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PubMed2006

Evaluation of different strategies for the development of amperometric biosensors for L-lactate.

Two strategies were investigated for the development of lactate biosensors based on sol-gel matrixes and polysulfone composite films, both containing L-lactate dehydrogenase (LDH). Firstly, reagentless disposable screen-printed electrodes (SPE's) with Meldola's Blue (MB) and the cofactor NAD(+) inside a sol-gel matrix were prepared. These showed relatively low sensitivities (260 microA/M). Secondly, mediator-modified-polysulfone-graphite composite films deposited over both cylindrical epoxy-graphite and SPE's. These electrodes showed enhanced performance characteristics: improved sensitivity (80 mA/M), detection limit (0.87 microM) and reproducibility (2%). Reagentless electrodes, incorporating NAD(+) in the polysulfone film, had a decreased sensitivity, although better than that achieved by the sol-gel electrodes. While sol-gel electrodes showed a linear range between 1.25 x 10(-4) and 2.48 x 10(-3)M, the epoxy-graphite composite electrodes based on polysulfone composite films allowed the detection of lactate at a linear range of lower concentrations from 1 x 10(-6) to 1.2 x 10(-5)M. Finally, the performance of the LDH-MB-polysulfone-composite film-based SPE's in a flow system was studied. Short response times were obtained (t<30s). Furthermore, repeatability and reproducibility values were notably improved, especially when working with electrodes covered with a polyamide layer prepared with N-(2-aminoethyl)-piperazine.

Biosensing Techniques↗

PubMed2007

A new principle photosynthesis capacity biosensor based on quantitative measurement of delayed fluorescence in vivo.

Delayed fluorescence (DF) is an excellent marker for evaluating plant photosynthesis. Compared with common methods for measuring the photosynthesis rate based on consumption of CO(2), DF technique can quantify the plant photosynthesis capacity more accurately and faster under its physiological status with less interference from the environment. We previously reported a method for measuring photosynthesis using DF of chloroplast [Wang, C.L., Xing, D., Chen, Q., 2004. Biosens. Bioelectron. 20, 454-459]. In the study, a novel fast and portable photosynthesis capacity biosensor system was developed, which was composed of light-emitting diode lattice as excitation light source, Channel Photomultiplier DC-Module to achieve DF, single-chip microcomputer as control center, hermetic dark sample chamber, battery power supply and CO(2), humidity and temperature controller. Compared with our previous work, the system was portable and can directly measure plant photosynthesis capacity in vivo in less than 10s. A database in the software to carry out data acquisition and processing was developed to translate maximal DF intensity to net photosynthesis rate (Pn). In addition, local-control and remote-control mode can be chosen in the system. To demonstrate the utility of the system, it was applied to evaluate maximum Pn of four different plant species samples (Queen Rape Myrtle (var. rubra), soybean (Lu Hei No. 1), maize (Jin Dan No. 39) and rice (Jing Dao No. 21)) in field. The results were compared with that using commercial photosynthesis system LI-6400 and the uncertainty was less than +/-5%. The new principle of photosynthesis measurement is a challenge and breakthrough to conventional method of gas exchange and may be a potential technique of next generation photosynthesis measurement.

Biosensing Techniques↗

PubMed2006

PhotoMEA: an opto-electronic biosensor for monitoring in vitro neuronal network activity.

PhotoMEA is a biosensor useful for the analysis of an in vitro neuronal network, fully based on optical methods. Its function is based on the stimulation of neurons with caged glutamate and the recording of neuronal activity by Voltage-Sensitive fluorescent Dyes (VSD). The main advantage is that it will be possible to stimulate even at sub-single neuron level and to record with high resolution the activity of the entire network in the culture. A large-scale view of neuronal intercommunications offers a unique opportunity for testing the ability of drugs to affect neuronal properties as well as alterations in the behaviour of the entire network. The concept and a prototype for validation is described here in detail.

Biosensing Techniques↗

PubMed2004

Silicon surface modification with a mixed silanes layer to immobilize proteins for biosensor with imaging ellipsometry.

One kind of surface modification method on silicon wafer was presented in this paper. A mixed silanes layer was used to modify silicon surface and rendered the surface medium hydrophobic. The mixed silanes layer contained two kinds of compounds, aminopropyltriethoxysilane (APTES) and methyltriethoxysilane (MTES). A few of APTES molecules in the layer was used to immobilize covalently human immunoglobulin G (IgG) on the silicon surface. The human IgG molecules immobilized covalently on the modified surface could retain their structures well and bind more antibody molecules than that on silicon surface modified with only APTES. This kind of surface modification method effectively improved the sensitivity of the biosensor with imaging ellipsometry.

Biosensing Techniques↗

PubMed2005

Advances in preservation methods: keeping biosensor microorganisms alive and active.

The ability of bacteria to sense their surroundings can be employed to measure the bioavailability and toxicity of pollutants. However, long-term maintenance of both viability and activity of the sensor bacteria is required for the development of cell-based devices for environmental monitoring. To meet these demands, various techniques to conserve such bacteria have been reported, including freeze drying, vacuum drying, continuous cultivation, and immobilisation in biocompatible polymers of organic or inorganic origin. Much effort has been invested in merging these bacterial preservation schemes with the construction of sensor cell arrays on platforms such as biochips or optic fibres, hopefully leading to effective miniaturised whole-cell biosensor systems. These approaches hold much promise for the future. Nevertheless, their eventual implementation in practical devices calls for significant enhancement of current knowledge on formulation of reporter microorganisms.

Bacteria↗

PubMed2005

A zucchini-peroxidase biosensor applied to dopamine determination.

A biosensor modified with peroxidase from crude extract of zucchini (Cucurbita pepo) was developed for the determination of dopamine in pharmaceutical samples. This enzyme catalyses the oxidation of dopamine to dopaminequinone, in presence of hydrogen peroxide, which the electrochemical reduction can be followed at a peak potential of -0.02 V. The recovery of dopamine from the samples ranged from 94.8% to 106% and a rectilinear analytical curve for dopamine concentration from 5.0 x 10(-4) to 3.0 x 10(-3) mol L-1 (r=0.9982) was obtained. The detection limit was 2.6x10(-5) mol L-1 and the relative standard deviation was less than 1.2% for 7.9 x 10(-4) mol L-1 dopamine in 0.1 mol L-1 phosphate buffer solution at pH 6.0 (n=10).

Biosensing Techniques↗

PubMed2005

Optical biosensor provides insights for bradykinin B(2) receptor signaling in A431 cells.

The spatial and temporal targeting of proteins or protein assemblies to appropriate sites is crucial to regulate the specificity and efficiency of protein-protein interactions, thus dictating the timing and intensity of cell signaling and responses. The resultant dynamic mass redistribution could be manifested by label free optical biosensor, and lead to a novel and functional optical signature for studying cell signaling. Here we applied this technology, termed as mass redistribution cell assay technology (MRCAT), to study the signaling networks of bradykinin B(2) receptor in A431 cells. Using MRCAT, the spatial and temporal relocation of proteins and protein assemblies mediated by bradykinin was quantitatively monitored in microplate format and in live cells. The saturability to bradykinin, together with the specific and dose-dependent inhibition by a B(2) specific antagonist HOE140, suggested that the optical signature is a direct result of B(2) receptor activation. The sensitivity of the optical signature to cholesterol depletion by methyl-beta-cyclodextrin argued that B(2) receptor signaling is dependent on the integrity of lipid rafts; disruption of these microdomains hinders the B(2) signaling. Modulations of several important intracellular targets with specific inhibitors suggested that B(2) receptor activation results in signaling via at least dual pathways - G(s)- and G(q)-mediated signaling. Remarkably, the two signaling pathways counter-regulate each other. Several critical downstream targets including protein kinase C, protein kinase A, and epidermal growth factor receptor had been identified to involve in B(2) signaling. The roles of endocytosis and cytoskeleton modulation in B(2) signaling were also demonstrated.

Biosensing Techniques↗

PubMed2006

Assessment of mucoadhesion by a resonant mirror biosensor.

The aim of this study was to add knowledge to the existing theories of mucoadhesion and to review mucoadhesive polymers based on their ability to form non-covalent bonds with mucus glycoprotein. Resonant mirror biosensor was used to study the candidate mucoadhesive polymers hydroxypropyl methylcellulose, carboxymethylcellulose, Carbopol, hyaluronate, alginate and chitosan. Bovine submaxillary mucin was chosen as substrate, representing the major glycosylated protein in mucus. For comparison, non-glycosylated bovine serum albumin was used as an alternative substrate. The results of this study reveal that there is a clear correlation between the ionization state of the polymer, which is dependent on the pH of the surrounding environment, and its binding behavior. Ionizable polymers need to be in their unionized state to be able to form non-covalent bonds with mucus glycoprotein. Acidic polymers display binding behavior only at pH around or lower than their corresponding pK(a) values and basic polymers vice versa. Chitosan was found to be the most mucoadhesive polymer. Unionizable polymers like hydroxypropyl methylcellulose did not display any affinity for mucus glycoprotein. Unionized amino- and carboxyl groups on polymers were found to be important structural feature of polymer for the formation of weak chemical bonds to mucus glycoproteins.

Acrylic Resins↗

PubMed2004

Off-line monitoring of bacterial stress response during recombinant protein production using an optical biosensor.

A surface plasmon resonance (SPR) biosensor was used to monitor the profiles of the heat-shock protein (DnaK) and the expression of a heterologous protein to map the dynamics of the cellular stress response in Escherichia coli. As expression system was used an E. coli strain overproducing human recombinant superoxide dismutase (rhSOD). Expression of DnaK showed complex patterns differing with strength of induction. The strong up-regulation of DnaK expression was observed in all cultivations which over-produced of rhSOD. Similar patterns were not observed in non-induced reference cultures. Differences in DnaK concentration profiles were correlated with induction strength. Presented data, carried out in shake flask and glucose limited fed-batch cultivation, show a good consistency with previously published transcriptional profiling results and provide complementary information to understand stress response related to overproduction of recombinant protein. The study also demonstrates the feasibility of using the SPR as a two channel protein array for monitoring of intracellular components.

Bioreactors↗

PubMed2004

Single molecule force spectroscopy on ligand-DNA complexes: from molecular binding mechanisms to biosensor applications.

Recent developments in single molecule force spectroscopy (SMFS) allow direct observation and measurements of forces that hold protein-DNA complexes together. Furthermore, the mechanics of double-stranded (ds) DNA molecules in the presence of small binding ligands can be detected. The results elucidate molecular binding mechanisms and open the way for ultra sensitive and powerful biosensor applications.

Binding Sites↗

PubMed2005

Amperometric biosensor for the detection of hydrogen peroxide using catalase modified electrodes in polyacrylamide.

A simple biosensor for the detection of hydrogen peroxide in organic solvents has been developed and coupled to a flow injection analysis (FIA) system. Catalase was entrapped in polyacrylamide gel and placed on the surface of platinum (working electrode) fixed in a Teflon holder with Ag-wire (auxiliary electrode), followed by addition of filter paper soaked in KCl. The entrapped catalase gel was held on the electrode using membranes. The effects of cellulose and polytetrafluroethylene (PTFE) membranes on the electrode response towards hydrogen peroxide have been studied. The modified electrode has been used to study the detection of hydrogen peroxide in solvents like water, dimethyl sulfoxide (DMSO), and 1,4-dioxane using amperometric techniques like cyclic voltammetry (CV) and FIA. The CV of modified catalase electrode showed a broad oxidation peak at -150 mV and a clear reduction peak at -212 mV in the presence of hydrogen peroxide. Comparison of CV with hydrogen peroxide in various solvents has been carried out. The electrode showed an irreversible kinetics with DMSO as the solvent. A flow cell has been designed in order to carry on FIA studies to obtain calibration plots for hydrogen peroxide with the modified electrode. The calibration plots in several solvents such as water, dimethyl sulfoxide, 1,4-dioxane have been obtained. The throughput of the enzyme electrode was 10 injections per hour. Due to the presence of membrane the response time of the electrode is concentration dependent.

Acrylic Resins↗

PubMed2006

Intracellular redox equilibrium and growth phase affect the performance of luciferase-based biosensors.

Light emission from the bacterial luciferase operon has been variously exploited during last two decades. The use of convenient inducible promoters has granted significant degrees of specificity to whole cell-based assays for high-throughput screening and environmental monitoring. Nevertheless, unexplained unspecific responses have been repeatedly reported. Here, we show that the impairment of the intracellular biochemical equilibrium interferes with the luminescence produced by Escherichia coli and Staphylococcus aureus strains carrying the lux operon under constitutive or inducible control. Compounds as trimethoprim and methotrexate, by indirectly inducing NADPH accumulation, enhance light emission. Conversely, molecules driving the cell toward an oxidized state, as dimethyl sulfoxide, inhibit luminescence. These findings fit into the accepted biochemical pathway for bioluminescence, where NADPH and reducing equivalents are necessary for the production of luciferase substrates, although they do not directly take part into the light-emitting reaction. Moreover, we investigated the influence of induction timing upon the bioluminescence response from inducible reporter systems and demonstrated a correlation between the emitted light and the growth phase at which induction is performed. Our results provide explanations for some unspecific responses recorded so far in whole cell-based luminescent biosensors and emphasize the intrinsic limitations of this kind of reporting system.

Biosensing Techniques↗

PubMed2006

Electrochemical biosensor for catechol using agarose-guar gum entrapped tyrosinase.

An electrochemical biosensor using tyrosinase was constructed for the determination of catechol. The enzyme was extracted from a plant source Amorphophallus companulatus and entrapped in agarose-guar gum composite biopolymer matrix. Catechol was determined by direct reduction of biocatalytically liberated quinone species at -0.1 V versus Ag/AgCl (3M KCl). The response was found to be linear and concentration dependent in the range of 6 x 10(-5) to 8 x 10(-4)M with a lower detection limit of 6 microM. It has reusability up to 20 cycles and a shelf life of more than 2 months when stored at 4 degrees C.

Benzoquinones↗

PubMed2005

Biosensor signal amplification of vesicles functionalized with glycolipid for colorimetric detection of Escherichia coli.

A supramolecular assembly of phospholipid-polymerized diacetylene vesicles functionalized with glycolipid can provide a molecular recognition function. The Escherichia coli-glycolipid binding event leads to a visible color change from blue to red, readily seen with the naked eye and quantified by absorption spectroscopy. The biosensor signal is amplified through a suitable increase of phospholipid content in the mixed lipid vesicles and pH of aqueous solutions.

Acetylene↗

PubMed2006

Sequential analysis of multiple analytes using a surface plasmon resonance (SPR) biosensor.

A sequential analysis method for the analysis of two analytes was developed using a surface plasmon resonance (SPR) biosensor. A sample with both analytes was introduced into the single sensing region and then each analyte was analyzed sequentially. Two detection models were devised for the samples with the following composition: (1) one target analyte resulting in a sensor response without any label and the other analyte with only additional label, (2) both target analytes requiring additional labels for detection. A standard curve for each model was prepared and applied for sequential analysis of anti-bovine serum albumin (anti-BSA) antibodies and horseradish peroxidase (HRP). The errors of the sequential analysis of Models 1 and 2 were found to be less than 6%, and this method was therefore acceptable for application. No cross-reaction arising from non-specific binding among the participating antigens and antibodies was shown to occur in Models 1 and 2. For optimization of the analyte binding capacity of immunoaffinity (IA), the concentration ratio of the molecular recognition element at the immobilization step was adjusted. Subsequently, from the measurement of the maximum sensor response (R(max)), optimization of the analyte binding capacity could be made. Using Model 2, the feasibility of sequential analysis was demonstrated by detecting levels of human chorionic gonadotropin (hCG) and human albumin (hA) in healthy human urine, since both proteins are known to be related to abortion and preterm delivery during early pregnancy.

Albumins↗

PubMed2005

Structural studies of an engineered zinc biosensor reveal an unanticipated mode of zinc binding.

Protein engineering was used previously to convert maltose-binding protein (MBP) into a zinc biosensor. Zn(2+) binding by the engineered MBP was thought to require a large conformational change from "open" to "closed", similar to that observed when maltose is bound by the wild-type protein. We show that although this re-designed MBP molecule binds Zn(2+) with high affinity as previously reported, it does not adopt a closed conformation in solution as assessed by small-angle X-ray scattering. High-resolution crystallographic studies of the engineered Zn(2+)-binding MBP molecule demonstrate that Zn(2+) is coordinated by residues on the N-terminal lobe only, and therefore Zn(2+) binding does not require the protein to adopt a fully closed conformation. Additional crystallographic studies indicate that this unexpected Zn(2+) binding site can also coordinate Cu(2+) and Ni(2+) with only subtle changes in the overall conformation of the protein. This work illustrates that the energetic barrier to domain closure, which normally functions to maintain MBP in an open concentration in the absence of ligand, is not easily overcome by protein design. A comparison to the mechanism of maltose-induced domain rearrangement is discussed.

Binding Sites↗

PubMed2006

Sensitivity enhancement by exchange mediated magnetization transfer of the xenon biosensor signal.

Hyperpolarized xenon associated with ligand derivatized cryptophane-A cages has been developed as a NMR based biosensor. To optimize the detection sensitivity we describe use of xenon exchange between the caged and bulk dissolved xenon as an effective signal amplifier. This approach, somewhat analogous to 'remote detection' described recently, uses the chemical exchange to repeatedly transfer spectroscopic information from caged to bulk xenon, effectively integrating the caged signal. After an optimized integration period, the signal is read out by observation of the bulk magnetization. The spectrum of the caged xenon is reconstructed through use of a variable evolution period before transfer and Fourier analysis of the bulk signal as a function of the evolution time.

Algorithms↗

PubMed2004

On-line monitoring of striatum glucose and lactate in the endothelin-1 rat model of transient focal cerebral ischemia using microdialysis and flow-injection analysis with biosensors.

In vivo studies on cerebral glucose and lactate metabolism following a brain insult require fast and sensitive monitoring techniques. Here we report on-line monitoring of ischemic events and metabolic changes following reperfusion in striatum of freely moving rats subjected to endothelin-1 (60-240 pmol) induced, transient focal cerebral ischemia using slow microdialysis (0.5 microl/min), fast sampling (every minute) and flow-injection analysis with biosensors for glucose and lactate. The high-time resolution provides detailed information on lactate rise times and duration of low glucose. In rats, developing large striatal lesions, lactate increased from 1.0 +/- 0.1 to 4.2 +/- 0.7 mM within 37 +/- 1 min, whereas glucose dropped from 0.3 +/- 0.1 mM to below detection levels (<0.05 mM) for a period of 80 +/- 18 min. The lactate increase measured over a 2-h period after endothelin-1 infusion was highly correlated with striatal infarct size. In some rats oscillatory changes are observed which cannot be detected in traditional assays. The here-described monitoring technique applied in a clinically relevant rat model is a sensitive tool to study post-ischemic energy metabolism, effects of therapeutic interventions and its relationship with histological outcome.

Animals↗