. This breakthrough towards robust and cost-effective detection was still hampered by the need for microstructured (and therefore expensive) substrates. We demonstrate that it is possible to utilize screen printed electrode structures for a chip-based electrical DNA detection. The electrode structures were produced on a glass substrate which made an additional optical readout possible. The screen printed structures show the required precision and are compatible with the applied biochemical protocols. A comparison with chip substrates produced by standard photolithography showed the same sensitivity and specificity for the screen printed chips. Screen printing of electrode structures for DNA-chip with electrical detection offers an interesting and cost-efficient possibility to produce DNA-chips with microstructured electrodes.
Keywords: Electrical DNA-chip; Horseradish peroxidase; Enzyme induced silver deposition; Screen printed electrodes;

Polyaniline (PAN) nano-particles, nano-fibrils, and nano-network have been synthesized via electrochemical polymerization of aniline using a three-step electrochemical deposition procedure on α-alanine (ALA)-monolayer functionalized glassy carbon electrode (GCE). The structure and properties of PAN nano-structures have been characterized using field emission scanning electron microscope (SEM), Fourier transform infrared spectra (FT-IR), and electrochemical techniques. The 3-dimensional (3D) PAN nano-network/ALA composite film coated GCE (PAN–ALA/GCE) leads to the direct electrochemistry of horse heart cytochrome c (Cyt c) immobilized on this electrode surface. The immobilized Cyt c maintains its activity, showing a surface-controlled electrode process with the electron transfer rate constant (k s) of 21.9 s−1 and the charge-transfer coefficient (α) of 0.37, and could be used for the electrocatalytic reduction of hydrogen peroxide (H2O2). The steady-state current response increases linearly with H2O2 concentration from 2.5 × 10−5 to 3.0 × 10−4  mol l−1. The detection limit (3δ) is 8.2 × 10−6  mol l−1.
Keywords: Polyaniline; Cytochrome c; α-Alanine; Hydrogen peroxide;

A novel and simple strategy for selective and sensitive determination of dopamine based on the boron-doped carbon nanotubes modified electrode by Chunyan Deng; Jinhua Chen; Mengdong Wang; Chunhui Xiao; Zhou Nie; Shouzhuo Yao (2091-2094).
The Boron-doped carbon nanotubes (BCNTs) modified glassy carbon (GC) electrode was obtained simply and used for highly selective and sensitive determination of dopamine (DA). Comparing with the bare GC and CNTs/GC electrodes, the BCNTs have higher catalytic activity toward the oxidation of DA and ascorbic acid (AA). Moreover, the voltammetric peaks of AA and DA were separated enough (ca. 238 mV) at the BCNTs/GC electrode, which is superior to that at the CNTs/GC electrode (ca. 122 mV). Thus, the selective determination of DA was carried out successfully in the presence of AA. A wide concentration range (2.0 × 10−8–7.5 × 10−5  M) and low detection limit (1.4 nM, S/N = 3) for the DA detection were obtained. The possibility of the BCNTs/GC electrode for the determination of DA in human blood serum has also been evaluated. The advantageous properties of this electrode for the DA determination lie in its excellent catalytic activity, selectivity and simplicity. The more edge plane sites presented on the BCNTs surface were partially responsible for its good analytical behavior.
Keywords: Boron-doped carbon nanotubes (BCNTs); Dopamine (DA); Ascorbic acid (AA); Differential pulse voltammetry (DPV);

A new label-free, homogenous, sensitive and economical one-step method to detect SNP genotyping of genomic DNA has been developed by combining allele-specific PCR technique with water-soluble cationic conjugated polyelectrolytes (CCP). The amplification of target DNA and fluorescence detection steps are combined into one-step. The target DNA fragment containing a G allele site acts as PCR template. For the G allele-specific forward primer whose 3′-terminal base is complementary to the G allele template, after the first step of reverse primer extension, G allele-specific primer perfectly anneals with newly formed strand and the extension reaction of forward primer starts. During the extension, dGTP-Fl and dUTP-Fl are incorporated into extension chain in the presence of Taq DNA polymerase and more fluorescein-labeled PCR amplicons are yielded. Upon adding the CCP, strong electrostatic interactions between DNA and CCP bring them close and efficient FRET from CCP to fluorescein occurs. For the C allele-specific forward primer, less fluorescein-labeled PCR amplicons are yielded and inefficient FRET occurs. By triggering the change of emission intensity of CCP and fluorescein, it is possible to assay the SNP genotypes. In contrast to previous reports, this method does not require designing dye-labeled primers, and gel electrophoresis and isolation step after PCR were avoided in this homogenous method. The genotyping of 50 ng genomic DNA from human lung cancer cell is easily detected using our new method. These qualities will make the new detection system ideal for SNP genotyping.
Keywords: PCR; Conjugated polyelectrolytes; DNA; SNP genotyping; FRET; Fluorescent sensor;

Surface plasmon resonance spectro-imaging sensor for biomolecular surface interaction characterization by Fabrice Bardin; Alain Bellemain; Gisèle Roger; Michael Canva (2100-2105).
Surface plasmon resonance (SPR) techniques have become, over the last ten years, powerful tools to study biomolecular surface interaction kinetics in real-time without any use of labels. The highest resolution is currently obtained using spectroscopic SPR systems through the measurement of the complete surface plasmon resonance curve in angular or spectral configuration. But, these systems are limited to a few independent channels (<10). In order to expand their capability to an array format, SPR sensors have also been developed in an imaging mode, allowing parallel monitoring of hundreds of sensing spots onto a camera. However, such sensors rely on the intensity variation measurement at a single position of the resonance spectrum, hence resulting in smaller resolution. We present in this work a SPR spectro-imaging system which aims at keeping the advantage of a mono-channel SPR sensor based on the full resonance curve measurement while introducing an additional spatial dimension (linear multi-spot array). The system is based on the illumination of a biochip through a vertical slit (y-dimension) by a white light source. The reflected light spectrum obtained through a diffracting grating is then imaged on the x-dimension of the camera. The complete spectral resonance curve of a full column of sensing spots can be monitored in parallel and in real-time. We demonstrate that data processing is key to reduce the noise and to improve the resolution. We report on the detection of signals with resolution comparable to the one obtained with a classical SPR mono-channel spectroscopic sensor (3.5 × 10−7 Refractive Index Unit), gaining an order of magnitude compared to SPR imaging sensors. Eventually, we show that short base DNA–DNA hybridizations with concentrations as low as 100 pM can be detected and discriminated in a few tens of minutes following injection by the SPR spectro-imaging system.
Keywords: Surface plasmon resonance imaging; Spectral SPR imaging; DNA microarray;

Development of an open stand-alone platform for regenerable automated microarrays by Katrin Kloth; Reinhard Niessner; Michael Seidel (2106-2112).
A novel automated chemiluminescence (CL) read-out system for analytical flow-through microarrays based on multiplexed immunoassays has been developed. The microarray chip reader (MCR 3) is designed as a stand-alone platform, with the goal to quantify multiple analytes in complex matrices of food and liquid samples for field analysis or for routine analytical laboratories. The CL microarray platform is a self-contained system for the fully automated multiplexed immunoanalysis: the microarray chip, the fluidic system and the software module enable automated calibration and determination of analyte concentrations during a whole working day. The detection of antibiotics in milk was demonstrated to validate this device. There are few quantitative multi-residue detection methods for routine analysis although the EU has defined maximum residue limits (MRLs) for a number of antibacterial reagents. Therefore, an automated multianalyte detection instrument is needed quantifying simultaneously antibiotics within some minutes. Also regeneration is required to avoid replacing the assay surface. The MCR 3 uses a microarray chip, which consists of two channels for parallel measurement and regeneration. The microarray chip is designed for parallel analysis of up to 13 different antibiotics in milk applying an indirect competitive microarray immunoassay (MIA). Microspotted antibiotics are directly coupled to epoxylated PEG surfaces. As an initial example, penicillin G is quantified in milk on the MCR 3. The penicillin G surface is regenerable for 47 measurement cycles per channel. A limit of detection (LOD) of 1.1 μg/L is achieved by an assay time of 6 min.
Keywords: Chemiluminescence; Analytical microarray; Multiplex immunoassay; Microfluidic; Automated microarray reader;

Sensor materials for the detection of proteases by Jacqueline L. Stair; Michael Watkinson; Steffi Krause (2113-2118).
The concept of generic and tunable sensor materials for the detection of proteases based on the thin film degradation of peptide cross-linked dextran hydrogels was explored. Hydrogel cross-links were formed via simple imine linkages between aldehyde groups in oxidized dextran and a peptide sequence susceptible to protease cleavage. Degradation of the hydrogel films was monitored in this study using a quartz crystal microbalance (QCM). The sensor material was developed using the protease/peptide pair of human neutrophil elastase (HNE) and Ala-Ala-Pro-Val-Ala-Ala-Lys (AAPVAAK). A direct relationship between the hydrogel degradation rate and protease activity was observed; HNE activities from 2.5 to 30 U ml−1 were detected using 25% cross-linked films. Film degradation was rapid and was complete in less than 10 min for HNE activities ≥10 U ml−1. An increase in the rate of degradation by a factor of 3.5 was achieved by increasing the cross-linking density from 25% to 75%. QCM admittance data fitted with a BVD equivalent circuit showed increases in film viscoelasticity upon enzyme addition. A second protease/peptide pair of cathepsin G and Ala-Ala-Pro-Phe-Phe-Lys (AAPFFK) was tested where 25% AAPFFK cross-linked hydrogels demonstrated a rapid response at 100 mU ml−1. Swapping the protease/peptide pairs to HNE/AAPFFK and cathepsin G/AAPVAAK showed low levels of cross-sensitivity further demonstrating the specificity of film degradation.
Keywords: Protease; Enzymatic degradation; Peptide cross-linked hydrogel; Quartz crystal microbalance (QCM); Human neutrophil elastase (HNE); Cathepsin G;

Kinetic insight into the mechanism of cholinesterasterase inhibition by aflatoxin B1 to develop biosensors by Tamara Hansmann; Benoît Sanson; Jure Stojan; Martin Weik; Jean-Louis Marty; Didier Fournier (2119-2124).
In this paper, the inhibition effect of aflatoxin B1 on different species of cholinesterases was investigated to unravel action mechanism. The inhibition curves of several cholinesterase mutants (obtained by spectrophotometric measurements of enzyme activity, pS curves) were analyzed. They showed that this toxin reversibly inhibits cholinesterases by binding to a peripheral site located at the entrance of the active site gorge without entering inside the site. Electric eel enzyme revealed the highest inhibition extent with a binding constant estimated to 0.35 μM. This binding prevents the entrance of substrate en route to the catalytic site and also decreases chemical steps of the reaction at the catalytic site: acetylation is reduced to the half and deacetylation is reduced to the third. Electric eel acetylcholinesterase was used to settle an amperometric biosensor. The best detection was obtained by using 0.3 mU enzyme on the electrode and 0.5 mM ATCh in the solution. The limit of detection was 3 μM corresponding to 20% inhibition.
Keywords: Acetylcholinesterase; Aflatoxin B1; Enzyme; Inhibition; Biosensor; Peripheral site;

A newly flow-through electrochemical immunosensor for monitoring IgG in human serum has been developed by using core-shell SiO2/Au nanocomposites and poly(amidoamine) G4 dendrimer as matrices. The ferrocenecarboaldehyde-labeled anti-IgG biomolecules were initially chemisorbed onto the nanoparticle surface, and then glucose oxidase (GOx), as a blocking reagent instead of bovine serum albumin (BSA), was backfilled onto the modified surface. The formation of the antibody–antigen complex by a simple one-step immunoreaction between the immobilized anti-IgG and IgG in sample solution introduced a barrier of direct electrical communication between the immobilized GOx and the base surface, and decreased the immobilized GOx toward the catalytic oxidation of glucose. The performance and factors influencing the performance of the immunosensor were evaluated. Under optimal conditions, the linear range of the developed immunosensor by using GOx as enhancer was from 5.0 × 10−6 to 9.6 × 10−4  mol/L with a detection limit of 8.0 × 10−7  mol/L IgG (at 3δ), while the detection limit by using BSA was 1.5 × 10−5  mol/L IgG (at 3δ) with the linear range from 3.5 × 10−5 to 1.2 × 10−3  mol/L. The selectivity, reproducibility and stability of the proposed immunosensor were acceptable. The IgG contents in 37 human serum samples obtained by the proposed method are identical with the data of clinical laboratory.
Keywords: Electrochemical immunosensor; Human IgG; Glucose oxidase; Bovine serum albumin; Biomimetic interface;

Real-time PCR microfluidic devices with concurrent electrochemical detection by Teh Huey Fang; Naveen Ramalingam; Dong Xian-Dui; Tan Swee Ngin; Zeng Xianting; Annie Tan Lai Kuan; Eric Yap Peng Huat; Gong Hai-Qing (2131-2136).
Electrochemistry-based detection methods hold great potential towards development of hand-held nucleic-acid analyses instruments. In this work, we demonstrate the implementation of in situ electrochemical (EC) detection method in a microfluidic flow-through EC-qPCR (FTEC-qPCR) device, where both the amplification of the target nucleic-acid sequence and subsequent EC detection of the PCR amplicon are realized simultaneously at selected PCR cycles in the same device. The FTEC-qPCR device utilizes methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements in the presence of PCR reagent components. Our EC detection method is advantageous, when compared to other existing EC methods for PCR amplicon analysis, since FTEC-qPCR does not require probe-modified electrodes, or asymmetric PCR, or solid-phase PCR. Key technical issues related to surface passivation, electrochemical measurement, PCR inhibition by metal electrode, bubble-free PCR, were investigated. By controlling the concentration of MB and the exposure of PCR mixture to the bare metal electrode, we successfully demonstrated electrochemical measurement of MB in solution-phase, symmetric PCR by amplifying a fragment of lambda phage DNA. The threshold cycle (C t) values for both the electrochemical and fluorescence-based assays decreased linearly with the increase of the input target quantity. The sensitivity of EC-based detection of PCR products is comparable to the sensitivity of an optical fluorescence detection system.
Keywords: Flow-through PCR; Microchip qPCR; Electrochemical qPCR; Methylene blue;

A biosensor for detection of viable Escherichia coli (E. coli) O157:H7 is developed using a quartz crystal microbalance with dissipation monitoring (QCM-D). The detection platform is based on the immobilization of affinity purified polyclonal antibodies onto gold-coated QCM-D quartz crystals via a cysteamine self-assembled monolayer. QCM-D measurements conducted over a broad range of bacterial cell concentrations show that the optimal biosensor response is the initial slope of the dissipation shift as a function of elapsed time (D slope). A highly log–log linear response in the initial D slope is obtained for detection of E. coli O157:H7 over a wide range of cell concentrations from 3 × 105 to 1 × 109  cells/mL. The prepared biosensor also exhibits a log–log linear working range from 107 to 109  cells/mL for E. coli K12 D21, a non-pathogenic model organism and further shows satisfactory selectivity using Bacillus subtilis. To our knowledge, this is the first study demonstrating the use of the initial D slope as a sensor response when using QCM-D technology.
Keywords: QCM-D; E. coli O157:H7; Dissipation slope; Cysteamine; Self-assembled monolayer;

Comparative study of SPR and ELISA methods based on analysis of CD166/ALCAM levels in cancer and control human sera by Hana Vaisocherová; Vitor M. Faca; Allen D. Taylor; Samir Hanash; Shaoyi Jiang (2143-2148).
Surface plasmon resonance (SPR), as a label free method for analysis of various analytes, has significantly advanced in recent years. However, assessment of the performance of SPR compared to label-based immunoassays such as the commonly used multiplexed enzyme-linked immunosorbent assay (ELISA) is limited, particularly for applications involving complex media. In this work, an optimized SPR assay was implemented and its performance compared with an ELISA assay for CD166/activated cell leukocyte adhesion molecule (ALCAM), as candidate pancreatic cancer marker, based on direct and amplified detection in buffer and in human serum samples from healthy individuals and subjects with cancer. ALCAM antibody was immobilized on the surface of a four-channel SPR sensor via physical adsorption onto charged amine-terminated alkanethiolates to mimic the ELISA plate surface. Excellent correlations between SPR and ELISA results were achieved in buffer and in human serum. SPR detected the target protein with a similar sensitivity to sandwich ELISA, with a detection limit below ng/mL. The detection time, sample consumption, throughput, signal referencing, and surface blocking and washing for detection in human serum were evaluated. It is demonstrated that SPR can distinguish between ALCAM levels in cancer and control sera using direct detection without the need for additional amplification steps.
Keywords: SPR; ELISA; ALCAM; CD166; Cancer biomarkers; Blood serum;

A novel H2O2 biosensor has been fabricated based on the direct electrochemistry and electrocatalysis of myoglobin (Mb) immobilized on silver nanoparticles doped carbon nanotubes film with hybrid sol–gel techniques. A pair of redox peaks with peak separation of 160 mV and formal potential of −0.295 V was observed at this composite film, corresponding to the direct electrochemistry of Mb. The heterogeneous rate constant was estimated to be 0.41 s−1. Under optimum conditions, the amperometric determination of H2O2 was performed with a linear range of 2.0 × 10−6–1.2 × 10−3  mol L−1 and a detection limit of 3.6 × 10−7  mol/L (S/N = 3). The Michealis–Menten constant was also estimated to be 1.62 mmol L−1. The proposed biosensor showed favorable reproducibility, stability, selectivity and accuracy, and has been used to determine H2O2 in real samples with favorable recoveries.
Keywords: Hydrogen peroxide; Myoglobin; Biosensor; Silver nanoparticles doped carbon nanotubes;

Development of an electrochemical immunoassay for rapid detection of E. coli using anodic stripping voltammetry based on Cu@Au nanoparticles as antibody labels by Xinai Zhang; Ping Geng; Huijie Liu; Yingqiao Teng; Yajun Liu; Qingjiang Wang; Wen Zhang; Litong Jin; Lai Jiang (2155-2159).
A sensitive electrochemical immunoassay for rapid detection of Escherichia coli has been developed by anodic stripping voltammetry (ASV) based on core–shell Cu@Au nanoparticles (NPs) as anti-E. coli antibody labels. The characteristics of Cu@Au NPs before and after binding with antibody were confirmed by transmission electron microscopy (TEM). After Cu@Au-labeled antibody reacted with the immobilized E. coli on Polystyrene (PS)-modified ITO chip, Cu@Au NPs were dissolved by oxidation to the metal ionic forms, and the released Cu2+ ions were determined at GC/Nafion/Hg modified electrode by ASV. The utilization of GC/Nafion/Hg modified electrode could enhance the sensitivity for Cu2+ detection with a concentration as low as 9.0 × 10−12  mol/L. Since Cu@Au NPs labels were only present when antibody reacted with E. coli, the amount of Cu2+ directly reflected the number of E. coli. The technique could detect E. coli with a detection limit of 30 CFU/mL and the overall analysis could be completed in 2 h. By introducing a pre-enrichment step, a concentration of 3 CFU/10 mL E. coli in surface water was detected by the electrochemical immunoassay.
Keywords: E. coli; Cu@Au nanoparticles; Anodic stripping voltammetry; Electrochemical immunoassay;

A novel and simple strategy for fabricating of DNA electrochemical biosensor was developed based on covalent coupling of probe NH2–ssDNA (S1) on Au electrode that had been functionalized by diazotization of assembled 4-aminothiophenol (4-ATP) monolayer. The thiol group of 4-ATP allowed the stable assembly of 4-ATP monolayer. The following diazotization reaction was directly performed to prepare functional diazo–ATP film for covalent coupling of probe S1. Remarkably, it was noting that the diazo–ATP provided a surface with high conductibility for electron transfer. The complementary ssDNA was determined by using differential pulse voltammetry. The linear range of the developed biosensor was from 1.57 × 10−9 to 4.52 × 10−7  M with a detection limit of 3.26 × 10−10  M. The fabricated biosensor possessed good selectivity and could be regenerated. The covalent immobilization of probe S1 by simple diazotization-coupling on self-assembled 4-ATP monolayer could serve as a versatile platform for DNA immobilization and biosensors fabricating.
Keywords: Biosensor; DNA; Covalent immobilization; Self-assembled monolayers; 4-Aminothiophenol;

A sensitive electrochemical DNA biosensor was successfully realized on polyaniline nanofibers (PANI), multi-walled carbon nanotubes (MWNT) and chitosan (CHIT) modified carbon paste electrode (CPE) based on the synergistic effect between PANI and MWNT nanoparticles in chitosan film. PANI and MWNT nanocomposites resulted in highly enhanced electron conductive and biocompatible nanostructured film, which was examined by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The immobilization of the probe DNA on the surface of electrode was largely improved due to the unique synergistic effect of PANI and MWNT. The DNA hybridization events were monitored with an EIS label-free detection strategy. Under the optimal conditions, the dynamic detection range of this DNA electrochemical biosensor was from 1.0 × 10−13 to 1.0 × 10−7  mol/L and a detection limit of 2.7 × 10−14  mol/L for the detection of DNA specific sequence of the phosphinothricin acetyltransferase gene (PAT, one of the important screening detection genes for the transgenic plants). Simultaneously, the polymerase chain reaction (PCR) amplification of the terminator of nopaline synthase gene (NOS) from the sample of one kind of genetically modified soybean was also detected satisfactorily.
Keywords: Carbon nanotubes; Polyaniline nanofibers; Electrochemical DNA biosensor; PAT gene; NOS gene;

Electrowetting-based pH- and biomolecule-responsive valves and pH filters by Shigeki Yamaguchi; Katsuya Morimoto; Junji Fukuda; Hiroaki Suzuki (2171-2176).
An electrowetting-based pH-responsive valve that uses a nonstandard electrochemical three-electrode system is proposed. The system comprises a gold valve electrode and an iridium electrode that act as the working and auxiliary electrodes, depending on the purpose, while an iridium oxide pH-sensitive electrode acts as the reference electrode. To make the valve open at pH higher than a threshold, the gold valve electrode is used as the working electrode and a voltage is applied to it with respect to the pH-sensitive reference electrode. To make the valve open at pH lower than the threshold, the gold valve electrode is used as the auxiliary electrode, while the iridium electrode is used as the working electrode. The wettability of the valve electrode is altered when a voltage is applied to it. When the pH of a solution crosses the threshold, the potential of the gold valve electrode exceeds a threshold potential because of the change in the potential of the pH-sensitive reference electrode. Consequently, the gold valve electrode becomes more hydrophilic, thereby allowing the solution to pass through the valve. Furthermore, by combining two valve electrodes, we realized a pH filter that allows solutions with pH within a limited range to pass through it. Urea- and glucose-responsive valves that opened at concentrations higher than the threshold could also be formed by immobilizing an enzyme on the pH-sensitive reference electrode.
Keywords: Valve; Flow channel; Electrowetting; Gold electrode; pH; Reference electrode;

A rationally designed oligoethylene glycol linker conjugate to testosterone was synthesised and covalently immobilized on a surface plasmon resonance (SPR) biosensor surface. The sensing surface was stable for more than 330 binding and regeneration cycles allowing a high degree of re-use. This surface was then used in the development of an ultrasensitive immunobiosensor system for testosterone in buffer utilizing both secondary antibody and gold nanoparticle signal enhancement. The mechanism for the increased sensitivity results from increased binding mass and a gold plasmon coupling effect. The addition of a secondary antibody with an attached gold nanoparticle increased the signal sensitivity of the assay 12.5-fold compared with primary antibody alone. In the enhanced format the assay had limits of detection (LOD) of 3.7 pg ml−1 with standard in running buffer, and 15.4 pg ml−1 in a stripped human saliva matrix. This immunobiosensor system has sufficient sensitivity to measure testosterone across the broad physiologically relevant range in male saliva (29–290 pg ml−1) in under 13 min allowing monitoring of testosterone in near real-time.
Keywords: Testosterone; Surface plasmon resonance; Linker; Conjugate; Saliva;

An automated, on-line system for protein quantification and identification, employing Surface Plasmon Resonance (SPR), enzymatic protein digestion, nanoLC and tandem-MS (MS/MS), has been developed. For the experiments recombinant human interferon-γ (rhIFN-γ) in buffer or diluted bovine plasma was used as a model protein. Upon injecting 90 μL of a 1 μg mL−1 solution of rhIFN-γ in diluted plasma at a flow rate of 10 μL min−1, 320 fmol of protein was reproducibly bound to the sensor surface. After desorption of the isolated protein from the SPR surface using 10 mM glycine pH 1.3, on-line digestion, nanoLC and MS/MS analysis, rhIFN-γ could be identified on basis of peptide masses and MS/MS fragmentation data. A sequence recovery of 66% was found when a pepsin micro reactor was used. For a trypsin micro reactor the sequence recovery was 50%. In the latter case, the desorbed protein solution was pH-tuned with a TRIS buffer for optimal enzyme activity. With the identified trypsin- and pepsin-produced peptides and because parts of their amino acid sequences overlap, the protein sequence can be largely elucidated showing the potential for the analysis of unknown proteins. The SPR-digestion-nanoLC-MS/MS platform provides unattended analysis of a sample within 60 min.
Keywords: Surface Plasmon Resonance; Immobilised enzyme reactor; Liquid chromatography; Mass spectrometry; Peptide mapping; Ligand fishing;

Microelectronic system for high-resolution mapping of extracellular electric fields applied to brain slices by U. Frey; U. Egert; F. Heer; S. Hafizovic; A. Hierlemann (2191-2198).
There is an enduring quest for technologies that provide – temporally and spatially – highly resolved information on electric neuronal or cardiac activity in functional tissues or cell cultures. Here, we present a planar high-density, low-noise microelectrode system realized in microelectronics technology that features 11,011 microelectrodes (3,150 electrodes per mm2), 126 of which can be arbitrarily selected and can, via a reconfigurable routing scheme, be connected to on-chip recording and stimulation circuits. This device enables long-term extracellular electrical-activity recordings at subcellular spatial resolution and microsecond temporal resolution to capture the entire dynamics of the cellular electrical signals. To illustrate the device performance, extracellular potentials of Purkinje cells (PCs) in acute slices of the cerebellum have been analyzed. A detailed and comprehensive picture of the distribution and dynamics of action potentials (APs) in the somatic and dendritic regions of a single cell was obtained from the recordings by applying spike sorting and spike-triggered averaging methods to the collected data. An analysis of the measured local current densities revealed a reproducible sink/source pattern within a single cell during an AP. The experimental data substantiated compartmental models and can be used to extend those models to better understand extracellular single-cell potential patterns and their contributions to the population activity. The presented devices can be conveniently applied to a broad variety of biological preparations, i.e., neural or cardiac tissues, slices, or cell cultures can be grown or placed directly atop of the chips for fundamental mechanistic or pharmacological studies.
Keywords: CMOS-based microelectrode array; Purkinje cells; Acute brain slice; Integrated circuits; Neurochip;

Preparation, characterization, and application of a new sensor for the determination of phosphate in blood serum is described by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and osteryoung square wave voltammetry (OSWV) in the presence of an appropriate redox reaction probe. The sensor was based on ionic adsorption of phosphate on Zr(IV) immobilized on gold–mercaptopropionic acid self-assembled monolayer (Au–MPA–Zr(IV) SAM) electrode. Parameters influencing the method were optimized. A linear range calibration curve from 1.0 × 10−7 to 1.0 × 10−6  M PO4 3− with a detection limit of 5.30 × 10−8  M and mean of relative standard deviations (R.S.D.) of 2.75% for n  = 4 was observed in the best conditions by OSWV. Possible interferences from the coexisting ions were also investigated. The results demonstrated that sensor could be used for the determination of phosphate in the presence of various ions. Regeneration of the surface was carried out successfully by 5-min sonication in 0.1 M KOH solution and then 1-min incubation in 1.0 × 10−3  M Zr(IV) with a good reproducibility, R.S.D. = 1.47% for n  = 4 by OSWV. The validity of the method and applicability of the sensor were successfully tested by detection of phosphate in blood serum after deproteinization of sample without interference from sample matrix. The long-term storage stability of the electrode was studied. The experimental data is presented and discussed from which the new sensor is characterized.
Keywords: Zirconium; Phosphate; Square wave voltammetry; Electrochemical impedance spectroscopy; Blood serum; Self-assembled monolayers;

Copper UPD as non-specific adsorption barrier in electrochemical displacement immunosensors by M.V. Duarte; P. Lozano-Sanchez; I. Katakis (2205-2210).
Non-specific adsorption events are responsible to a large extent for the lack of reliability and applicability of electrochemical immunosensors. In the particular case of displacement-based immunosensors, as an approach to achieve reagentless, labelless and easy to use immunosensors, the hindering effect of then non-specific adsorption is amplified when the system presents a low affinity constant between biorecognition element and target. The application of Copper UPD as non-specific adsorption barrier in combination with the use of self-assembled monolayers (SAM) to provide efficient binding of biomolecules to the immunosensor electrode surface is shown to be a very promising mechanism to construct protein resistant surfaces with no harming effects on the electrochemical transducing mechanism. The electrochemical immunodetection of TCA (2,4,6-Trichloroanisole) has been chosen as example for a real case study. A monoclonal antibody to detect the target TCA and an appropriate sub-optimum antigen were used. In addition to a rational strategy for displacement immunosensor development, the decrease of non-specific adsorption phenomena by introducing Copper UPD is reported here. With such strategy an electrochemical displacement immunosensor with a limit of detection of 200 ppb and response time of 10 min is achieved.
Keywords: Electrochemical displacement immunosensors; Non-specific adsorption; Copper electrodeposition; Underpotential deposition; 2,4,6-Trichloroanisole (TCA);

A novel amperometric biosensor was fabricated based on the immobilization of cholesterol oxidase (ChOx) into a cross-linked matrix of chitosan (Chi)–room-temperature ionic liquid (IL) (1-butyl-3-methylimidazolium tetrafluoroborate). Initially, the surface of bare electrode (indium tin oxide coated glass) was modified with the electrodeposition of Au particles onto thiol (–SH) functionalized multi-walled carbon nanotubes (MWNTs). The biosensor electrode is designated as MWNT(SH)–Au/Chi–IL/ChOx. Scanning electron microscopy image of MWNT(SH)–Au/Chi–IL/ChOx reveals that Chi–IL exists as the interconnected wires covering the Au particles on the surface of MWNT(SH)–Au. Cyclic voltammetry and chronoamperometry were used for the electrochemical determination of cholesterol at the biosensor electrode, MWNT(SH)–Au/Chi–IL/ChOx. The presence of Au particles in the matrix of CNTs provides an environment for the enhanced electrocatalytic activities. The MWNT(SH)–Au/Chi–IL/ChOx biosensor exhibited a linear response to cholesterol in the concentration range of 0.5–5 mM with a correlation coefficient of 0.998, good sensitivity (200 μA M−1), a low response time (∼7 s), repeatability (R.S.D value of 1.9%) and long term stability (20 days with a decrease of 5% response). The synergistic influence of MWNT(SH), Au particles, Chi and IL contributes to the excellent performance for the biosensor.
Keywords: Cholesterol; Gold nanoparticles; Ionic liquid; Chitosan; Multi-wall carbon nanotubes; Amperometry;

This work reports on a dynamically configurable micro-array surface plasmon resonance biochip platform. The platform comprises a digital electrowetting-on-dielectric (EWOD) microfluidic device tailored to surface plasmon resonance imaging (SPRi). We demonstrate its application for simultaneous immobilization of different DNA probes at the designated detection sites on-chip from sub-μL volume solutions in combination with multichannel label-free real-time detection of subsequent hybridization reactions. Successful on-chip DNA probe dilution and immobilization is also demonstrated using SPRi hybridization detection. Furthermore, active control of the immobilized probe density and orientation is achieved under an applied potential using the electric interface of the EWOD device. For low probe densities, under negative applied potential, the DNA hybridization efficiency is enhanced compared to passive probe immobilization, yielding a two-fold SPR signal increase within only 8 min of hybridization. EWOD microfluidic platform coupled with SPRi promises to dramatically increase the speed of detection and quantification of biomolecular interactions while reducing reagent consumption. The proposed system would enable the development of high-throughput, rapid and ultrasensitive detection of biomolecules beyond DNA microarray applications.
Keywords: Digital microfluidics; Electrowetting-on-dielectric; Surface plasmon resonance imaging; DNA biochips; DNA hybridization detection;

To immobilize laccase (Lac) from Trametes versicolor that shows its maximum enzymatic activity in acidic aqueous solutions, the biopolymer chitosan (CS) was chemically modified with glutaraldehyde (GA) to form GA functionalized CS (GAfCS), which was then allowed to react with Lac to form a Lac–GAfCS composite that is robust in weakly acidic solutions (two-step protocol), as confirmed by quartz crystal microbalance and durability tests. The Lac–GAfCS–multiwalled carbon nanotubes (MWCNTs)/glassy carbon (GC) electrode exhibited good catalytic activity towards O2 reduction in the presence of 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS), and the pH-dependent enzymatic activity of the immobilized Lac towards O2 reduction was examined. A glucose/air biofuel cell was fabricated, with the Lac–GAfCS–MWCNTs/GC electrode as the biocathode and a glucose oxidase (GOx)–GAfCS–MWCNTs/GC electrode as the bioanode in a Nafion membrane-separated acetate buffer solution (pH 5.0). The biofuel cell output a maximum power density of 9.6 μW/cm2, an open-circuit cell voltage of 0.19 V, and a short-circuit current density of 114 μA/cm2, respectively, as measured with an electrochemical noise (ECN) apparatus. Furthermore, the Lac–GAfCS–MWCNTs/GC electrode was applied to determine catechol in Britton–Robinson buffer solution (pH 3.0), with a linear range of 0.1–50 μM and a limit of detection of 20 nM. In comparison with the direct use of GA for one-pot Lac–GA-CS or Lac–GA crosslinking to immobilize Lac, the use of macromolecular GAfCS in the proposed two-step protocol was proven to be less harmful to the enzymatic activity and thus more suitable for immobilizing the enzyme to construct the biofuel cell and biosensor. This work may be helpful for exploiting the popular biocompatible CS as an acid-resistant film matrix for many other biotechnology applications, and the proposed two-step crosslinking protocol is recommended for high-activity immobilization of other biomolecules.
Keywords: Laccase; Chitosan; Multiwalled carbon nanotubes; Two-step crosslinking with glutaraldehyde; Biofuel cell; Phenolic biosensor;

STD sensor based on nucleic acid functionalized nanostructured polyaniline by Renu Singh; Rachna Prasad; G. Sumana; Kavita Arora; Seema Sood; R.K. Gupta; B.D. Malhotra (2232-2238).
STD (sexually transmitted disease, Gonorrhoea) sensor based on nucleic acid probe (from Opa, a multi-copy gene of Neisseria gonorrhoeae) functionalized nanostructured-polyaniline coated onto indium–tin-oxide-coated glass plate has been fabricated using avidin–biotin as cross-linking agent. This DNA functionalized electrode can specifically detect upto 0.5 × 10−15  M of complementary target within 60 s of hybridization time at 25 °C by differential pulse voltammetry (DPV) using methylene blue as electro-active DNA hybridization indicator. This highly sensitive and specific nucleic acid functionalized nanostructured-polyaniline electrode can distinguish presence of N. gonorrhoeae from Neisseria meningitidis and Escherichia coli culture and spiked samples from the urethral swabs of the patients.
Keywords: Sexually transmitted disease; Neisseria gonorrhoeae; DNA biosensor; Polyaniline; Methylene blue; Electrochemical characterization;

A novel automated discontinuous venous blood monitoring system for ex vivo glucose determination in humans by R. Schaller; F. Feichtner; H. Köhler; M. Bodenlenz; J. Plank; A. Wutte; J.K. Mader; M. Ellmerer; R. Hellmich; H. Wedig; R. Hainisch; T.R. Pieber; L. Schaupp (2239-2245).
Intensive insulin therapy reduces mortality and morbidity in critically ill patients but imposes great demands on medical staff who must take frequent blood samples for the determination of glucose levels. A solution to this resourcing problem would be provided by an automated blood monitoring system. The aim of the present clinical study was to evaluate such a system comprising an automatic blood sampling unit linked to a glucose biosensor. Our approach was to determine the correlation and system error of the sampling unit alone and of the combined system with respect to reference levels over 12 h in humans. Two venous cannulae were inserted to connect the automatic and reference systems to the subjects. Blood samples were taken at 15 and 30 min intervals. The median Pearson coefficient of correlation between manually and automatically withdrawn blood samples was 0.982 for the sampling unit alone and 0.950 for the complete system. The biosensor had a linear range up to 20 mmol l−1 and a 95% response time of <2 min. Clark Error Grid analysis showed that 96.93% of the data (228 data pairs) was in zone A and 3.07% in zone B. Insulin Titration Error Grid analysis suggested an acceptable treatment in 99.56% of cases. Implementation of a “Keep Vein Open” saline infusion into the automated blood sampling system reduced blood withdrawal failures through occluded catheters fourfold. In summary, automated blood sampling from a peripheral vein coupled with automatic glucose determination is a promising alternative to frequent manual blood sampling.
Keywords: Glucose; Sensors; Monitoring; Blood; Automated; Ex vivo;

Signal amplification of electrochemical immunosensor for the detection of human serum IgG using double-codified nanosilica particles as labels by Zhaoyang Zhong; Mengxia Li; Debing Xiang; Nan Dai; Ying Qing; Dong Wang; Dianping Tang (2246-2249).
A simple and sensitive method for in situ amplified electrochemical immunoassay of human serum IgG has been developed by using double-codified nanosilica particles as labels based on horseradish peroxidase-doped nanosilica particles (HRP–SiO2) with the conjugation of anti-IgG antibodies (anti-IgG–SiO2–HRP). With the sandwich-type immunoassay format, the linear range of the developed immunosensor by using anti-IgG–SiO2–HRP as tracer and hydrogen peroxide (H2O2) as enzyme substrate is 0.01–15 nmol/L IgG with a detection limit of 5.0 pmol/L, while the assay sensitivity by directly using HRP-labeled anti-IgG as secondary antibodies is 1.0–10 nmol/L with a detection limit of 0.1 nmol/L IgG. The reproducibility, stability and specificity of the proposed immunoassay method were acceptable. The IgG concentrations of the clinical serum specimens assayed by the developed immunosensor show consistent results in comparison with those obtained by commercially available enzyme-linked immunosorbent assay (ELISA) method.
Keywords: Double-codified nanosilica particles; Electrochemical immunosensor; Human IgG; Sandwich-type immunoassay;

Detecting effects of low levels of cytochalasin B in 3T3 fibroblast cultures by analysis of electrical noise obtained from cellular micromotion by Douglas C. Lovelady; Jennifer Friedman; Sonali Patel; David A. Rabson; Chun-Min Lo (2250-2254).
We performed micromotion experiments using electric cell–substrate impedance sensing (ECIS) on a confluent layer of 3T3 fibroblasts exposed to different low levels of the toxin cytochalasin B. This toxin is know to affect actin polymerization and to disrupt cytoskeletal structure and function in cells, changing the morphology of confluent cell cultures and altering the nature of the cellular micromotion, which is measured by ECIS as changes in impedance. By looking at several measures to characterize the long- and short-term correlations in the noise of the impedance time series, we are able to detect the effects of the toxin at concentrations down to 1  μ M; there are intriguing hints that the effects may be discernible at levels as low as 0.1  μ M. These measures include the power spectrum, the Hurst and detrended-fluctuation-analysis exponents, and the first zero and first 1 / e crossings of the autocorrelation function. While most published work with ECIS uses only average impedance values, we demonstrate that noise analysis provides a more sensitive probe.
Keywords: ECIS; Noise analysis; Toxin assay; Cytochalasin B;

A novel label-free multi-throughput optical biosensor based on localized surface plasmon resonance by Haowen Huang; Chaocai He; Yunlong Zeng; Xiaodong Xia; Xianyong Yu; Pinggui Yi; Zhong Chen (2255-2259).
A novel and sensitive multi-throughput localized surface plasmon resonance (MLSPR) biosensor was developed for the first time. Various gold nanorods with different aspect ratios were used to fabricate the optical sensor. Five kinds of gold nanorods with different aspect ratios were chosen to construct five throughputs of MLSPR. Various LSPR peaks imply that different acceptor–ligand pairs can be detected simultaneously in the wavelength range from 530 to 940 nm. The biosensor immobilized on glass slides was applied to label-free detection between acceptor and ligand. The MLSPR-based optical biosensor can be used to detect three antigen–antibody pairs simultaneously. The biosensor proposed herein is easy to fabricate, and its operation procedure is convenient as labeling procedure is unnecessary.
Keywords: Localized surface plasmon resonance; Label-free; Gold nanorods; Longitudinal plasmon wavelength;

Surface-enhanced Raman scattering imaging of HER2 cancer markers overexpressed in single MCF7 cells using antibody conjugated hollow gold nanospheres by Sangyeop Lee; Hyangah Chon; Moonkwon Lee; Jaebum Choo; Soon Young Shin; Young Han Lee; Im Joo Rhyu; Sang Wook Son; Chil Hwan Oh (2260-2263).
Antibody-conjugated hollow gold nanospheres (HGNs) have been used for the SERS imaging of HER2 cancer markers overexpressed in single MCF7 cells. SERS mapping images show that HGNs have much better homogeneous scattering properties than silver nanoparticles. The results demonstrate the potential feasibility of HGNs as highly sensitive and homogeneous sensing probes for biological imaging of cancer markers in live cells.
Keywords: Hollow gold nanosphere; Surface-enhanced Raman scattering; Cellular imaging; Cancer marker; Homogeneous sensing probe;

A novel biosensor for the highly sensitive detection of aflatoxin M1 (AFM1) in milk was developed. This biosensor is based on surface plasmon-enhanced fluorescence spectroscopy (SPFS) which was advanced through the excitation of long range surface plasmons (LRSPs). In SPFS, the binding of fluorophore-labeled molecules to the sensor surface is probed with surface plasmons (SPs) and the emitted fluorescence light is detected. This approach takes advantages of the enhanced intensity of electromagnetic field occurring upon the resonant excitation of SPs which directly increases the fluorescence signal. For the detection of AFM1, LRSP-enhanced fluorescence spectroscopy was combined with an inhibition immunoassay in which a derivative of AFM1 was immobilized on the sensor surface and antibodies against AFM1 were used as recognition elements. The developed biosensor allowed for the detection of AFM1 in milk within 53 min at concentrations as low as 0.6 pg mL−1. The achieved limit of detection was about two orders of magnitude lower than the maximum AFM1 residue level in milk stipulated by the European Commission legislation.
Keywords: Surface plasmon resonance; Long range surface plasmons; Fluorescence spectroscopy; Inhibition immunoassay; Aflatoxin;

Selenium nanoparticles (Se NPs) were prepared based on the reduction of selenious acid (H2SeO3), by employing sodium alginate (SA) as a template. The real-time monitoring of the drug-inducing apoptosis process of human hepatic cancer cells Bel7402 was performed with the quartz crystal microbalance (QCM) measurement. The anti-tumor effect of adriamycin (ADM) used in combination with Se NPs was investigated. It is found that both drugs were able to inhibit cell proliferation in a dose-dependent way and the combined treatment with ADM and Se NPs was more effective in inhibiting cell growth than each of the two drugs alone. The cytotoxic effects of drug combination were evaluated with the modified Bürgi formula (Jin equation) based the Δf 0 responses. The grades gradually changed from apparent synergism to simple addition with the drug-treatment time increasing but the drug combination with lower concentrations still exhibited synergism after 24 h, suggesting a potential application in cancer therapy.
Keywords: Quartz crystal microbalance; Cytotoxicity; Adriamycin; Selenium nanoparticles; Synergistic effect; Hepatic cancer cells Bel7402;

Electrochemiluminescence detection of NADH and ethanol based on partial sulfonation of sol–gel network with gold nanoparticles by Liu Deng; Lihua Zhang; Li Shang; Shaojun Guo; Dan Wen; Fuan Wang; Shaojun Dong (2273-2276).
We developed a stable, sensitive electrochemiluminescence (ECL) biosensor based on the synthesis of a new sol–gel material with the ion-exchange capacity sol–gel to coimmobilize the Ru(bpy)3 2+ and enzyme. The partial sulfonated (3-mercaptopropyl)-trimethoxysilane sol–gel (PSSG) film acted as both an ion exchanger for the immobilization of Ru(bpy)3 2+ and a matrix to immobilize gold nanoparticles (AuNPs). The AuNPs/PSSG/Ru(bpy)3 2+ film modified electrode allowed sensitive the ECL detection of NADH as low as 1 nM. Such an ability of AuNPs/PSSG/Ru(bpy)3 2+ film to promote the electron transfer between Ru(bpy)3 2+ and the electrode suggested a new, promising biocompatible platform for the development of dehydrogenase-based ECL biosensors. With alcohol dehydrogenase (ADH) as a model, we then constructed an ethanol biosensor, which had a linear range of 5 μM to 5.2 mM with a detection limit of 12 nM.
Keywords: Electrochemiluminescence; NADH; Alcohol dehydrogenase; (3-mercaptopropyl)-trimethoxysilane; Sol–gel; Gold nanoparticle;

One-step membrane-based competitive immunochromatographic assay using colloidal gold–antibody probe for rapid detection of indomethacin (IDM) in water samples was developed. A positive reaction as a result of the remaining gold–antibody conjugate combining with antigen coated on the membrane was obvious by visual detection. Under optimal conditions, the visual detection limit was found to be 0.1 ng mL−1, which was about 10–1000 times lower than those in published literatures, demonstrating the high sensitivity of the assay. The stability test indicated the immunochromatographic strips could be used for 8 weeks at room temperature without significant loss of activity. Real water samples were detected by the assay and confirmed by enzyme-linked immunosorbent assay. The assay time for indomethacin detection was less than 10 min, suitable for rapid testing on-site.
Keywords: Immunochromatographic assay; Colloidal gold particle; Antibody; Indomethacin;

Quantitative analysis of enhanced light irradiance in waveguide-based fluorescent microarrays by Gabriel Sagarzazu; Mélanie Bedu; Lucio Martinelli; Nicolas Pelletier; Viatcheslav I. Safarov; Claude Weisbuch; Thierry Gacoin; Henri Benisty (2281-2284).
Probing microarray assays in the presence of a hybridization mix retrieves precious information on hybridization kinetics. However, in common detection schemes, useful surface signals compete with the high supernatant background from labelled targets in the mix. A known solution consists in exciting specifically the microarray surface with evanescent fields. Configurations using planar optical waveguides to produce such fields are shown here to present also a dramatic excitation irradiance enhancement at the guide/surrounding matter interface. We compare theoretically and experimentally a guided excitation with a classical external excitation. A full electromagnetic analysis predicts an irradiance increase higher than 104 for adequately tailored waveguides. We deposited high-index TiO2 sol–gel waveguides on glass substrates according to best simulations. Quantitative enhancement analysis exploiting actual biological fluorescent spots perfectly confirms the irradiance amplification effect of a thin waveguide. The impact of amplification on the design of biochip readers is discussed since it leaves ample margin for simple and low-cost light couplers, advantageous in affordable readers and sensor systems.
Keywords: Fluorescence; Microarray; Waveguide; Evanescent field;

We developed a simple strategy for designing a highly sensitive electrochemical biosensor for organophosphate pesticides (OPs) based on acetylcholinesterase (AChE) immobilized onto Au nanoparticles–polypyrrole nanowires composite film modifid glassy carbon electrode (labeled as AChE–Au–PPy/GCE). Where, the generated Au nanoparticles (AuNPs) were homogenously distributed onto the interlaced PPy nanowires (PPy NWs) matrix, constructing a three-dimensional porous network. This network-like nanocomposite not only provided a biocompatible microenvironment to keep the bioactivity of AChE, but also exhibited a strong synergetic effect on improving the sensing properties of OPs. The combination of AuNPs and PPyNWs greatly catalyzed the oxidation of the enzymatically generated thiocholine product, thus increasing the detection sensitivity. On the basis of the inhibition of OPs on the enzymatic activity of AChE, the conditions for OPs detection were optimized by using methyl parathion as a model OP compound. The inhibition of methyl parathion was proportional to its concentration ranging from 0.005 to 0.12 and 0.5 to 4.5 μg mL−1. The detection limit was 2 ng mL−1. The developed biosensor exhibited good reproducibility and acceptable stability. This study provides a new promise tool for analysis of organophosphate pesticides.
Keywords: Polypyrrole nanowires; Gold nanopaticles; Acetylcholinesterase; Methyl parathion; Biosensor;

We report a liquid crystal (LC)-based sensor for real-time and label-free identification of phospholipase-like toxins. Beta-bungarotoxin exhibits Ca2+-dependent phospholipase A2 activity whereas alpha-bungarotoxin and myotoxin II do not exhibit any phospholipase activity. The sensor can selectively identify beta-bungarotoxin, when it hydrolyzes a phospholipid monolayer self-assembled at aqueous–LC interface, through orientational responses of LCs. As a result, optical signals that reflect the spatial and temporal distribution of phospholipids during the hydrolysis can therefore be generated in a real-time manner. The sensor is very sensitive and requires less than 5 pg of beta-bungarotoxin for the detection. When phospholipase A2 inhibitors are introduced together with beta-bungarotoxin, no orientational response of LCs can be observed. In addition, the regeneration of the sensor can be done without affecting the sensing performance. This work demonstrates a simple and cost-effective LC-based sensor for identifying phospholipase-like toxins and for screening compound libraries to find potential toxin inhibitors.
Keywords: Liquid crystal; Beta-bungarotoxin; Phospholipase; Real-time detection; Toxin inhibitor;

Discrimination of specific and non-specific bindings by dielectrophoretic repulsion in on-chip magnetic bio-assays by Chengxun Liu; Randy De Palma; Gunter Reekmans; Wim Laureyn; Tim Stakenborg; Liesbet Lagae (2294-2297).
Affinity binding is the principle used in a large number of bio-assays. Aside from specific bindings, non-specific bindings usually deteriorate assays by giving false positive signals and restrict the detection limit. Currently, the assay specificity is mainly dependent on the effectiveness of a suitable surface chemistry. We report an approach to discriminate specific and non-specific bindings with dielectrophoretic (DEP) forces for on-chip magnetic bio-assays. Conjugated to the analytes, magnetic particles were used as the agents for DEP force generation. Due to a weaker binding strength, the non-specifically bound particles were removed while specific bindings remained intact. Analytical and finite element calculations were also performed to study all relevant forces. Furthermore, the removal of magnetic particles was also assessed by measuring the magnetic signal using magnetoresistive sensors. This technique can not only be used to improve the specificity of the on-chip bio-assays but also be developed as a tool of force spectroscopy for the study of bio-molecular binding physics.
Keywords: Lab-on-a-chip; Dielectrophoresis; Magnetic biosensor; Binding force; Magnetic particles;

G proteins, further divided into four subfamilies (Gs, Gq, G12 and Gi) based on their Gα subunits, are the primary components activated by G protein-coupled receptors (GPCRs). Current GPCR assays are limited to the evaluation of selective Gα signaling and do not allow comprehensive screening for orphan GPCRs without a known coupled Gα. Therefore, our aim was to design a chimeric reporting system that covers responses from all Gα subfamilies simultaneously. Because Gs activates cAMP response element (CRE)-driven genes whereas Gq and G12 activate serum response element (SRE)-driven genes, we therefore incorporated 2× CRE and 5× SRE (2CRE5SRE) into a promoter for driving luciferase expression. To further report Gi signals, a 2CRE5SRE-driven chimeric Gqi, in which the C-terminus of Gq is replaced by that of Gi, was integrated to switch the responses of Gi-coupled GPCRs to the Gq signaling. The novel reporter system showed a strong signal amplification when activated by neuromedin U receptor 1 (mainly activates Gq), neuromedin U receptor 2 (mainly activates Gi) or luteinizing hormone receptor (mainly through the Gs and Gq pathways). In addition, 293T cells stably carrying our reporter construct showed a similar sensitivity to the radioactive cAMP assay when revealing the constitutive signal from gain-of-function mutants of luteinizing hormone receptor. To our knowledge, this is the first reporting system capable of covering the Gs, Gq, G12 and Gi signals and revealing the phenomena of constitutively active GPCRs. Such a universal platform will benefit future high-throughput screening and drug designs for any GPCR.
Keywords: G protein-coupled receptors; Luciferase; SRE; CRE; Gqi;

The development of a MIP-optosensor for the detection of monoamine naphthalenes in drinking water by Angel Valero-Navarro; Alfonso Salinas-Castillo; Jorge F. Fernández-Sánchez; Antonio Segura-Carretero; Ricardo Mallavia; Alberto Fernández-Gutiérrez (2305-2311).
To enhance the advantages of fluorescent flow-through sensing for drinking water we have designed a novel sensing matrix based on molecularly imprinted polymers (MIPs). The synergic combination of a tailor-made MIP recognition with a selective room temperature fluorescence detection is a novel concept for optosensing devices and is assessed here for the simple and selective determination of pollutants in water.We describe a simple approach to preparing synthetic receptors for monoamine naphthalene compounds (MA-NCs) using non-covalent molecular imprinting techniques and naphthalene as template. We examine in detail the binding characteristics of the imprinted polymer and describe the flow-through sensor of MA-NCs by solid-surface fluorescence. Its detection limits for recognizing 1-naphthylamine (1-NA) and 2-naphthylamine (2-NA) separately are 26 ng mL−1 and 50 ng mL−1, respectively, and it also determines 1-NA and 2-NA simultaneously with a detection limit of 45 ng mL−1.All the instrumental, chemical and flow variables were carefully optimized and an interference study was carried out to demonstrate its applicability and selectivity. Finally, we applied it to the analysis of 1-NA and 2-NA in tap and mineral waters, obtaining a 98% average recovery rate.
Keywords: Molecular imprinting; Fluorescence optosensor; Flow injection; Naphthalene compounds; Water analysis;