Quantitative analysis of calibration dependence of biosensors

Three-parameter Hill's equation, which is used in enzyme kinetics, was shown to applicable to calibration curves of both potentiometric (glucose, pesticides, urea, etc.) and amperometric (surfactants, biphenyl, etc.) biosensors. Possible causes of errors of analyte concentration measurements are discussed.     

Quantitative analysis of hydrogen peroxide with special emphasis on biosensors

Bioprocess and Biosystems Engineering - Determination of hydrogen peroxide (H2O2) has become essential in pharmaceutical, biological, clinical and environmental studies. The conventional detection...     

Market analysis of biosensors for food safety

This paper is presented as an overview of the pathogen detection industry. The review includes pathogen detection markets and their prospects for the future. Potential markets include the medical, military, food, and environmental industries. Those industries combined have a market size of $563 million for pathogen detecting biosensors and are expected to grow at a compounded annual growth rate of 4.5%. The food market is further segmented into different food product industries. The overall food-pathogen testing market is expected to grow to $192 million and 34 million tests by 2005. The trend in pathogen testing emphasizes the need to commercialize biosensors for the food safety industry as legislation creates new standards for microbial monitoring. With quicker detection time and reusable features, biosensors will be important to those interested in real time diagnostics of disease causing pathogens. As the world becomes more concerned with safe food and water supply, the demand for rapid detecting biosensors will only increase.     

Quantitative analysis of cell cycle phase durations and PC12 differentiation using fluorescent biosensors

Cell cycle analysis typically relies on fixed time-point measurements of cells in particular phases of the cell cycle. The cell cycle, however, is a dynamic process whose subtle shifts are lost by fixed time-point methods. Live-cell fluorescent biosensors and time-lapse microscopy allows the collection of temporal information about real time cell cycle progression and arrest. Using two genetically-encoded biosensors, we measured the precision of the G1, S, G2, and M cell cycle phase durations in different cell types and identified a bimodal G1 phase duration in a fibroblast cell line that is not present in the other cell types. Using a cell line model for neuronal differentiation, we demonstrated that NGF-induced neurite extension occurs independently of NGF-induced cell cycle G1 phase arrest. Thus, we have begun to use cell cycle fluorescent biosensors to examine the proliferation of cell populations at the resolution of individual cells and neuronal differentiation as a dynamic process of parallel cell cycle arrest and neurite outgrowth.     

Analysis of calibration methodologies for solvent effects in drug discovery studies using evanescent wave biosensors

Recent improvements in sensitivity have enabled direct binding studies of small molecules with evanescent wave biosensors, which monitor binding by measuring refractive index changes close to the sensing surface. The universal solvent for small molecules, dimethylsulfoxide has a high refractive index; consequently, on ligate addition a large non-specific solvent effect is seen which can mask the specific signal. It has been previously noted that different sensor surfaces can respond differently to the same buffer change. The difference is proposed to arise from differences in buffer space and contraction and swelling of the surface hydrogel. Within this paper, a number of calibration approaches are investigated and tested using warfarin binding to human serum albumin as a model system. A number of recommendations are made for accurate referencing for non-specific effects. Changes to the ionic strength of the running buffer had little effect, whilst changes to the charge density of the carboxylmethyl dextran significantly affected how well the control surface reflects the non-specific signal. An amended 'calibration method' can be used, however, it is an additional complex step that was found to overcorrect in the presence of non-specific binding. Matching immobilisation levels between control and active surface significantly reduces solvent differences allowing accurate correction providing solvent compositional changes are minimised in experimental design. Under these circumstances, the traditional method of simple subtraction of the control from the active response is the most appropriate method of correction.     

Glucose biosensors as models for the development of advanced protein-based biosensors

Glucose sensing is used as a model to explore the advantages and problems deriving from the use of either enzymes or sugar binding proteins to develop stable fluorescence biosensors. We report on a novel approach to address the problem of substrate consumption by sensors based on enzymes, namely the utilization of apo-enzymes as non-active forms of the protein which are still able to bind the substrate/ligand. We also review studies in which derivatization of a naturally thermostable sugar-binding protein with a fluorescent probe allows quantitative monitoring of glucose binding even after immobilization on a solid support.     

Sensitivity enhancement of spectral surface plasmon resonance biosensors for the analysis of protein arrays

A novel method for sensitivity enhancement of spectral surface plasmon resonance (SPR) biosensors was presented by reducing the refractive index of the sensing prism in the analysis of protein arrays. Sensitivity of spectral SPR biosensors with two different prisms (BK-7, fused silica) was analyzed by net shifts of resonance wavelength for specific interactions of GST–GTPase binding domain of p21-activated kinase-1 and anti-GST on a mixed thiol surface. Sensitivity was modulated by the refractive index of the sensing prism of the spectral SPR biosensors with the same incidence angle. The sensitivity of a spectral SPR biosensor with a fused silica prism was 1.6 times higher than that with a BK-7 prism at the same incidence angle of 46.2°. This result was interpreted by increment of the penetration depth correlated with evanescent field intensity at the metal/dielectric interface. Therefore, it is suggested that sensitivity enhancement is readily achieved by reducing the refractive index of the sensing prism of spectral SPR biosensors to be operated at long wavelength ranges for the analysis of protein arrays.     

Quantitative methods for the analysis of zoosporic fungi

Quantitative estimations of zoosporic fungi in the environment have historically received little attention, primarily due to methodological challenges and their complex life cycles. Conventional methods for quantitative analysis of zoosporic fungi to date have mainly relied on direct observation and baiting techniques, with subsequent fungal identification in the laboratory using morphological characteristics. Although these methods are still fundamentally useful, there has been an increasing preference for quantitative microscopic methods based on staining with fluorescent dyes, as well as the use of hybridization probes. More recently however PCR based methods for profiling and quantification (semi- and absolute) have proven to be rapid and accurate diagnostic tools for assessing zoosporic fungal assemblages in environmental samples. Further application of next generation sequencing technologies will however not only advance our quantitative understanding of zoosporic fungal ecology, but also their function through the analysis of their genomes and gene expression as resources and databases expand in the future. Nevertheless, it is still necessary to complement these molecular-based approaches with cultivation-based methods in order to gain a fuller quantitative understanding of the ecological and physiological roles of zoosporic fungi.     

Quantitative analyses of individual sugars in mixture using FRET‐based biosensors

Molecular biosensors were developed and applied to measure individual sugars in biological mixtures such as bacterial culture broths. As the sensing units, four sugar‐binding proteins (SBPs for allose, arabinose, ribose, and glucose) were selected from the Escherichia coli genome and connected to a cyan fluorescent protein and yellow fluorescent protein via dipeptide linkers (CFP‐L‐SBP‐YFP). The putative sensors were randomized in the linker region (L) and then investigated with regard to the intensity of fluorescence resonance energy transfer on the binding of the respective sugars. As a result, four representatives were selected from each library and examined for their specificity using 16 available sugars. The apparent dissociation constants of the allose, arabinose, ribose, and glucose sensors were estimated to be 0.35, 0.36, 0.17, and 0.18 μM. Finally, the sugar sensors were applied to monitor the consumption rate of individual sugars in an E. coli culture broth. The individual sugar profiles exhibited a good correlation with those obtained using an HPLC method, confirming that the biosensors offer a rapid and easy‐to‐use method for monitoring individual sugars in mixed compositions. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Quantitative analysis of the energy requirements for development of obesity

OBJECTIVE: Obesity is typically developed over long time and reflected in an energy imbalance, which is too small to be measured and controlled. Our objective is to formulate a mathematical model for the relation between the change in body mass and the values of the energy intake and the energy expenditure, controlled by the physical activity factor PAF. DATA AND THEORY: The uncontrolled components of energy expenditure increases as result of body mass increase: expenditure of a larger mass and expenditure to convert matter in intake into tissue. Both contributions depend on the fraction of fat in the added tissue. Based on data from the literature, the fraction of fat in added tissue and the energy required to convert energy into tissue are estimated and included in the model. RESULTS: Application of the theory shows that an increase in body mass of 1 kg/year corresponds to an energy imbalance of 71 kJ/d for men. Of this imbalance, 82% are stored as new tissue, while 18% are used for energy conversion. If a man in steady state changes energy intake by 0.1 MJ/d, keeping the physical activity factor constant, then the corresponding increase in steady-state body mass is 1.77 kg/PAF, and it will take 320/PAF days before half the change of body mass has taken place. A typical value for PAF is 1.8. CONCLUSION: Energy-based theoretical relations between the various factors involved in energy balance help identifying and quantifying the components of the energy balance and understanding their relations during development of obesity. The inclusion of increased energy expenditure to convert food energy to tissue changes previous estimates of the energy imbalance by about 20 percent.     

Amperometric biosensors/sequential injection analysis system for simultaneous determination of S- and R-captopril

Two amperometric biosensors based on L- and D-amino acid oxidase, respectively, are proposed for the simultaneous detection of S- and R-captopril in a sequential injection analysis system (SIA). The linear concentration ranges are: 0.4-1.6 micromol/l (S-captopril) and 120-950 nmol/l (R-captopril) with detection limits of 0.2 and 15 nmol/l, respectively. The biosensors/SIA system can be used reliably on-line in synthesis process control, for the simultaneous assay of S- and R-captopril with a frequency of 34 samples/h.     

A single-fractal analysis of cellular analyte-receptor binding kinetics utilizing biosensors

A fractal analysis of a confirmative nature only is presented for cellular analyte-receptor binding kinetics utilizing biosensors. Data taken from the literature can be modeled by using a single-fractal analysis. Relationships are presented for the binding rate coefficient as a function of the fractal dimension and for the analyte concentration in solution. In general, the binding rate coefficient is rather sensitive to the degree of heterogeneity that exists on the biosensor surface. It is of interest to note that examples are presented where the binding coefficient, k exhibits an increase as the fractal dimension (D(f)) or the degree of heterogeneity increases on the surface. The predictive relationships presented provide further physical insights into the binding reactions occurring on the surface. These should assist in understanding the cellular binding reaction occurring on surfaces, even though the analysis presented is for the cases where the cellular "receptor" is actually immobilized on a biosensor or other surface. The analysis suggests possible modulations of cell surfaces in desired directions to help manipulate the binding rate coefficient (or affinity). In general, the technique presented is applicable for the most part to other reactions occurring on different types of biosensor or other surfaces.     

Flow injection analysis and in-line biosensors for bioprocess control: a comparison.

Miniaturization will unify the different approaches chosen for the application of biosensors in bioprocess control. The most versatile system, which in our opinion is flow injection analysis will be the method of choice for the introduction of biosensors in bioprocess control. A lot of experience will be gained for the future development of miniaturized total chemical analysis systems.     

Ionic solution and nanoparticle assisted MALDI-MS as bacterial biosensors for rapid analysis of yogurt

Bacterial analysis from food samples is a highly challenging task because food samples contain intensive interferences from proteins and carbohydrates. Three different conditions of yogurt were analyzed: (1) the fresh yogurt immediately after purchasing, (2) the yogurt after expiry date stored in the refrigerator and (3) the yogurt left outside, without refrigeration. The shelf lives of both these yogurt was compared in terms of the decrease in bacterial signals. AB which initially contained 10(9) cells/mL drastically reduced to 10(7) cells/mL. However, Lin (Feng-Yin) yogurt which initially (fresh) had 10(8) cells/mL, even after two weeks beyond the expiry period showed no marked drop in bacterial count. Conventional MALDI-MS analysis showed limited sensitivity for analysis of yogurt bacteria amidst the complex milk proteins present in yogurt. A cost effective ionic solution, CrO(4)(2-) solution was used to enable the successful detection of bacterial signals (40-fold increased in sensitivity) selectively without the interference of the milk proteins. 0.035 mg of Ag nanoparticles (NPs) were also found to improve the detection of bacteria 2-6 times in yogurt samples. The current approach can be further applied as a rapid, sensitive and effective platform for bacterial analysis from food.     

Surface plamon resonance imaging of DNA based biosensors for potential applications in food analysis

The adsorption processes of oligonucleotides immobilised onto suitable photolithographic patterned gold substrates have been investigated in aqueous buffer solution by using a home made surface plasmon resonance (SPR) imaging equipment. A rapid self-assembled method for the construction of DNA chips to be used in SPR imaging experiments have been followed. The immobilised DNA molecules (probes) adopted in our SPR experiments anchored to a gold surface via thiol group were 5'thiol-modified containing a (CH(2))(15) tail. The hybridisation processes taking place with its complementary sequence have been observed and characterized by monitoring phenomena by a SPR imaging system. The two analysed oligonucleotides (probes and target) are of interest in plant gene biotechnological application and differing for the presence at the 5'-end of a poly T16 spacer. Dynamic investigation of smallest changes in SPR imaging pictures performed in liquid phase in the presence of DNA complementary probes have been performed. Quantitative information in terms of threshold of sensitivity has been extracted by using a specific images treatment.