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Monomeric sarcosine oxidase (mSOx) fusion with the silaffin peptide, R5, designed previously for easy protein production in low resource areas, was used in a biosilification process to form an enzyme layer electrode biosensor. mSOx is a low activity enzyme (10–20 U/mg) requiring high amounts of enzyme to obtain an amperometric biosensor signal, in the clinically useful range <1 mM sarcosine, especially since the Km is >10 mM. An amperometric biosensor model was fitted to experimental data to investigate dynamic range. mSOx constructs were designed with 6H (6×histidine) and R5 (silaffin) peptide tags and compared with native mSOx. Glutaraldehyde (GA) cross‐linked proteins retained ∼5 % activity for mSOx and mSOx‐6H and only 0.5 % for mSOx‐R5. In contrast R5 catalysed biosilification on (3‐mercaptopropyl) trimethoxysilane (MPTMS) and tetramethyl orthosilicate (TMOS) particles created a 'self‐immobilisation' matrix retaining 40 % and 76 % activity respectively. The TMOS matrix produced a thick layer (>500 μm) on a glassy carbon electrode with a mediated current due to sarcosine in the clinical range for sarcosinemia (0–1 mM). The mSOx‐R5 fusion protein was also used to catalyse biosilification in the presence of creatinase and creatininase, entrapping all three enzymes. A mediated GC enzyme linked current was obtained with dynamic range available for creatinine determination of 0.1–2 mM for an enzyme layer ∼800 nm. [ABSTRACT FROM AUTHOR]

Lanthanide metal organic frameworks (MOFs), Eu(BTC) and TbEu(BTC) were assessed to follow uptake of deoxynucleotide triphosphate (dNTPs) in loop mediated isothermal amplification (LAMP) using BST2.0. dNTPs could be determined below 150 μM. A ratiometic measurement of Tb3+:Eu3+ fluorescence in TbEu(BTC) gave a limit of detection of 2.08 μM for dNTPs. dNTP uptake slowed above 8 mM Mg2+. This correlated with end‐point and real‐time LAMP, using PicoGreen and SYBR green respectively. For the P.Mal‐Lau 18S rRNA primer set, with P. malariae a detection limit of 102.5 copies was obtained. Cross reactivity between P. malariae and P. knowlesi was shown with 18S rRNA primer sets. [ABSTRACT FROM AUTHOR]

Microbubbles and microcapsules of silane-polycaprolactone (SiPCL) have been filled with a fluorescent acridium salt (lucigenin) as a model for a drug-loaded delivery vehicle. The uptake and delivery were studied and compared with similar microbubbles and microcapsules of silica/mercaptosilica (S/M/S). Positively charged lucigenin was encapsulated through an electrostatic mechanism, following a Type I Langmuir isotherm as expected, but with an additional multilayer uptake that leads to a much higher loading for the SiPCL system (~280 µg/2.4 x 109 microcapsules compared with ~135 µg/2.4 x 109 microcapsules for S/M/S). Whereas the lucigenin release from the S/M/S bubbles and capsules loaded below the solubility limit is consistent with diffusion from a monolithic structure, the SiPCL structures show distinct release patterns; the Weibull function predicts a general trend for diffusion from normal Euclidean space at short times tending toward diffusion out of fractal spaces with increasing time. As a slow release system, the dissolution time (Td) increases from 1 to 2 days for the S/M/S and for the low concentration, loaded SiPCl vehicles to ~10 days for the high loaded microcapsules. However, Td can be reduced on insonation to 2 days, indicating the potential to gain control over the local enhanced release with ultrasound. This was tested for a docetaxel model and its effect on C4-2B prostate cancer cells, showing improved cell toxicity for concentrations below the normal EC50 in solution. [ABSTRACT FROM AUTHOR]

The fabrication of an enzyme-degradable polymer/silica hybrid microbubble is reported that produces an ultrasound contrast image. The polymer, a triethoxysilane end-capped polycaprolactone (SiPCL), is used to incorporate enzyme-degradable components into a silica microbubble synthesis, and to impart increased elasticity for enhanced acoustic responsiveness. Formulations of 75, 85, and 95 wt % SiPCL in the polymer feed produced quite similar ratios of SiPCL and silica in the final bubble but different surface properties. The data suggest that different regions of the microbubbles were SiPCL-rich: the inner layer next to the polystyrene template core and the outer surface layer, thereby creating a sandwiched silica-rich layer of the bubble shell. Overall, the thickness of the microbubble shell was dependent on the starting TEOS concentration and the reaction time. Despite the layered structure, the microbubble could be efficiently degraded by lipase enzyme, but was stable without enzyme. The ultrasound contrast showed a general trend of increase in image intensity with SiPCL feed ratio, although the 95 wt % SiPCL bubbles did not produce a contrast image, probably due to bubble collapse. At higher normalized peak negative acoustic pressure (mechanical index, MI), a nonlinear frequency response also emerges, characterized by the third harmonic at around 3f0, and increases with MI. The threshold MI transition from linear to nonlinear response increased with decrease in SiPCL. [ABSTRACT FROM AUTHOR]

Spores of Bacillus megaterium QM B1551 are encased in a morphologically distinctive exosporium. We demonstrate here that genes encoded on the indigenous pBM500 and pBM600 plasmids are required for exosporium assembly and or stability in spores of this strain. Bioinformatic analyses identified genes encoding orthologues of the B. cereus-family exosporium nap and basal layer proteins within the B. megaterium genome. Transcriptional analyses, supported by electron and fluorescent microscopy, indicate that the pole-localized nap, identified here for the first time in B. megaterium QM B1551 spores, is comprised of the BclA1 protein. The role of the BxpB protein, which forms the basal layer of the exosporium in B. cereus spores, is less clear since spores of a null mutant strain display an apparently normal morphology. Retention of the localized nap in bxpB null spores suggests that B. megaterium employs an alternative mechanism to that used by B. cereus spores in anchoring the nap to the spore surface. [ABSTRACT FROM AUTHOR]

Mycobacterium acquire iron by producing siderophores called mycobactins, with an extremely high affinity complexation of Fe3+. The iron complex shows distinctive electrochemistry predicting 18 orders of magnitude greater affinity of the mycobactin for Fe3+ than Fe2+. A heterogeneous standard rate constant, ks of the order of 10−5 cm s−1 confirms quasi reversible electrochemistry and based on the equilibrium in the presence of excess solution Fe3+/Fe2+ the oxidation and reduction peaks for the siderophore complex could be calibrated for ferric mycobactin J (FeMJ). FeMJ spiked urine collected from cows showed a matrix effect on the current peak height. For example, 240±15 µM FeMJ was estimated in 600 µM spiked urine. However, in the presence of excess solution Fe(acac)3 the same sample yielded an estimated 580±25 µM FeMJ. [ABSTRACT FROM AUTHOR]

Taking advantage of BRET, a mutant firefly luciferase with higher pH- and thermo-stability than the wild-type could be coupled with the red-emitting fluorescent protein of mCherry in both a fused and unfused format. The BRET pair allows >40% of the light emitted to be red shifted over 600 nm to the mCherry acceptor wavelength. Taking the expected quantum yield for mCherry (0.22), a good fit to predicted light transfer is shown, with no other losses. Two measurements are considered for ATP determination: (a) a ratiometric technique for ATP measurement using both donor and acceptor emission intensities, making the calibration slope independent of protein concentration in a broad range. This measurement was limited by the BRET efficiency and the low quantum yield of the mCherry acceptor, but this detection limit might be improved with other fluorescent proteins with higher quantum yield. The fused BRET pair also resulted in a small increase in the BRET ratio. (b) An ATP dependent shift in the wavelength maximum using just the acceptor mCherry emission was also proposed for ATP determination. This did not require a high BRET efficiency and only uses emission above 600 nm to obtain the acceptor emission maximum, but not its intensity; it is independent of protein concentration across a broad range. This offers a novel and robust method for determination of ATP between 10−11 to 10−5 M with an easy baseline calibration with ATP concentration >10−4 M. [ABSTRACT FROM AUTHOR]

Semiconductor quantum dots (QDs) have been shown to be highly sensitive to electron or charge transfer processes, which may alter their optical properties. This feature can be exploited for different sensing applications. Here, we demonstrate that QD-anthraquinone conjugates can function as electron transfer-based pH nanosensors. The attachment of the anthraquinones on the surface of QDs results in the reduction of electron hole recombination, and therefore a quenching of the photoluminescence intensity. For some anthraquinone derivatives tested, the quenching mechanism is simply caused by an electron transfer process from QDs to the anthraquinone, functioning as an electron acceptor. For others, electron transfer and energy transfer (FRET) processes were found. A detailed analysis of the quenching processes for CdSe/ZnS QD of two different sizes is presented. The photoluminescence quenching phenomenon of QDs is consistent with the pH sensitive anthraquinone redox chemistry. The resultant family of pH nanosensors shows pKa ranging ∼5–8, being ideal for applications of pH determination in physiological samples like blood or serum, for intracellular pH determination, and for more acidic cellular compartments such as endosomes and lysosomes. The nanosensors showed high selectivity towards many metal cations, including the most physiologically important cations which exist at high concentration in living cells. The reversibility of the proposed systems was also demonstrated. The nanosensors were applied in the determination of pH in samples mimicking the intracellular environment. Finally, the possibility of incorporating a reference QD to achieve quantitative ratiometric measurements was investigated. [ABSTRACT FROM AUTHOR]

An enzyme-linked analytical nanosphere sensor (ANSor) is described, responding to enzyme-substrate turnover in the vicinity of a quantum dot (QD) due to cohnmobilized enzyme and pH sensitive ligand. QD capping by mercapto-alkanoic acids were rejected as a pH sensitive ligand, but with the use of a layer-by-layer assembly on mercaptopropionic capped QDs and an intermediate poly(allylamine hydrochloride) layer, anthraquinone sulfonate (calcium red, CaR) was introduced to modify the pH in the immobilized system > 8. QD-CaR absorption shows speciral overlap with QDroo emission at all pHs and gives a complex pH dependent fluorescence resonance energy transfer (FREL) efficiency, due to excited state proton transfer (χex = 540 nm; χem = 585 nm). In contrast QD615-CaR with spectral overlap between the QD and CaR gave a strong and reproducible pH response. QD-urease and QD-creatinine deiminase conjugates could be linked with pH changes produced by enzyme degradation of urea and crealinine, respectively. Close coupling between the pH sensitive QD and enzyme conjugate maximized signal compared with solution based assays: QD-urease and QD-CD bioconjugates were tested in model biological media (Dulbecco's modified Eagle's Medium and fetal calf serum) and in urine, showing a response in 3-4 min. [ABSTRACT FROM AUTHOR]

A new fluorescent nanosensor family for Zn2+ determination is reported based on azamacrocycle derivatization of CdSe/ZnS core/shell quantum dot nanoparticles. They are the first zinc ion sensors using QD nanoparticles in a host-guest and receptor-fluorophore system. Three azamacrocycles are demonstrated as receptors: TACN (1,4,7-triazacyclononane), cyclen (1,4,7,10-tetraazacyclododecane), and cyclam (1,4,8,11-tetraazacyclotetradecane). Azamacrocycles conjugated to QDs via an amide link interact directly with one of the photoinduced QD charge carriers, probably transferring the hole in the QD to the azamacrocycle, thereby disrupting the radiative recombination process. When zinc ion enters the azacrown, the lone pair electrons of the nitrogen atom become involved in the coordination and the energy level is no longer available for the hole-transfer mechanism, switching on the QD emission and a dramatic increase of the fluorescence intensity results, allowing the detection of low concentrations of zinc ions. Using this operating principle, three zinc ion sensors based on CdSe-ZnS core-shell QD nanoparticles showed a very good linearity in the range 5-500 μM, with detection limits lower than 2.4 μM and RSDs ∼3% (n = 10). In addition, the versatility of the sensors was demonstrated, since different sizes (and colors) of QDs can be employed and will respond to zinc in a similar way. In a study of interferences, the zinc-sensitive QDs showed good selectivity in comparison with other physiologically important cations and other transition metals tested. The results from fetal calf serum and samples mimicking physiological conditions suggested very good applicability in the determination of zinc ion in physiological samples. [ABSTRACT FROM AUTHOR]

Carboxy-terminated polydiacetylene vesicles are known to undergo dramatic color transitions in response to exposure to external stimuli such as pH, temperature, and receptor- ligand binding. FHR spectroscopy was used to identify the breakdown in the interfacial hydrogen-bonding interactions of the carboxylic acid headgroups of polymerized 10,12-tricosadiynoic acid (TRCDA) vesicles in aqueous solution during pH chromic transition. The headgroup structure was monitored as the chromic transition takes place and the dissociation dependence of the PKa was determined. Due to the attenuated acidity of the interfacially confined carboxy groups, which exhibit PKa values in the range 9.5-9.9, it was found that the deprotonation-triggered blue-red chromic transition occurred in the pH range 9.0-10.1 and that the mechanism of the transition required interaction with the surface carboxyl group, which is of importance in the design of a biochromic mechanism using PDA assemblies. Transmission electron microscopy and FTIR spectroscopy revealed that the surface ionization and the pH-induced chromogenic transition was also accompanied by a dramatic vesicle- planar morphological transition alongside subtle changes to the alkyl chain conformation and packing. A two-step mechanism was implicated as causing the chromic transition that first involves surface deprotonation and then specific cation binding, which can aid the design of sensitive surface-ligand chemistry for new PDA structures. [ABSTRACT FROM AUTHOR]

A polymyxin B (PMB) modified copolymer of ethyl acrylate and 2-hydroxyethyl methacrylate is reported that shows antibacterial activity towards E. coli. The polymer is designed for biosensor interfaces to provide protection against microbial contamination. However, leaching of a biomolecule from its solid support is a well-known problem in immobilization chemistry. It has been proposed that antimicrobial activity results from release of the antibiotic from the polymer matrix, but in the work reported here, bactericidal action was seen for tresyl activated polymers even when no polymyxin B could be detected in the supernatant and where covalent attachment of PMB was indicated. Further evidence for the in situ action of PMB on the polymer was provided using a novel method of investigation, subjecting the polymer, deposited on an Ion-Sensitive Field-Effect Transistor (ISFET) to a step change in ion-concentration (Ion-step). With the ion-step method activation of the polymer and the grafting of Polymyxin-B were followed. A comparison of absorbed PMB on a nonactivated polymer and PMB on the tresyl activated polymer suggested that if leaching of PMB from the latter occurred, it was below the detection limit and well below the minimum inhibitory concentration (MIC) for antimicrobial action. Bactericidal action was thus attributed to PMB attached to the polymer. [ABSTRACT FROM AUTHOR]

The exosporium of Bacillus megaterium QM B1551 spores is morphologically distinct from exosporia observed for the spores of many other species. Previous work has demonstrated that unidentified genes carried on one of the large indigenous plasmids are required for the assembly of the Bacillus megaterium exosporium. Here, we provide evidence that pBM600-encoded orthologues of the Bacillus subtilis CotW and CotX proteins, which form the crust layer in spores of that species, are structural components of the Bacillus megaterium QM B1551 spore exosporium. The introduction of plasmid-borne cotW and orthologous cotX genes to the PV361 strain, which lacks all indigenous plasmids and produces spores that are devoid of an exosporium, results in the development of spores with a rudimentary exosporium-type structure. Additionally, purified recombinant CotW protein is shown to assemble at the air-water interface to form thin sheets of material, which is consistent with the idea that this protein may form a basal layer in the Bacillus megaterium QM B1551 exosporium. [ABSTRACT FROM AUTHOR]

Bacillus stearothermophilus large fragment (BSTLF) DNA polymerase is reported, isolated on silica via a fused R5 silica-affinity peptide and used in nucleic acid diagnostics. mCherry (mCh), included in the fusion construct, was shown as an efficient fluorescent label to follow the workflow from gene to diagnostic. The R5 immobilisation on silica from cell lysate was consistent with cooperative R5-specific binding of R52-mCh-FL-BSTLF or R52-mCh-H10-BSTLF fusion proteins followed by non-specific protein binding (including E. coli native proteins). Higher R5-binding could be achieved in the presence of phosphate, but phosphate residue reduced loop-mediated isothermal amplification (LAMP) performance, possibly blocking sites on the BSTLF for binding of β- and γ-phosphates of the dNTPs. Quantitative assessment showed that cations (Mg2+ and Mn2+) that complex the PPi product optimised enzyme activity. In malaria testing, the limit of detection depended on Plasmodium species and primer set. For example, 1000 copies of P. knowlesi 18S rRNA could be detected with the P.KNO-LAU primer set with Si-R52-mCh-FL-BSTLF , but 10 copies of P. ovale 18S rRNA could be detected with the P.OVA-HAN primer set using the same enzyme. The Si-immobilised BSTLF outperformed the commercial enzyme for four of the nine Plasmodium LAMP primer sets tested. Si-R52-mCh-FL-BSTLF production was transferred from Cambridge to Accra and set up de novo for a trial with clinical samples. Different detection limits were found, targeting the mitochondrial DNA or the 18S rRNA gene for P. falciparum. The results are discussed in comparison with qPCR and sampling protocol and show that the Si-BSTLF polymerase can be optimised to meet the WHO recommended guidelines. [ABSTRACT FROM AUTHOR]

Upconversion nanoparticles (UCNPs) should be particularly well suited for measurement inside cells because they can be imaged down to submicrometer dimensions in near real time using fluorescence microscopy, and they overcome problems, such as photobleaching, autofluorescence, and deep tissue penetration, that are commonly encountered in cellular imaging applications. In this study, the performance of an UCNP modified with a pH-sensitive dye (pHAb) is studied. The dye (emission wavelength 580 nm) was attached in a polyethylene imine (PEI) coating on the UCNP and excited via the 540-nm UCNP emission under 980-nm excitation. The UC resonance energy transfer efficiencies at different pHs ranged from 25 to 30% and a Förster distance of 2.56 nm was predicted from these results. Human neuroblastoma SH-SY5Y cells, equilibrated with nigericin H+/K+ ionophore to equalize the intra- and extracellular pH‚ showed uptake of the UCNP-pHAb conjugate particles and, taking the ratio of the intensity collected from the pHAb emission channel (565–630 nm) to that from the UCNP red emission channel (640–680 nm), produced a sigmoidal pH response curve with an apparent pKa for the UCNP-pHAb of ~ 5.1. The UCNP-pHAb were shown to colocalize with LysoBrite dye, a lysosome marker. Drug inhibitors such as chlorpromazine (CPZ) and nystatin (NYS) that interfere with clathrin-mediated endocytosis and caveolae-mediated endocytosis, respectively, were investigated to elucidate the mechanism of nanoparticle uptake into the cell. This preliminary study suggests that pH indicator–modified UCNPs such as UCNP-pHAb can report pH in SH-SY5Y cells and that the incorporation of the nanoparticles into the cell occurs via clathrin-mediated endocytosis. [ABSTRACT FROM AUTHOR]

Upconversion nanoparticles (UCNPs) can provide a vehicle for chemical imaging by coupling chemically sensitive dyes and quenchers. The mechanism for coupling of two anthraquinone dyes, Calcium Red and Alizarin Red S, was investigated as a function of pH. The green emission band of the UCNPs was quenched by a pH-dependent inner filter effect (IFE) while the red emission band remained unchanged and acted as the reference signal for ratiometric pH measurements. Contrary to previous expectation, there was little evidence for a resonance energy transfer (RET) mechanism even when the anthraquinones were attached onto the UCNPs through electrostatic attraction. Since the UCNPs are point emitters, only emitters close to the surface of the UCNP are within the expected Förster distance and UC-RET is <10%. The theoretical and experimental analysis of the interaction between UCNPs and pH-sensitive quenchers will allow the design of UCNP pH sensors for determination of pH via IFE. [ABSTRACT FROM AUTHOR]

The first CdSe/ZnS quantum dot photoluminescence lifetime-based pH nanosensor has been developed. The average lifetime of mercaptopropionic acid-capped QD nanosensors showed a linear response in the pH range of 5.2–6.9. These nanosensors have been satisfactorily applied for pH estimation in simulated intracellular media, with high sensitivity and high selectivity toward most of the intracellular components. [ABSTRACT FROM AUTHOR]

Colloidosomes have attracted great interest in recent years because of their capability for storage and delivery of small molecules for medical and pharmaceutical applications. However, traditional polymer shell colloidosomes leak low molecular weight drugs due to their intrinsic shell permeability. Here, we report aqueous core colloidosomes with a silver shell, which seals the core and makes the shell impermeable. The silver-coated colloidosomes were prepared by reacting l-ascorbic acid in the microcapsule core with silver nitrate in the wash solution. The silver shell colloidosomes were then modified by using 4,4'-dithiodibutyric acid and cross-linked with rabbit Immunoglobulin G (IgG). Label-free surface plasmon resonance was used to test the specific targeting of the functional silver shell with rabbit antigen. To break the shells, ultrasound treatment was used. The results demonstrate that a new type of functional silver-coated colloidosome with immunoassay targeting, nonpermeability, and ultrasound sensitivity could be applied to many medical applications. [ABSTRACT FROM AUTHOR]

A novel type of self-plasticizing polyacrylate-based membrane was developed for all-solid-state ion-selective potentiometric electrodes. The membrane composition contains a conducting polymer (CP): poly(3,4-ethylenedioxythiophene) end capped with methacrylate groups, chemically grafted with the membrane during the photo-polymerization step. This composition results in ion-selective membranes with the following advantages: lower electrical resistance compared to the CP-free membrane, facile ion-to-electron transduction between the membrane and the electrode support, controlled low activity of analyte ions, and high concentration of interferent ions (incorporated with the CP) within the membrane, potentially resulting in improved analytical parameters. Ca2+- and K+-selective membranes were chosen as model systems to study the effect of pretreatment and CP content on the potentiometric sensor's characteristics. For Ca2+ sensors, reproducible and stable Nernstian characteristics were obtained within the range from 0.1 to 10-9 M CaCl2, without a time-consuming preconditioning step. For K+-selective sensors, the influence on Nernstian response range was observed for varying KCl concentrations in the conditioning solution, with the lowest detection limit found close to 10-8 M KCl. Mass spectrometry coupled with laser ablation studies of the membranes revealed that in this case the detection limit is not related to primary ion content in the membrane contacting a sample solution, but is affected by interfering ion concentration close to the membrane surface. [ABSTRACT FROM AUTHOR]

Carrier-based ion-selective electrodes have long been in routine analytical use, yet they still attract an important focus for further research, for example trying to push their capabilities into new dimensions. In the normal construction, the polymeric/ionophore membrane interfaces with the sample and, on the backside, with an aqueous electrolyte in which an internal reference electrode is placed. At low sample activities, zero current ion fluxes across the membrane due to extraction processes at the membrane interfaces can cause analyte ion activity in the phase boundary to be significantly higher than in the sample bulk. This influences the measured potential, so that consideration of these processes and their management has become a prime objective to attain lower detection limits and optimum selectivity.