Device for measuring soft tissue interface pressures

This paper describes the construction and performance of a simple pressure sensing device with a continuous electrical output. It was constructed utilizing a commercially available transducer, an electropneumatic sensor capsule and a 1 m long tube. The transducer used was a piezo-resistive pressure-sensitive device producing an output voltage proportional to the applied pressure. This low cost, high accuracy device is temperature compensated and shows good linearity and negligible hysteresis. The sensor cell has a good thickness-to-diameter ratio and is sufficiently flexible to conform to most contours of the body. The tubing that conveys the pressure transmitting fluid also serves as a means of keeping the transducer distant from the measuring site. The device showed a highly satisfactory performance under laboratory conditions and has proven to be robust and reliable when used for clinical studies.     

A fluorescence polarization assay for native protein substrates of kinases

Protein phosphorylation is the mediator of many important cellular processes of signal transduction and cell regulation. Phosphorylation often occurs on multiple sites within a single protein, whereby the results of individual phosphorylations are not well defined. This is partially due to the lack of tools for analyzing specific phosphorylation states in a quantitative manner. We have developed a high-throughput, rapid, and quantitative method for the determination of the phosphorylation status of peptides and, more importantly, native protein substrates of kinases using a competitive fluorescence-based approach. We have applied our method to measuring the phosphorylation activity of the Wee1 and Myt1 kinases. Our technique allows one to monitor the bis-phosphorylation status of the Cdk2 protein using an antibody specific for bis-phosphorylated Cdk2 and a fluorescently labeled bis-phosphorylated Cdk2 peptide. We have used this assay to screen a library of 16 general kinase inhibitors against Wee1 and Myt1 activity. None of the inhibitors inhibited Wee1, but both staurosporine and K-252a inhibited Myt1, with IC(50) values of 9.2+/-3.6 and 4.0+/-1.3 microM, respectively.     

Single-molecule fluorescence characterization in native environment

Single-molecule detection (SMD) with fluorescence is a widely used microscopic technique for biomolecule structure and function characterization. The modern light microscope with high numerical aperture objective and sensitive CCD camera can image the brightly emitting organic and fluorescent protein tags with reasonable time resolution. Single-molecule imaging gives an unambiguous bottom-up biomolecule characterization that avoids the "missing information" problem characteristic of ensemble measurements. It has circumvented the diffraction limit by facilitating single-particle localization to ~1 nm. Probes developed specifically for SMD applications extend the advantages of single-molecule imaging to high probe density regions of cells and tissues. These applications perform under conditions resembling the native biomolecule environment and have been used to detect both probe position and orientation. Native, high density SMD may have added significance if molecular crowding impacts native biomolecule behavior as expected inside the cell.     

Laser-assisted fluorescence microscopy for measuring cell membrane dynamics.

Membranes of living cells are characterized by laser-assisted fluorescence microscopy, in particular a combination of microspectrofluorometry, total internal reflection fluorescence microscopy (TIRFM), fluorescence lifetime imaging (FLIM) and Forster resonance energy transfer (FRET) spectroscopy. The generalized polarization (GP, characterizing a spectral shift which depends on the phase of membrane lipids) as well as the effective fluorescence lifetime (tau(eff)) of the membrane marker laurdan were revealed to be appropriate parameters for membrane stiffness and fluidity. GP decreased with temperature, but increased during cell growth and was always higher for the plasma membrane than for intracellular membranes. Microdomains of different fluorescence lifetimes tau(eff) were observed at temperatures above 30 degree C and disappeared during cell aging. Non-radiative energy transfer was used to detect laurdan selectively in close proximity to a molecular acceptor (DiI) and may present a possibility for measuring membrane dynamics in specific microenvironments.     

Fluorometric device for direct measuring of culture fluorescence within fermenters

A fluorometric device for direct measuring of culture fluorescence within the fermenter is described. The calibration by quinine sulfate as well as the response to a step increase of the input is presented.     

Particle concentration fluorescence immunoassay for measuring interleukin-6 receptor numbers

Analysis of the number of receptors per cell and the affinity of the ligand/receptor interaction has provided considerable insight into the functioning of numerous cytokines. Interleukin-6 (IL-6) is a multifunctional cytokine which may have considerable clinical relevance in inflammatory or immunodeficiency diseases. Using particle concentration fluorescence immunoassay (PCFIA) technology, an assay is described which calculates the receptor number and affinity on small numbers of human cells. Resting B cells are shown to lack IL-6 receptors but activation of B cells induces up to 1,300 receptors per cell, with Kd of 1 x 10(-11) to 2 x 10(-11) M. Other recombinant mediators do not alter the binding of labeled IL-6 to the cells. PCFIA avoids the use of radioactivity and requires very small numbers of cells (2 x 10(4) per well). Potential application to the study of regulatory mechanisms and to clinical situations where small samples of blood are available is feasible.     

A quantitative stopped-flow fluorescence assay for measuring polymerase elongation rates

The measurement of nucleic acid polymerase elongation rates is often done via a lengthy experimental process involving radiolabeled substrates, quenched elongation experiments, electrophoretic product separation, and band quantitation. In this work, we describe an alternative real-time stopped-flow assay for obtaining kinetic parameters for elongation of extended sequences. The assay builds on our earlier PETE (polymerase elongation template element) assay designed for high-throughput screening purposes [S.P. Mestas, A.J. Sholders, O.B. Peersen, A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity, Anal. Biochem. 365 (2007) 194-200] and relies on measuring how long it takes a polymerase to reach the end of a defined length template. Using poliovirus polymerase and self-priming hairpin RNA substrates with 6- to 26-nt-long templating regions, we demonstrate that the assay can be used to determine V_max rates for elongation and apparent K_m values for nucleotide triphosphate (NTP) use. Modeling the reaction kinetics as a series of irreversible steps allows us to numerically fit the entire time-based dataset by properly accounting for the temporal distribution of intermediate species. This enables us to determine average elongation rates over heterogeneous templating regions that mimic viral genome substrates. The assay is easily extendable to other RNA and DNA polymerases, can accommodate secondary structures in the template, and can in principle be used for any enzyme traversing along an extended substrate.     

Capillary electrophoresis with laser-induced native fluorescence detection for profiling body fluids

Laser-induced native fluorescence detection with a KrF excimer laser (λ=248 nm) was used to investigate the capillary electrophoretic (CE) profiles of human urine, saliva and serum without the need for sample derivatization. All separations were carried out in sodium phosphate and/or sodium tetraborate buffers at alkaline pH in a 50-μm I.D. capillary. Sodium dodecyl sulfate was added to the buffer for micellar electrokinetic chromatography (MEKC) analysis of human urine. Although inherently a pulsed source, the KrF excimer laser was operated at a high pulse repetition rate of 553, 1001 or 2009 Hz to simulate a continuous wave excitation source. Detection limits were found to vary with pulse rate, as expected, in proportion to average excitation power. The following detection limits (3σ) were determined in free solution CE: tryptophan, 4 nM; conalbumin, 10 nM; α-lactalbumin, 30 nM. Detection limits for indole-based compounds and catecholamine urinary metabolites under MEKC separation conditions were in the range 7–170 nM.     

A novel method for measuring photosynthesis using delayed fluorescence of chloroplast

Photosynthesis is the most important chemical reaction in the world. The measurement of plant photosynthesis rate plays an important role in agriculture. Light-induced delayed fluorescence (DF) in plants is an intrinsic label of the efficiency of charge separation at P680 in photosystem II (PS II). In this paper, we have developed a biosensor that can accurately measure the plant photosynthesis ability by means of DF. Compared with common methods for measuring the photosynthesis rate based on consumption of CO2, the proposed technique can quantify the plant photosynthesis ability with less influence of the environment. The biosensor is an all-weather measuring instrument, it has its own illumination power and utilizes intrinsic DF as the measurement marker. The current investigation has revealed that, there is a good correspondence between the results measured by the biosensor and that by commercially available portable photosynthesis system under controlled conditions. We thus conclude that DF is an excellent marker for evaluating plant photosynthesis ability under its biological status with less interferences of the environment.     

Synchronous scan fluorescence spectroscopy of proteins and human eye lenses

Scanning both the excitation and emission monochromators synchronously while recording the fluorescence spectrum results in a considerable decrease in the apparent band width and shift in the peak position. We demonstrate the potential of this approach in the studies on proteins and their interactions as well as fluorophores in condensed media. We have chosen crystallins, eye lens proteins and human lenses. Synchronous scan spectra of alpha-, beta- and gamma-crystallins are clearly distinguishable and appear to provide specific signatures. The spectrum of the mixed solution could be simulated by the linear combination of components indicating that these proteins might not have any specific interaction in the dilute solutions. Synchronous spectra of the human lenses, both normal and cataractous, show several distinguishable features.     

Comparative fluorescence studies of native and crosslinked glucose oxidase

The binding characteristics of flavin adenine dinucleotide (FAD) to apoenzyme preparations obtained from native and intramolecularly crosslinked glucose oxidase were determined and compared. The dissociation constants K_diss as well as rates of recombination of FAD with the two apoenzyme preparations, were independently evaluated from fluorescence quenching of either the tryptophans of FAD. The K_diss values thus obtained were <10^?19M for native glucose oxidase and 4 ± 1 × 10^?7M for the crosslinked enzyme. The recombination of apo glucose oxidase with FAD, which is presumably diffusion controlled, is followed by an apparent first order decrease in fluorescence intensity of both the protein tryptophans and FAD, with a rate constant around 0.2 min^?1. This could be related to conformational changes which occur immediately after binding of FAD to the apoenzyme, an interpretation which is supported by the markedly different results obtained in the analogous experiments with the crosslinked enzyme. A model for the conformational characteristics of glucose oxidase, based on this study, is proposed.     

Limitations of the pulse-modulated technique for measuring the fluorescence characteristics of algae

Precise measurements of the minimal fluorescence yield (F_o) and maximal fluorescence yield (F_m) of a dark-adapted sample are prerequisites for the quantification of other fluorescence parameters. The pulse amplitude-modulated chlorophyll fluorometer (PAM 101 Chlorophyll Fluorometer, Heinz Walz, Effeltrich, Germany) and saturating pulse technique have frequently been used in measuring F_o and F_m and in resolving the contributions of photochemical and nonphotochemical quenching to the total fluorescence yield. The extent to which instrument-dependent factors may affect the accurate measurement of F_o and F_m is addressed. It is shown that the increase in pulse amplitude-modulated measuring beam intensity at 1.6 and 100 kHz was nonlinear at higher light intensity settings. The implications of this for measurements of F_o (1.6 kHz) and F_m (100 kHz) are discussed. It is also demonstrated that underestimation of F_m may result due to saturation of the PAM 101 photodiode by scattered infrared light associated with intense light pulses. In addition, it is shown how sample-dependent factors may affect measurements of F_o and F_m in samples with low chlorophyll concentrations, in particular, dilute algal suspensions of Phaeodactylum tricornutum and Chlamydomonas reinhardtii. A technique is presented for the accurate measurement of F_o in algal suspensions (<8 μg chlorophyll a mL⁻¹). The importance of examining the saturating pulse transient and F_m level as a function of the damping setting, pulse width, and pulse intensity, and in the presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea is discussed.     

Dual labeling of a binding protein allows for specific fluorescence detection of native protein

Up-Converting Phosphor Technology (UPT) is based on lanthanide-containing, submicrometer-sized, ceramic particles that can absorb infrared light and emit visible light. Biological matrices do not up-convert; hence, there is no contribution to test background from sample autofluorescence. Up-converting phosphors do not photobleach and are inert to common assay interferants such as hemoglobin. A reader called UPlink has been developed to interrogate lateral flow test strips that utilize UPT labels. The reader contains a miniaturized, 1-W, infrared laser with peak emission at 980 nm. Preliminary assays that use up-converting phosphor labels, including tests for a drugs of abuse panel and Escherichia coli O157:H7, have been developed. In a "sandwich" assay format, 10(3) org/mL E. coli O157:H7 organisms were detectable in a negative control background of 10(9) other organisms per milliliter of culture medium. Coefficients of variation in concentrations tested from 0 to 10(7) org/mL were all < or =10%. In a competitive inhibition assay format, a multiplexed test simultaneously detected amphetamine, methamphetamine, phencyclidine, and opiates in saliva. For all assays, the percent displacement at 10 ng/mL was > or =40% demonstrating performance comparable with lab-based, commercially available EIAs. All assays were complete in 10 min. The development of rapid tests using UPT creates new applications for on-site testing with sensitivity not available using other label technologies.     

Pitfalls in measuring fluorescence polarization in mitogen-stimulated T-lymphocytes

Fluorescence polarization measurements with the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) were performed to detect changes in the fluidity of plasma membranes from T-lymphocytes stimulated with mitogens. When the cells were incubated with succinyl-concanavalin A an increase in fluorescence polarization was observed. This, however, could be shown to be due to the interaction of the mitogen with the label DPH and did not reflect changes in the plasma membrane. In purified plasma membranes a decrease rather than an increase of fluorescence polarization was observed.