Optical fiber enzymatic biosensor for reagentless measurement of ethylene dibromide

A reagentless enzymatic optical biosensor has been constructed to measure the concentration of ethylene dibromide (EDB, 1,2‐dibromoethane), a US EPA Priority Pollutant. This biosensor is based on the haloalkane dehalogenase DhaA, which generates protons as a product of the dehalogenation of EDB. The resulting pH change is detected as a shift in the fluorescence intensity of fluoresceinamine. When layers of fluoresceinamine and Rhodococcus sp. GJ70 expressing DhaA were immobilized on the tip of an optical fiber, the resulting changes in fluorescence were proportional to the EDB concentration in the range 1–10 μg/L and nonlinear (saturation‐type trend) for concentrations up to 10 mg/L. EDB concentrations as low as 1 μg/L could be detected in aqueous solutions. Both the pH and buffer capacity of the sample had significant effects on the sensor's performance. EDB biosensors were active for at least 37 d, although their sensitivity decreased after 7 d. The biosensor's potential to measure continuously and in situ could make it useful for environmental or water treatment process monitoring systems.     

Internal transport properties of macroporous sugar polyacrylate hydrogels: microsphere diffusion described by phenomenological laws

We have determined the internal transport properties of heterogeneous, macroporous hydrogels based on the regioregular sugar polyacrylate poly(6-acryloyl-beta-O-methyl-galactopyranoside). This was accomplished by measuring the diffusive flux of variously sized polystyrene microspheres and combining these results with solutions of phenomenological transport laws (the Navier-Stokes equations and Fick's Law with an assumption of first-order irreversible sphere capture by the gel polymer). This enabled calculation of gel properties such as average pore diameters (ca. 11.76 microm) and the diffusivities of the polystyrene spheres in the gel. These values range from 76% to 83% of that in free solution and correlate closely with the equilibrium solution content of the gel (82.3%). This approach has also enabled calculation of the sphere capture rates (2.4 x 10(-3) to 9.6 x 10(-5) s(-1)). These low capture rates indicate that the gel is extremely non-adhesive towards the spheres, and a linear correlation with sphere form drag area (r(2) = 1) was found. The pore sizes of the hydrated gel were observed via DIC light microscopy and the visible effective diameters corresponded very closely to the calculated values (11.66 vs. 11.76 microm). The diffusion/capture of inert spheres in the hydrogel can thus be described in a non-destructive manner by straightforward application of phenomenological transport laws. This result is significant in that these laws were intended to describe macroscopic ensembles of very large numbers of particles in continuous media, not small numbers (i.e., hundreds) in discontinuous media.     

Fetal and Newborn Calf Thymus as a Source of Chromatin Proteins: Purification of HMG-1 and HMG-2

The high mobility group (HMG) non-histone chromosomal proteins were first isolated from calf thymus' but were later found in numerous organs of many vertebrates.' The proteins can be extracted from calf thymus 1 with 0.35 M NaCl and they are quite soluble in 2% trichloroacetic acid. We have shown that members of the HMG-1 family (i.e., HMG-1, HMG-2, and HMG-E) exhibit a preferential affinity for single-stranded DNA at roughly physiological ionic trength. Members of this family have other intriguing properties (see references 6 and 7 for recent reviews), including the ability to assemble nucleosomes in vitroe8 The architecture of the proteins strongly suggests that they are designed to interact simultaneously with histones and with DNA through physically distinct domains^{6, 9}.     

Critical role of peripheral drug actions in experience‐dependent changes in nucleus accumbens glutamate release induced by intravenous cocaine

Recent studies reveal that cocaine experience results in persistent neuroadaptive changes within glutamate (Glu) synapses in brain areas associated with drug reward. However, it remains unclear whether cocaine affects Glu release in drug‐naive animals and how it is altered by drug experience. Using high‐speed amperometry with enzyme‐based and enzyme‐free biosensors in freely moving rats, we show that an initial intravenous cocaine injection at a low self‐administering dose (1 mg/kg) induces rapid, small and transient Glu release in the nucleus accumbens shell (NAc), which with subsequent injections rapidly becomes a much stronger, two‐component increase. Using cocaine‐methiodide, cocaine's analog that does not cross the blood–brain barrier, we confirm that the initial cocaine‐induced Glu release in the NAc has a peripheral neural origin. Unlike cocaine, Glu responses induced by cocaine‐methiodide rapidly habituate following repeated exposure. However, after cocaine experience this drug induces cocaine‐like Glu responses. Hence, the interoceptive actions of cocaine, which essentially precede its direct actions in the brain, play a critical role in experience‐dependent alterations in Glu release, cocaine‐induced neural sensitization and may contribute to cocaine addiction.

     

Applications of commercial biosensors in clinical,food, environmental,and biothreat/biowarfare analyses

The lack of specific, low-cost, rapid, sensitive, and easy detection of biomolecules has resulted in the development of biosensor technology. Innovations in biosensor technology have enabled many biosensors to be commercialized and have enabled biomolecules to be detected onsite. Moreover, the emerging technologies of lab-on-a-chip microdevices and nanosensors offer opportunities for the development of new biosensors with much better performance. Biosensors were first introduced into the laboratory by Clark and Lyons. They developed the first glucose biosensor for laboratory conditions. Then in 1973, a glucose biosensor was commercialized by Yellow Springs Instruments. The commercial biosensors have small size and simple construction and they are ideal for point-of-care biosensing. In addition to glucose, a wide variety of metabolites such as lactate, cholesterol, and creatinine can be detected by using commercial biosensors. Like the glucose biosensors (tests) other commercial tests such as for pregnancy (hCG), Escherichia coli O157, influenza A and B viruses, Helicobacter pylori, human immunodeficiency virus, tuberculosis, and malaria have achieved success. Apart from their use in clinical analysis, commercial tests are also used in environmental (such as biochemical oxygen demand, nitrate, pesticide), food (such as glutamate, glutamine, sucrose, lactose, alcohol, ascorbic acid), and biothreat/biowarfare (Bacillus anthracis, Salmonella, Botulinum toxin) analysis. In this review, commercial biosensors in clinical, environmental, food, and biowarfare analysis are summarized and the commercial biosensors are compared in terms of their important characteristics. This is the first review in which all the commercially available tests are compiled together.     

Potential diagnostic applications of biosensors: current and future directions

This review describes recent advances in biosensors of potential clinical applications. Biosensors are becoming increasingly important and practical tools in pathogen detection, molecular diagnostics, environmental monitoring, food safety control as well as in homeland defense. Electrochemical biosensors are particularly promising toward these goals arising due to several combined advantages including low-cost, operation convenience, and miniaturized devices. We review the clinical applications of electrochemical biosensors based on a few selected examples, including enzyme-based biosensors, immunological biosensors and DNA biosensors.     

DNA‐Functionalized Plasmonic Nanomaterials for Optical Biosensing

Plasmonic nanomaterials, especially Au and Ag nanomaterials, have shown attractive physicochemical properties, such as easy functionalization and tunable optical bands. The development of this active subfield paves the way to the fascinating biosensing platforms. In recent years, plasmonic nanomaterials–based sensors have been extensively investigated because they are useful for genetic diseases, biological processes, devices, and cell imaging. In this account, a brief introduction of the development of optical biosensors based on DNA‐functionalized plasmonic nanomaterials is presented. Then the common strategies for the application of the optical sensors are summarized, including colorimetry, fluorescence, localized surface plasmon resonance, and surface‐enhanced resonance scattering detection. The focus is on the fundamental aspect of detection methods, and then a few examples of each method are highlighted. Finally, the opportunities and challenges for the plasmonic nanomaterials–based biosensing are discussed with the development of modern technologies.     

量子点生物传感体系用于胆碱酯酶抑制剂检测的研究进展

量子点是一种具有独特性质的纳米材料,近年来被广泛应用于传感领域。对高毒性胆碱酯酶抑制剂类化合物进行灵敏检测一直是传感领域的一个研究热点。我们就偶联标记型和非标记型两种传感策略,对量子点生物传感体系用于胆碱酯酶抑制剂检测的研究工作进行了综述。     

Broad-spectrum protein biosensors for class-specific detection of antibiotics

The dramatically increasing prevalence of multi-drug-resistant human pathogenic bacteria and related mortality requires two key actions: (i) decisive initiatives for the detection of novel antibiotics and (ii) a global ban for use of antibiotics as growth promotants in stock farming. Both key actions entail technology for precise, high-sensitive detection of antibiotic substances either to detect and validate novel anti-infective structures or to enforce the non-use of clinically relevant antibiotics. We have engineered prokaryotic antibiotic response regulators into a molecular biosensor configuration able to detect tetracycline, streptogramin, and macrolide antibiotics in spiked liquids including milk and serum at ng/mL concentrations and up to 2 orders of magnitude below current Swiss and EC threshold values. This broad-spectrum, class-specific, biosensor-based assay has been optimized for use in a storable ready-to-use and high-throughput-compatible ELISA-type format. At the center of the assay is an antibiotic sensor protein whose interaction with specific DNA fragments is responsive to a particular class of antibiotics. Binding of biosensor protein to the cognate DNA chemically linked to a solid surface is converted into an immuno-based colorimetric readout correlating with specific antibiotics concentrations.     

Stabilization of glucose oxidase in alginate microspheres with photoreactive diazoresin nanofilm coatings

The nanoassembly and photo-crosslinking of diazo-resin (DAR) coatings on small alginate microspheres for stable enzyme entrapment is described. Multilayer nanofilms of DAR with poly(styrene sulfonate) (PSS) were used in an effort to stabilize the encapsulation of glucose oxidase enzyme for biosensor applications. The activity and physical encapsulation of the trapped enzyme were measured over 24 weeks to compare the effectiveness of nanofilm coatings and crosslinking for stabilization. Uncoated spheres exhibited rapid loss of activity, retaining only 20% of initial activity after one week, and a dramatic reduction in effective activity over 24 weeks, whereas the uncrosslinked and crosslinked {DAR/PSS}-coated spheres retained more than 50% of their initial activity after 4 weeks, which remained stable even after 24 weeks for the two and three bilayer films. Nanofilms comprising more polyelectrolyte layers maintained higher overall activity compared to films of the same composition but fewer layers, and crosslinking the films increased retention of activity over uncrosslinked films after 24 weeks. These findings demonstrate that enzyme immobilization and stabilization can be achieved by using simple modifications to the layer-by-layer self-assembly technique.     

An improved bioluminescence‐based signaling assay for odor sensing with a yeast expressing a chimeric olfactory receptor

The goal of this work was to improve the bioluminescence‐based signaling assay system to create a practical application of a biomimetic odor sensor using an engineered yeast‐expressing olfactory receptors (ORs). Using the yeast endogenous pheromone receptor (Ste2p) as a model GPCR, we determined the suitable promoters for the firefly luciferase (luc) reporter and GPCR genes. Additionally, we deleted some genes to further improve the sensitivity of the luc reporter assay. By replacing the endogenous yeast G‐protein α‐subunit (Gpa1p) with the olfactory‐specific Gα_olf, the optimized yeast strain successfully transduced signal through both OR and yeast Ste2p. Our results will assist the development of a bioluminescence‐based odor‐sensing system using OR‐expressing yeast. Biotechnol. Bioeng. 2012; 109: 3143–3151. © 2012 Wiley Periodicals, Inc.     

Two‐ and three‐photon absorption cross‐section characterization for high‐brightness,cell‐specific multiphoton fluorescence brain imaging

We demonstrate an accurate quantitative characterization of absolute two‐ and three‐photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high‐brightness, cell‐specific two‐ and three‐photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two‐photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep‐tissue experiments.     

Modulating the affinity and the selectivity of engineered calmodulin EF-Hand peptides for lanthanides

A set of engineered peptides (33 amino acids long) corresponding to the helix-turn-helix (EF-Hand) motif of the metal-binding site I of the protein calmodulin from paramecium tetraurelia have been synthesized. A disulfide bridge has been introduced in the native sequence in order to stabilize a native-like conformation. The calcium-binding carboxylate residues in positions 20, 22, 24, and 31 were mutated into other amino acids and the influence of such mutations on the binding affinity of the peptides for calcium and lanthanides have been studied. It was shown that the binding affinity for terbium ions can be modulated with dissociation constants ranging from 40 nmolar to 40 mmolar. The study of the influence of the mutations on the terbium affinity showed that the residue in position 24 played a key role on the capability of the peptides to bind lanthanides and that the affinity could be enhanced by mutations on non-coordinating positions. Such peptides with high affinity for lanthanides may facilitate the development of new highly sensitive biosensors to monitor the metal pollution in the environment.     

Threshold‐based spraying decision programmes for the red spider mite Tetranychus marianae on eggplant

The red spider mite, Tetranychus marianae McGregor (Acari: Tetranychidae), has been an important pest of eggplant Solanum melongena L. (Solanaceae) and other vegetables in the Mariana Islands. The damage due to T. marianae has been severe and caused huge economic losses. Because no threshold levels are available for T. marianae, many growers are applying up to 12 chemical applications per eggplant cropping period. This is not only expensive, but also results in lower yields because of extensive foliar damage and development of resistance in mites to chemicals. To diminish the calendar‐based chemical applications and to preclude damage to foliage and fruit quality, this study was undertaken for the development of a threshold level for optimum timing of chemical applications for T. marianae. In the direction of this aim, an attempt was made to generate different threshold levels by applying chemical spray (Sun‐spray 6E, 5 ml/l) within 12 h after reaching the threshold levels at 2, 4, 6, 8 and 10 mites/leaf, as well as current recommended calendar‐based sprays and non‐sprayed control in a replicated block design for the dry and wet seasons of 2010 at two locations (Yigo and Inaranjan) in Guam (USA). Based on T. marianae‐infested leaves, incidence of T. marianae and yield levels, the plots sprayed at 2 or 4 mites/leaf in the dry season and 2–8 mites/leaf during the wet season had significantly lower leaf damage and incidence of T. marianae compared with a greater number of mites/leaf in calendar‐based sprays and control plots. At the greater threshold levels, the mean yield was significantly reduced in comparison with the mean yield obtained when plots were sprayed at a threshold of 2–8 mites/leaf. Therefore, it is concluded that the optimum threshold chemical spray for T. marianae on eggplant is 4 mites/leaf during the dry season and 8 mites/leaf in the wet season.     

Remote photonic sensing of cerebral hemodynamic changes via temporal spatial analysis of acoustic vibrations

A novel photonic method for remote monitoring of task‐related hemodynamic changes in human brain activation is presented. Physiological processes associated with neural activity, such as nano‐vibrations due to blood flow and tissue oxygenation in the brain, are detected by remote sensing of nano‐acoustic vibrations using temporal spatial analysis of defocused self‐interference random patterns. Temporal nanometric changes of the speckle pattern due to visual task‐induced hemodynamic responses were tracked by this method. Reversing visual checkerboard stimulation alternated with rest epochs, and responsive signals were identified in occipital lobe using near‐infrared spectroscopy. Temporal vibrations associated with these hemodynamic response functions were observed using three different approaches: (a) single spot illumination at active and control areas simultaneously, (b) subspots cross‐correlation‐based analysis, and (c) multiwavelength measurement using a magnitude‐squared wavelet coherence function. Findings show remote sensing of task‐specific neural activity in the human brain.     

Time-resolved fluorescence spectroscopy and molecular dynamics simulations point out the effects of pressure on the stability and dynamics of the porcine odorant-binding protein

The effects of hydrostatic pressure on the structure and stability of porcine odorant-binding protein (pOBP) in the presence and absence of the odorant molecule 2-isobutyl-3-methoxypyrazine (IBMP) were studied by steady-state and time-resolved fluorescence spectroscopy as well as by molecular dynamics simulation. The authors found that the application of moderate values of hydrostatic pressure to pOBP solutions perturbed the microenvironment of Trp(16) and disrupted its highly quenched complex with Met(39). In addition, compared with the protein in the absence of IBMP, the MD simulations experiments carried out at different pressures highlighted the role of this ligand in stabilizing the Trp(16)/Met(39) interaction even at 2000 bar. The obtained results will assist for the tailoring of this protein as specific sensing element in a new class of fluorescence-based biosensors for the detection of explosives.