A molecular rotor as viscosity sensor in aqueous colloid solutions

BACKGROUND: Molecular rotors exhibit viscosity-dependent quantum yield, allowing non-mechanical determination of fluid viscosity. We analyzed fluorescence in the presence of viscosity-modulating macromolecules several orders of magnitude larger than the rotor molecule. METHOD OF APPROACH: Fluorescence of aqueous starch solutions with a molecular rotor in solution was related to viscosity obtained in a cone-and-plate viscometer. RESULTS: In dextran solutions, emission intensity was found to follow a power-law relationship with viscosity. Fluorescence in hydroxyethylstarch solutions showed biexponential behavior with different exponents at viscosities above and below 1.5 mPa s. Quantum yield was generally higher in hydroxyethylstarch than in dextran solutions. The power-law relationship was used to backcalculate viscosity from intensity with an average precision of 2.2% (range of -5.5% to 5.1%). CONCLUSIONS: This study indicates that hydrophilic molecular rotors are suitable as colloid solution viscosity probes after colloid-dependent calibration.     

Fluorescence characteristics and photophysical parameters of light-harvesting chlorophyll <Emphasis Type="Italic">a/b</Emphasis> complex aggregates

Fluorescence characteristics and molecular photophysical parameters of light-harvesting chlorophyll a/b complexes isolated from pea were studied in relation to their aggregation state. The aggregate size was varied by changing the Triton X-100 concentration from 0 to 0.23 mM at a chlorophyll concentration of 2.45 μg/ml. Molecular photophysical parameters were determined with laser fluorimetry. Dispersion of large aggregates into smaller ones drastically decreased the intensity of low-temperature (77 K) fluorescence at 700 nm, reduced the singlet-singlet annihilation rate by more than two orders of magnitude, and prolonged the fluorescence lifetime from 0.16 to 3.2 ns.     

Quantification of random motility and chemotaxis bacterial transport coefficients using individual-cell and population-scale assays

A number of individual-cell and population-scale assays have been introduced to quantify bacterial motility and chemotaxis. The transport coefficients reported in the literature, however, span several orders of magnitude, making it difficult to ascertain to what degree variations in bacterial species/strain, growth medium, growth and experimental conditions, and experiment type contribute to the reported differences in coefficient values. We quantified the random motility of Escherichia coli AW405 using the capillary assay, stopped-flow diffusion chamber (SFDC), and tracking microscope. We obtained good agreement for the random motility coefficient between these assays when using the same bacterial strain and consistent growth and experimental conditions. Chemotaxis of E. coli toward the attractant alpha-methylaspartate was quantified using the SFDC and capillary assay. Good agreement for the chemotactic sensitivity coefficient between the SFDC and the capillary assay was obtained across a limited attractant concentration range. Three different mathematical models were considered for analyzing capillary assay data to obtain a chemotactic sensitivity coefficient. These models differed by their treatment of the bacterial concentration in the chamber and the attractant concentration at the mouth. Results from our study indicate that the capillary assay, the most commonly used bacterial random motility and chemotaxis assay, can be used to accurately quantify bacterial transport coefficients over a limited range of attractant concentrations, provided experiments are performed carefully and appropriate mathematical models are used to interpret the experimental data.     

A new technique for assaying cytochrome P450 enzyme activity in a single cell

A new microspectrofluorometric technique for measuring the ethoxyresorufin-O-deethylase (EROD) activity of cytochrome P450 (CYP)1A1 in single living cells is described. The system, which uses a perfusion chamber and an HPLC pump, allowed cells to be stained, fixed, blocked, and washed by injecting each treatment solution into the on-line carrier stream of buffer from the sampling block of the HPLC pump. After addition of the substrate 7-ethoxyresorufin, the fluorescence intensity of the metabolite resorufin was measured in individual cells. Fluorescence intensity steeply increased to a unique peak for each cell and then decreased to the basal level. Furthermore, CYP1A1 in each cell was stained with its antibody and quantified using the fluorescence intensity of an FITC-conjugated secondary antibody. EROD activity was normalized using the FITC fluorescence. The results show that the initial slopes and peak values of resorufin production by the cells were dependent on the CYP1A1 level. Treatment of hepatocytes with two nonspecific P450 inhibitors, cimetidine and SKF-525A, suppressed EROD activity.     

Detection of 100 molecules of product formed in a fucosyltransferase reaction

We present the detection of 100 molecules of enzyme substrateand product. A fucosyltransferase and a fucosidase were usedto add and remove, respectively, a monosaccharide from a syntheticoligosaccharide fluorescently labelled with tetramethylrhodamine.The reaction was followed by use of capillary zone electrophoresis,to separate the product and reactant, with laser-induced fluorescencedetection. These are the most sensitive enzyme assays reportedto date and six orders of magnitude more sensitive than anyreported for these two enzymes. This simple technology allowshigh-sensitivity determination of the activity of any enzymefor which a fluorescent substrate can be synthesized, and bringswithin reach the ability to assay glycosyltransferase activitiesin single cells. capillary electrophoresis enzyme assay fucosidase fucosyltransferase glycosyltransferase assay laser-induced fluorescence yoctomole analysis     

A Critical Review of Discrete Soil Sample Data Reliability: Part 2—Implications

Part 2 of this study investigates the implications of random, small-scale contaminant concentration variability in soil for reliance on discrete soil sample data to guide environmental investigations. Random variability around an individual point limits direct comparison of discrete sample data to risk-based screening levels. “False negatives” can lead to premature termination of an investigation or remedial action. Small-scale distributional heterogeneity of contaminants in soil is expressed as artificial, seemingly isolated “hot spots” and “cold spots” in isoconcentration maps. Surgical removal of hot spots can lead to erroneous conclusions regarding the magnitude of remaining contamination. The field precision of an individual discrete sample data set for estimation of means for a contaminant in a risk assessment is not directly testable. Omission of “outlier” data in order to force data to fit a geostatistical model distorts estimates of mean concentrations and introduces error into a risk assessment. The potential for such errors was pointed out in early USEPA guidance but largely ignored or misunderstood. Decision Unit and Multi Increment sample investigation methods, long known to the agricultural and mining industries, were specifically developed to overcome these inherent shortcomings of discrete sampling methods and provide more reliable and defensible data for environmental investigations.     

Utilization of the non-covalent fluorescent dye, NanoOrange, as a potential clinical diagnostic tool: Nanomolar human serum albumin quantitation

The commercially available dye, NanoOrange, has been investigated as a potential tool for clinical diagnostics due to its low cost, ease of use, and ability to detect nanomolar concentrations of protein. Virtually non-fluorescent in dilute aqueous solutions, NanoOrange fluorescence is enhanced by at least an order of magnitude upon non-covalent interaction with proteins. These features, coupled with the requirement for high throughput assays in the clinical laboratory has prompted the development of two orthogonal NanoOrange approaches. Human serum albumin (HSA) was used as a model protein for the development of both 96-well microplate and capillary electrophoresis laser-induced fluorescence (CE–LIF) assay formats. Dye performance in five commonly used buffers of various concentrations and pH indicated considerable flexibility in assay buffer selection, with optimal performance at pH 9.0. A salt concentration study indicated that increasing NaCl concentration generally decreases fluorescence emission and can be minimized by pre-diluting biological samples to a final salt concentration of 20–80 mM. Titration of protein with NanoOrange resulted in optimal HSA–NanoOrange complex formation utilizing 1× and 2× NanoOrange in the 96-well microplate and CE–LIF approaches, respectively. A NanoOrange binding model based on rapid signal enhancement and zero order fluorescence emission kinetics is proposed. The utilization of NanoOrange in CE–LIF based human serum analysis results in a signal-to-background ratio improvement of up to two orders of magnitude.     

A Critical Review of Discrete Soil Sample Data Reliability: Part 1—Field Study Results

Part 1 of this study summarizes data for a field investigation of contaminant concentration variability within individual, discrete soil samples (intra-sample variability) and between closely spaced, “co-located” samples (inter-sample variability). Hundreds of discrete samples were collected from three sites known respectively to be contaminated with arsenic, lead, and polychlorinated biphenyls. Intra-sample variability was assessed by testing soil from ten points within a minimally disturbed sample collected at each of 24 grid points. Inter-sample variability was assessed by testing five co-located samples collected within a 0.5-m diameter of each grid point. Multi Increment soil samples (triplicates) were collected at each study site for comparison. The study data demonstrate that the concentration of a contaminant reported for a given discrete soil sample is largely random within a relatively narrow (max:min <2X) to a very wide (max:min >100X) range of possibilities at any given sample collection point. The magnitude of variability depends in part on the contaminant type and the nature of the release. The study highlights the unavoidable randomness of contaminant concentrations reported in discrete soil samples and the unavoidable error and inefficiency associated with the use of discrete soil sample data for decision making in environmental investigations.     

Single Molecule Detection Technologies in Miniaturized High Throughput Screening: Fluorescence Correlation Spectroscopy

Fluorescence assay technologies used for miniaturized high throughput screening are broadly divided into two classes. Macroscopic fluorescence techniques (encompassing conventional fluorescence intensity, anisotropy [also often referred to as fluorescence polarization] and energy transfer) monitor the assay volume- and time-averaged fluorescence output from the ensemble of emitting fluorophores. In contrast, single-molecule detection (SMD) techniques and related approaches, such as fluorescence correlation spectroscopy (FCS), stochastically sample the fluorescence properties of individual constituent molecules and only then average many such detection events to define the properties of the assay system as a whole. Analysis of single molecular events is accomplished using confocal optics with an illumination/detection volume of approximately 1 fl (10(-15) L) such that the signal is insensitive to miniaturization of HTS assays to 1 μl or below. In this report we demonstrate the general applicability of one SMD technique (FCS) to assay configuration for target classes typically encountered in HTS and confirm the equivalence of the rate/equilibrium constants determined by FCS and by macroscopic techniques. Advantages and limitations of the current FCS technology, as applied here, and potential solutions, particularly involving alternative SMD detection techniques, are also discussed.     

High-resolution semi-quantitative real-time PCR without the use of a standard curve

The repeatability and sensitivity of a simple, adaptable, semi-quantitative, real-time RT-PCR assay was investigated. The assay can be easily and rapidly applied to quantitate relative levels of any gene product without using standards, provided that amplification conditions are specific for the PCR product of interest. Using the LightCycler real-time PCR machine, a serial 10-fold dilution series (spanning four orders of magnitude) of a 379-bp cDNA template was amplified, and the PCR product was detected using SYBR Green I chemistry. The experiment was repeated on a subsequent day. The experimental design was such that the data lent itself to analysis using an appropriate method for testing repeatability. It was found that, within a single assay, for samples assayed in triplicate, a difference of 23% may be reliably detected. Furthermore, when all of the factors that contribute to variability in the assay are taken into account, such as day-to-day variation in pipetting and amplification efficiency, a 52% difference in target template can be detected using a sample size of 4. The assay was found to be linear over at least four orders of magnitude.     

Fluorescence polarization immunoassay employing immobilized antibody

The use of an antibody immobilized on latex or silver colloid in fluorescence polarization immunoassay (FPI) is assessed. In FPI it is possible to detect antigens of high molecular weight because the molecular weight of the antibody is effectively increased. In the assay for rabbit immunoglobulin G a limit of detection lower by two orders of magnitude and an assay range wider by one order of magnitude can be obtained in comparison with conventional FPI. The detection limit is 10(-10) mol l-1 and the total assay time for one sample is 8 min. This assay combines a low detection limit with a short assay time.     

Single-molecule detection on a protein-array assay platform for the exposure of a tuberculosis antigen

Based on a single-molecule sensitive fluorescence-linked immunosorbent assay, an analytical platform for the detection of lipoarabinomannan (LAM), a lipopolysaccharide marker of tuberculosis, was established that is about 3 orders of magnitude more sensitive than comparable current ELISA assays. No amplification step was required. Also, no particular sample preparation had to be done. Since individual binding events are detected, true quantification was possible simply by counting individual signals. Utilizing a total internal reflection configuration, unprocessed biological samples (human urine and plasma) to which LAM was added could be analyzed without the requirement of sample purification or washing steps during analysis. Samples containing about 600 antigen molecules per microliter produced a distinct signal. The methodology developed can be employed for any set of target molecules for which appropriate antibodies exist.     

A Quantitative Microtiter Plate Nuclease Assay Based on Ethidium/DNA Fluorescence

A microtiter plate assay was developed to quantitate the nuclease activity of the extracellularSerratia marcescensendonuclease under different buffer conditions. Substrate cleavage was followed as decrease in ethidium/DNA fluorescence using a uv-transilluminator and a video documentation system. Time courses of DNA cleavage were recorded and cleavage rates determined very precisely within a factor of 1.2. The assay has a linear dynamic range covering three orders of magnitude of nuclease activity and can be carried out very quickly within a few minutes. It can also be used with RNA as substrate. With appropriate modifications it should be possible to adapt this assay for other enzymatic reactions which are accompanied by changes in absorbance or fluorescence.     

Direct assessment and distribution of regional portal blood flow in the pig by means of fluorescent microspheres.

Measurement of regional organ blood flow by means of fluorescent microspheres (FM) is an accepted method. However, determination of regional portal blood flow (RPBF) cannot be performed by microspheres owing to the entrapment of the spheres in the upstream capillary bed of the splanchnic organs. We hypothesized that an adequate experimental setting would enable us to measure RPBF by means of FM and to analyze its distribution within the pig liver. A mixing chamber for the injection of FM was developed, and its capability to distribute FM homogeneously in the blood was evaluated in vitro. The chamber was implanted into the portal vein of six anesthetized pigs (23.5 +/- 2.9 kg body wt). Three consecutive, simultaneous injections of FM of two different colors into the chamber were performed. Reference portal blood samples were collected by means of a Harvard pump. At the end of the experiment, the liver was explanted and fixed in formalin before dissection. FM were isolated from the tissue samples by an automated process, and fluorescence intensity was determined. Comparison of 5,458 single RPBF values, determined by simultaneously injected FM, revealed good agreement (bias 2.5%, precision 12.7%) and high correlation (r = 0.97, r2 = 0,95, slope = 1.04, intercept = 0.05). Median RPBF was 1.07 +/- 0.78 ml x min(-1) x g(-1). Allocation of the blood flow values to the anatomic regions of the liver revealed a significantly higher RPBF (P = 0.01) in the liver tissue located close to the diaphragm compared with the rest of the organ and a significantly lower RPBF (P = 0.01) in the left liver lobe compared with the median and right lobes. The results show that the model presented makes it possible to measure RPBF by means of FM reliably and that RPBF is distributed heterogeneously in the porcine liver.     

A Quantitative Comparison of Stimulus—Response Relationships of Vibrissa-Activated Neurons in Subnuclei Oralis and Interpolaris of the Rat's Trigeminal Sensory Complex: Receptive Field Properties and Threshold Distributions

Electrical activity of single vibrissa-activated neurons was recorded in pars interpolaris and pars oralis of the nucleus of the trigeminal spinal tract of rats. Stimuli consisted of quantitatively controlled deflections of individual mystacial vibrissae. The evoked spike trains were analyzed as point-process time series with a variety of quantitative procedures. Most second-order trigeminal neurons were directionally sensitive. About one-third of interpolaris neurons and over half of oralis neurons responded to axial push of the hair shaft. About half of the neurons of both oralis and interpolaris had receptive fields that included more than one vibrissa. The upper-quartile receptive field size of neurons of interpolaris was about half that of oralis, although the distributions overlapped. In interpolaris, almost one-fifth of the sample discharged in the absence of an intentionally applied stimulus; in oralis, over one-third of the sample displayed background activity. The ranges of angular displacement thresholds of both samples exceeded three orders of magnitude. The distributions differed quantitatively. They were, nevertheless, similar in shape, and exhibited considerable overlap. The median threshold of oralis neurons was about half that of interpolaris neurons, and about one-fifth that of first-order neurons. About one-fourth of the second-order neurons exhibited a nonmonotonic relationship between pulse displacement and the number of evoked spikes. Neurons of interpolaris tended to be more severely nonmonotonic than those of oralis, and some “turned off” at stimulus magnitudes well above threshold. Nevertheless, the number of spikes evoked in either entire sample was a monotonically increasing function of pulse displacement. The range of angular velocity thresholds observed in both second-order samples exceeded three orders of magnitude. As with the angular displacement thresholds, the distributions of angular velocity thresholds were quantitatively different, although their shapes and extremes were similar, and they overlapped extensively. The observed differences of stimulus-response relationships of the neurons in interpolaris and oralis reflect differences in the ways different trigeminal nuclei receive, process, and distribute information to the circuits in which they participate.     

A microfluorometric assay for the measurement of de novo DNA synthesis in individual cells

A microfluorometric assay has been developed for the quantitation of de novo DNA synthesis. The assay reliably detects newly synthesized DNA in individual cells and provides a relative measure of the proportion of new DNA in each cell. When combined with microscopic techniques for cell identification, selected subpopulations can be examined in samples containing a complex mixture of interacting cell types. The assay employs the quenching of Hoechst-33258 fluorescence by bromodeoxyuridine incorporated into newly synthesized DNA. The ability of BUdR to quench H-33258 fluorescence is lost after a brief exposure of ultraviolet fluorescence excitation. Therefore, quenched and unquenched fluorescence intensity measurements from the same cell can be compared. Increases in fluorescence intensity occur only in cells that have synthesized DNA after the addition of BUdR. In addition the change in fluorescence intensity is proportional to the degree of BUdR substitution within the range of the assay and provides a relative measure of DNA synthesis.     

Plasmonic Enhancement of Fluorescence and Raman Scattering by Metal Nanotips

We report modifications to the optical properties of fluorophores in the vicinity of noble metal nanotips. The fluorescence from small clusters of quantum dots has been imaged using an apertureless scanning near-field optical microscope. When a sharp gold tip is brought close to the sample surface, a strong distance-dependent enhancement of the quantum dot fluorescence is observed, leading to a simultaneous increase in optical resolution. These results are consistent with simulations of the electric field and fluorescence enhancement near plasmonic nanostructures. Highly ordered periodic arrays of silver nanotips have been fabricated by nanosphere lithography. Using fluorescence lifetime imaging microscopy, we have created high-resolution spatial maps of the lifetime components of vicinal fluorophores; these show an order of magnitude increase in decay rate from a localized volume around the nanotips, resulting in a commensurate enhancement in the fluorescence emission intensity. Spatial maps of the Raman scattering signal from molecules on the nanotips shows an enhancement of more than five orders of magnitude.     

Chemiluminescence determination of pharmacologically active compounds by capillary electrophoresis.

A simple and rapid capillary electrophoresis with direct chemiluminescence method has been developed for the determination of five natural pharmacologically active compounds including rutin, protocatechuic aldehyde, chlorogenic acid, luteolin and protocatechuic acid. The luminol as a component of the separation electrolyte buffer was introduced at the head of the separation capillary. The separation of five compounds was carried out in a fused-silica capillary with 15.0 mmol/L tetraborate, 1.0 mmol/L SDS and 0.42 mmol/L luminol (pH 8.5). The analytes was determined by enhancing the chemiluminescence of luminol with 0.13 mmol/L K3Fe(CN)6 in 0.05 mol/L NaOH, which was introduced at the post-column stage. The voltage applied was 16 kV. Under the optimum conditions, the analytes were separated within 10 min. The excellent linearity was obtained over two to three orders of magnitude with a detection limit (signal:noise = 3) of 0.012-0.055 micromol/L for all five analytes. The method was successfully used in the analysis of pharmaceutical and biological samples, and the assay results were satisfactory.     

A rapid, sensitive and automated method for detection of Salmonella species in foods using AG-9600 AmpliSensor Analyzer

The AG-9600 AmpliSensor Analyzer is an automated fluorescence-based system for detection of polymerase chain reaction (PCR) products. The principle of the AmpliSensor PCR assay involves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to a target sequence within a 284-bp amplified fragment of the Salmonella invA gene. Since the assay is homogenous, the data can be obtained by direct measurement of fluorescence of the amplification mixture. The accumulation of the amplified product, reflected by the fluorescence index, is monitored cycle by cycle by the AG-9600 Analyzer. The detection limit of the assay was less than 2 colony-forming units (cfu) per PCR reaction using a pure culture of Salmonella typhimurium. In post-spiking experiments in which Salmonella was added to the overnight pre-enriched samples (chicken carcass rinses, ground beef, ground pork and raw milk), the detection limit of the assay was 2–6 cfu per PCR reaction. In pre-spiking experiments in which Salmonella was added to the samples prior to overnight pre-enrichment, the detection limit was less than 3 cfu per 25 g or 25 ml of food. The assay was up to 2 orders of magnitude more sensitive than detection by ethidium bromide-stained agarose gel electrophoresis. To further evaluate assay performance, 54 naturally contaminated chicken carcass rinses, 65 raw milk and six ground pork samples were tested in the study. Thirty-eight Salmonella- positive samples confirmed by the Modified Semi-solid Rappaport-Vassiliadis (MSRV) culture assay were found positive using the AmpliSensor assay. Two chicken carcass rinses found positive using the assay were MSRV-negative. In addition, relative quantification using the AmpliSensor assay was linear up to 3 logs of initial target concentration in artificially contaminated food samples.