Development of catheter-type optical oxygen sensor and applications to bioinstrumentation

A catheter-type optical oxygen sensor based on phosphorescence lifetime was developed for medical and animal experimental use. Since the sensor probe should have biocompatibility and high oxygen permeability in vivo, we focused attention on acceptable polymer materials for contact lenses as the substrates of probes. Pd-porphyrin was doped in silicone-based polymer, and was fixed at the edge of an optical fiber inserted in a catheter tube. The shape of the probe was 600 μm in diameter and 100 μm in thickness, and the probe had high oxygen permeability of Dk value 455. In accuracy evaluation, there found an excellent correlation between the pO₂ values measured through phosphorescence lifetime using the oxygen sensors and those measured as the calibrating data using oxygen electrodes. The response time required to achieve 90% from reversible default value to be from 150 to 0 mmHg, and from 0 to 150 mmHg was 15.43 and 7.52 s, respectively. In addition, other properties such as temperature and pH dependency, response, and durability of our optical oxygen sensor were investigated. In animal experiments, the catheter-type oxygen sensor was inserted via the femoral artery of a rat, and arterial oxygen pressure was monitored under asphyxiation. The sensor was valid in the range of oxygen concentration sufficient for biometry, and expected to be integrated with an indwelling needle.     

Investigation on light-addressable potentiometric sensor as a possible cell-semiconductor hybrid

This article reports an investigation on light-addressable potentiometric sensor (LAPS) to be used as a possible biological cell-semiconductor hybrid that will enable us to make an interface between the physical and biological system. To increase the surface potential sensitivity, we used a LAPS structure with single insulator (SiO2) coated with poly-L-ornithine and laminin (PLOL) on Si. Efficient culturing of PC-12 and nerve cells of Lymnaea stagnalis on PLOL-coated Si3N4 and SiO2 was achieved. The thickness of the PLOL layer was found to be about 4 nm by the atomic force microscope (AFM) measurement. Using the advantage of this thin layer of PLOL, we compared the performance of a novel structure to the previously reported "PLOL-coated Si3N4/SiO2/Si" structure. Due to high insulating capacitance, the photocurrent response of the novel LAPS was found to be very steep. As a result, higher sensitivity was achieved. This steepness did not degrade during 10 days when the sensor surface was kept in contact with the cell culture medium and environment. The thickness of PLOL layer, its ability to improve the biological cell adhesion, enhanced sensitivity, and experiment with simulated neural action potential (AP) applied to the novel LAPS show a good promise for LAPS to be a biological cell-semiconductor hybrid.     

Immobilised activated sludge based biosensor for biochemical oxygen demand measurement

A biochemical oxygen demand (BOD) sensor, based on an immobilised mixed culture of microorganisms in combination with a dissolved oxygen electrode, has been developed for the purpose of on-line monitoring of the biological treatment process for waste and wastewater. The sensor was designed for easy replacement of the biomembrane, thereby making it suitable for short-term use. The drawbacks of activated sludge based sensor, such as short sensor lifetime, were thereby circumvented. The sensor BOD measurements were carried out in the kinetic mode using a flow injection system, resulting in 25 s for one measurement followed by 4–8 min recovery time. Based on the results of normalised sensor responses, the OECD synthetic wastewater was considered to be a more suitable calibration solution in comparison with the GGA solution. Good agreement was achieved between the results of the sensor BOD measurement and those obtained from BOD₅ analysis of a wastewater sample from a food-processing factory. Reproducibility of responses using one sensor was below ±5.6% standard deviation. Reproducibility of responses using different sensors was within acceptable bias limits, viz. ±15% standard deviation.     

A novel colorimetric fluoride sensor based on a semi‐rigid chromophore controlled by hydrogen bonding

A novel semi‐rigid latent chromophore E1, containing an amide subunit activated by an adjacent semi‐rigid intramolecular hydrogen‐bonding (IHB) unit, was designed for the detection of fluoride ion by the ‘naked‐eye’ in CH₃CN. Comparative studies on structural analogs (E2, E3, and E4) provided significant insight into the structural and functional role of the amide N–H and IHB segment in the selective recognition of fluoride ions. The deprotonation of the amide N–H followed by the enhancement of intramolecular charge transfer (ICT) induced the colorimetric detection of E1 for fluoride ion. Copyright © 2015 John Wiley & Sons, Ltd.     

Label-free fluorescent sensor for lead ion detection based on lead(II)-stabilized G-quadruplex formation

A label-free fluorescent DNA sensor for the detection of lead ions (Pb²⁺) based on lead(II)-stabilized G-quadruplex formation is proposed in this article. A guanine (G)-rich oligonucleotide, T30695, was used as a recognition probe, and a DNA intercalator, SYBR Green I (SG), was used as a signal reporter. In the absence of Pb²⁺, the SG intercalated with the single-stranded random-coil T30695 and emitted strong fluorescence. While in the presence of Pb²⁺, the random-coil T30695 would fold into a G-quadruplex structure and the SG could barely show weak fluorescence, and the fluorescence intensity was inversely proportional to the involving amount of Pb²⁺. Based on this, a selective lead ion sensor with a limit of detection of 3.79 ppb (parts per billion) and a detection range from 0 to 600 ppb was constructed. Because detection for real samples was also demonstrated to be reliable, this simple, low-cost, sensitive, and selective sensor holds good potential for Pb²⁺ detection in real environmental samples.     

Detection of glucose using immobilized bienzyme on cyclic bisureas–gold nanoparticle conjugate

A highly sensitive electrochemical glucose sensor has been developed by the co-immobilization of glucose oxidase (GOx) and horseradish peroxidase (HRP) onto a gold electrode modified with biocompatible cyclic bisureas–gold nanoparticle conjugate (CBU–AuNP). A self-assembled monolayer of mercaptopropionic acid (MPA) and CBU–AuNP was formed on the gold electrode through a layer-by-layer assembly. This modified electrode was used for immobilization of the enzymes GOx and HRP. Both the HRP and GOx retained their catalytic activity for an extended time, as indicated by the low value of Michaelis–Menten constant. Analytical performance of the sensor was examined in terms of sensitivity, selectivity, reproducibility, lower detection limit, and stability. The developed sensor surface exhibited a limit of detection of 100 nM with a linear range of 100 nM to 1 mM. A high sensitivity of 217.5 μA mM⁻¹ cm⁻² at a low potential of −0.3 V was obtained in this sensor design. Various kinetic parameters were calculated. The sensor was examined for its practical clinical application by estimating glucose in human blood sample.     

Rapid evaluation of a protein-based voltage probe using a field-induced membrane potential change

The development of a high performance protein probe for the measurement of membrane potential will allow elucidation of spatiotemporal regulation of electrical signals within a network of excitable cells. Engineering such a probe requires a functional screen of many candidates. Although the glass-microelectrode technique generally provides an accurate measure of a given test probe, throughputs are limited. In this study, we focused on an approach that uses the membrane potential changes induced by an external electric field in a geometrically simple mammalian cell. For quantitative evaluation of membrane voltage probes that rely on the structural transition of the S1–S4 voltage sensor domain and hence have non-linear voltage dependencies, it was crucial to introduce exogenous inwardly rectifying potassium conductance to reduce cell-to-cell variability in resting membrane potentials. Importantly, the addition of the exogenous conductance drastically altered the profile of the field-induced potential. Following a site-directed random mutagenesis and the rapid screen, we identified a mutant of a voltage probe Mermaid, exhibiting positively shifted voltage sensitivity. Due to its simplicity, the current approach will be applicable under a microfluidic configuration to carry out an efficient screen. Additionally, we demonstrate another interesting aspect of the field-induced optical signals, ability to visualize electrical couplings between cells.     

Implicit membrane treatment of buried charged groups: Application to peptide translocation across lipid bilayers

The energetic cost of burying charged groups in the hydrophobic core of lipid bilayers has been controversial, with simulations giving higher estimates than certain experiments. Implicit membrane approaches are usually deemed too simplistic for this problem. Here we challenge this view. The free energy of transfer of amino acid side chains from water to the membrane center predicted by IMM1 is reasonably close to all-atom free energy calculations. The shape of the free energy profile, however, for the charged side chains needs to be modified to reflect the all-atom simulation findings (IMM1-LF). Membrane thinning is treated by combining simulations at different membrane widths with an estimate of membrane deformation free energy from elasticity theory. This approach is first tested on the voltage sensor and the isolated S4 helix of potassium channels. The voltage sensor is stably inserted in a transmembrane orientation for both the original and the modified model. The transmembrane orientation of the isolated S4 helix is unstable in the original model, but a stable local minimum in IMM1-LF, slightly higher in energy than the interfacial orientation. Peptide translocation is addressed by mapping the effective energy of the peptide as a function of vertical position and tilt angle, which allows identification of minimum energy pathways and transition states. The barriers computed for the S4 helix and other experimentally studied peptides are low enough for an observable rate. Thus, computational results and experimental studies on the membrane burial of peptide charged groups appear to be consistent. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.