Filter Fluorometer Research Articles

Filter Fluorometer Calibration Without the Fluorometer.

We recently described a lightweight, low-power, waterproof filter fluorometer using a 180° backscatter geometry for chlorophyll-a (chl-a) detection. Before it was constructed it was modeled to ensure it would have satisfactory performance. This manuscript repeats the modeling process that allows the calibration slope and detection limit for a fluorescent analyte in water to be estimated from system component performance and conventional spectrofluorometry alone. These values are validated by comparison to the experimental result of calibration from the completed instrument. Our model yields a calibration slope of 8.22 mV-L/µg for dissolved chl-a, consistent with the experimentally measured slope of 8.21 mV-L/µg. The detection limit modeled from this slope and an estimate of the baseline noise of the instrument was 0.15 µg/L chl-a, while the measured detection limit using real blank samples was 0.18 µg/L, in 0.1 s differential measurements.

Endoscopic filter fluorometer for detection of accumulation of Protoporphyrin IX to improve photodynamic diagnostic (PDD)

Abstract Photodynamic diagnostic (PDD) is an optical enhancement option for the endoscope to support the detection of cancer, for example in the bladder. In real application PDD efficiency suffers due to the complex accumulation of the photosensitizing drug inside the tumor and the associated processes of heme syntheses to create the fluorescent components needed. To optimize the diagnostic outcome of PDD it would be helpful to predict the optimal time for diagnosis based on measurable precursors. In a previous cell study, we proposed a new filter fluorometer to image the accumulation of the precursors Coproporphyrin III (CP-III) and Uroporphyrin III (UP-III) that metabolize to Protoporphyrin IX (PP-IX) later. This accumulation process can be used to predict the optimal time slot for diagnostic imaging. Therefore, a new filter system was designed to distinguish between CP-III and PP-IX. In this work we tested this filter system in combination with a standard PDD endoscopic imaging system. Goal of this study was to prove the technical feasibility in a non-patient setup to prepare a later clinical study.

Endoscopic filter fluorometer for emission detection of Protoporphyrin IX and its direct precursors in PDT and PDD

Abstract Photodynamic therapy (PDT) is a potential option for treatment of cancer since it can be performed non- invasive for superficial cancers or minimal-invasive with low traumatization. But PDT is intrinsically inefficient due to the complex accumulation of the photosensitizing drug inside the tumor and the processes of heme syntheses to create the needed cell killing components. To optimize the outcome of PDT and increase acceptance as viable option it is necessary to predict the optimal time for the start of the treatment based on measurable precursors. A former cell study proposed a new filter fluorometer in a complex and sensitive setup. In this work we now designed and tested a simplified system that can be used in combination with standard endoscopic imaging systems. This system will be used as base to prove viability of this approach for a future clinical study.

Fiber-optic filter fluorometer for emission detection of Protoporphyrin IX and its direct precursors – A preliminary study for improved Photodynamic Therapy applications

Abstract In this work we present first results of a laboratory manufactured filter-fluorometer to study differences in intensity and position of the main peaks of three porphyrins that appear during the Heme-Synthesis. Porphyrins play a major role in Photodynamic Therapy (PDT) for cancer treatment. Within the Heme-Synthesis, Porphyrins such as Protoporphyrin IX (PPIX) and its two precursors Coproporphyrin III (CPIII) and Uroporphyrin III (UPIII) represent photochemical agents that can interact with light to show fluorescence or generate Reactive Oxygen Species (ROS) to destroy cells. A major problem that arises is determining the ideal time slot to begin treatment after drug application. Our work is meant to show a way to solve this problem by looking at concentration changes of precursors appearing in Heme-Synthesis and using these changes to predict the occurence of PPIX inside the mitochondria.

Simple and Inexpensive 3D Printed Filter Fluorometer Designs: User-Friendly Instrument Models for Laboratory Learning and Outreach Activities

In this work, a versatile and user-friendly selection of stereolithography (STL) files and computer-aided design (CAD) models are shared to assist educators and students in the production of simple and inexpensive 3D printed filter fluorometer instruments. These devices are effective resources for supporting active learners in the exploration of instrument design and performance. In contrast to sophisticated commercial-grade fluorometers, the models provided here are open source, customizable for a variety of applications, and easily assembled by students in activities that directly confront the “black box” perception of analytical instrumentation. In order to aid beginner CAD and 3D printer users, these models are compatible with accessible software packages, such as 123D Design and Inventor Professional. Additionally, CAD tutorials and extensive slicer settings are supplied to assist educators and students in modifying digital designs and obtaining reliable 3D prints. The provided filter fluorometer mod...

Focus on sensors and detectors

Focus on sensors and detectors

Spectrophotometric and Colorimetric Determination of Protein Concentration

This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measurement at 280 nm is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein. An alternate protocol uses absorbance at 205 nm to calculate the protein concentration. Both methods can be used to quantitate total protein in crude lysates and purified or partially purified protein. Use of a spectrofluorometer or a filter fluorometer to measure the intrinsic fluorescence emission of a sample solution is also described. The measurement is compared with the emissions from standard solutions to determine the concentration of purified protein. The Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to an unknown protein, is also presented, as is the Lowry method, which measures colorimetric reaction of tyrosyl residues in an unknown.

Hand-held thermal-regulating fluorometer

This article relates to the construction of a portable, low cost, thermal-regulating light-emitting diode (LED)-based, handheld fluorometer. This regulated fluorometer is based on both a low thermal mass infrared heater, and an orthogonal geometry LED-based filter fluorometer. Power is supplied through an external power supply and data is collected in real time through standard serial interfaces of personal computers or personal digital assistants. Thermal regulation is automatically maintained using temperature sensor feedback control. Optical excitation relies on LED light source(s) and optical detection is made through an adjustable integrating photodetector. With such a handheld system, applications requiring temperature sensitive photometric measurements for real-time analyte detection can be more easily performed in the field.

Spectrophotometric Determination of Protein Concentration

This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measured at 280 nm (A(280)) is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein (a(280)). Alternatively, absorbance measured at 205 nm (A(205)) is used to calculate the protein concentration. The A(280) and A(205) methods can be used to quantify total protein in crude lysates and purified or partially purified protein. A spectrofluorometer or a filter fluorometer can be used to measure the intrinsic fluorescence emission of a sample solution; this value is compared with the emissions from standard solutions to determine the sample concentration. The fluorescence emission method is used to quantify purified protein. This simple method is useful for dilute protein samples and can be completed in a short amount of time. There are two colorimetric methods: the Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to the protein of interest, and the Lowry method, which measures colorimetric reaction of tyrosyl residues in the protein sample.

Evaluation of a compact bench top immunoassay analyzer for automatic and near continuous monitoring of a sample for environmental contaminants

A compact bench top immunoassay analyzer is evaluated and shown to possess sufficient automation to allow continuous unattended sampling and measuring while still achieving the theoretical (antibody affinity based) detection limit for analyte. The system is comprised of antigen coated particles in a disposable flow cell held at the focus of a filter fluorometer. Capture of fluorescently labeled antibody from the flow stream is inhibited by analyte in the sample, allowing analyte concentrations to be determined from the fluorescent intensity. The disposable cell was designed to allow easy end user changing of test specificity, e.g. for selection of any member of a panel of environmental contaminants. Standard curves are shown for six analytes of environmental interest, dioxin F114 (2,3,4,7,8-PeCDF), the pesticide Fenitrothion, three coplanar PCBs, including the most toxic, PCB 126, and estradiol. In each case the curves are constructed using antibody concentrations at or below the K d of the antibody, assuring that the sensitivity shown is limited by the antibody itself rather than the analyzer. The dynamic range for the six analytes investigated ranged from a low of 5 to 340 pM for fenitrothion to a high of 0.8 to 59 nM for dioxin F114, and is correlated to the antibody K d in every case. Data is also shown for 17 consecutive samples, including both high and low values, measured completely automatically over a period of hours. With further development and characterization, the bench top analyzer is expected to fill an important niche in environmental testing.

Spectrophotometric Determination of Protein Concentration

This unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measured at 280 nm (A(280)) is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein (a(280)). Alternatively, absorbance measured at 205 nm (A(205)) is used to calculate the protein concentration. The A(280) and A(205) methods can be used to quantify total protein in crude lysates and purified or partially purified protein. A spectrofluorometer or a filter fluorometer can be used to measure the intrinsic fluorescence emission of a sample solution; this value is compared with the emissions from standard solutions to determine the sample concentration. The fluorescence emission method is used to quantify purified protein. This simple method is useful for dilute protein samples and can be completed in a short amount of time. There are two colorimetric methods: the Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to the protein of interest, and the Lowry method, which measures colorimetric reaction of tyrosyl residues in the protein sample.

RNA Quantitation by Fluorescence-Based Solution Assay: RiboGreen Reagent Characterization

We describe the development of a sensitive fluorescence-based solution assay for RNA using a new dye, RiboGreen RNA quantitation reagent. RiboGreen reagent exhibits >1000-fold fluorescence enhancement and high quantum yield (0.65) upon binding nucleic acids, with excitation and emission maxima near those of fluorescein. Unbound dye is essentially nonfluorescent and has a large extinction coefficient (67,000 cm−1M−1). The RiboGreen assay allows detection of as little as 1.0 ng/ml RNA in a standard fluorometer, filter fluorometer, or fluorescence microplate reader—surpassing the sensitivity achieved with ethidium bromide by 200-fold. The linear quantitation range for RiboGreen reagent extends over three orders of magnitude in RNA concentration. Using 750 nM RiboGreen reagent, we quantitated 20 ng/ml to 1.0 μg/ml RNA. By diluting the reagent to 75 nM, we could quantitate 1.0 to 50 ng/ml RNA. Both assay ranges exhibited linear fluorescence increases versus RNA concentration (r2= 0.999). Assay linearity was maintained in the presence of salts, protein, urea, ethanol, chloroform, agarose, and some detergents. Several different RNA types yielded similar signal intensities and detection sensitivities. The assay is easy to use, rapid, and readily adaptable for automation.

Some Advances in Fluorometric Techniques for Water Tracing

A common technique for ground water tracing uses continuous or discrete analysis of fluorescent tracer dyes to produce tracer breakthrough curves which can be analysed to determine ground water trajectory, velocity and dispersivity. Some fluorescent dyes are excellent tracers, but there remain significant constraints in fluorometry with respect to installation, control and data acquisition using standard filter fluorometers as well as with general analytical protocols. This paper identifies some of the constraints associated with the widely used Turner Designs Model 10 Series filter fluorometer and technical and operational solutions which can significantly improve the precision, accuracy and time resolution of tracer data.

Quenched BODIPY Dye-Labeled Casein Substrates for the Assay of Protease Activity by Direct Fluorescence Measurement

We have prepared casein conjugates of two BODIPY dyes for use as fluorogenic protease substrates in homogeneous assays. Both conjugates are labeled to such an extent that the dyes are efficiently quenched in the protein, yielding virtually nonfluorescent substrate molecules. These fluorogenic substrates release highly fluorescent BODIPY dye-labeled peptides upon protease digestion, with fluorescence increases proportional to enzyme activity. These quenched substrates are suitable for the continuous assay of enzymatic activity using standard fluorometers, filter fluorometers, or fluorescence microplate readers using either fluorescein excitation and emission wavelengths to measure BODIPY FL casein hydrolysis or Texas Red wavelengths to detect proteolysis of BODIPY TR-X casein. Most current techniques for detecting protease activity, such as the fluorescein thiocarbamoyl casein (FTC–casein) protease assay, require extensive manipulation, including separation steps, and are therefore labor intensive and error-prone. In comparison, we found the BODIPY dye-labeled casein protease assays to be simple and precise and to have greater sensitivity and a broader dynamic range of detection than the FTC-casein assay. We were able to sensitively detect the activities of a wide variety of enzymes with these new substrates, including serine, acid, sulfhydryl, and metalloproteases. We also found the assay suitable for quantitating protease inhibitor concentrations and for real-time analysis of proteolysis.

Spectrophotometric and Colorimetric Determination of Protein Concentration

AbstractThis unit describes spectrophotometric and colorimetric methods for measuring the concentration of a sample protein in solution. Absorbance measurement at 280 nm is used to calculate protein concentration by comparison with a standard curve or published absorptivity values for that protein. An alternate protocol uses absorbance at 205 nm to calculate the protein concentration. Both methods can be used to quantitate total protein in crude lysates and purified or partially purified protein. Use of a spectrofluorometer or a filter fluorometer to measure the intrinsic fluorescence emission of a sample solution is also described. The measurement is compared with the emissions from standard solutions to determine the concentration of purified protein. The Bradford colorimetric method, based upon binding of the dye Coomassie brilliant blue to an unknown protein, is also presented, as is the Lowry method, which measures colorimetric reaction of tyrosyl residues in an unknown.

High-efficiency filter fluorometer for capillary electrophoresis and its application to fluorescein thiocarbamyl amino acids

Fluorescence detection has produced excellent detection limits in capillary electrophoresis. Laser excitation produces the highest sensitivity detection. lie perceived difficulties associated with the use of the laser has discouraged applications of fluorescence to capillary electrophoresis. In particular, difficulties in wavelength selection limit the choice of chemistry available in capillary clectrophoresis. We report the design of a filter fluorometer based on a compact 75 W xenon arc lamp for capillary electrophoresis; the instrument produces detection limits (3σ) of 20 zeptomol (1 zeptomol = 10 −21 mol) for fluorescein and 200 zeptomol for fluorescein labeled amino acids.

Continuous liquid-phase fluorometry coupled to a diffusion scrubber for the real-time determination of atmospheric formaldehyde, hydrogen peroxide and sulfur dioxide

An aqueous scrubber liquid is pumped through a filament-filled narrow bore microporous hydrophobic membrane tube while sample air flows around it. A constant fraction of the analyte gas, dependent on its diffusion coefficient and the effective mass accommodation coefficient in the scrubber liquid and on the diffusion scrubber dimensions and sampling rate, is collected in the liquid. One or more reagents are added to the effluent liquid for specific determination of the gas of interest. The Hantzsch reaction, peroxidase-mediated oxidation of p-hydroxyphenylacetic acid by peroxide, and bisulfite addition to 9- N-acridinylmaleimide, all direct adaptations of previously described continuous flow aqueous analytical procedures, are used for the determination of HCHO, H 2O 2 and SO 2, respectively. A permeation-based SO 2 source and previously described Henry's Law-based porous membrane sources of HCHO and H 2O 2 are integral to the respective instruments for calibration. Programmed inert valves allow the instruments to perform any sequence of zero, calibrate and sample functions. Each instrument occupies a standard two-tier (50×100 cm) laboratory cart and has been successfully field tested. With a filter fluorometer as detector, LODs are 100 pptv HCHO, 30 pptv H 2O 2 and 175 pptv SO 2. There is no significant dependence on relative humidity. Specific interference testing ( SO 2 O 3 for HCHO and H 2O 2, O 3 H 2O 2H 2SCH 3SH for SO 2) reveals no major interferences.

The separation of d/l amino acid pairs by high-performance liquid chromatography after precolumn derivatization with optically active naphthylethyl isocyanate

A method for determining the optical purity of amino acids using HPLC and precolumn derivatization is described. (+)-1-(1-Naphthyl)ethyl isocyanate reacts with racemic amino acids, in high yield, to form naphthylethyl carbamoyl derivatives. The resulting diastereoisomeric pairs were separated on reversed-phase C 18 columns and detected fluorometrically. Excitation maxima for naphthylethyl carbamoyl aspartic acid were 235 and 297 nm. The emission maximum was at 333 nm. Using a filter fluorometer with a zinc or cadmium lamp, less than 1 pmol of a d amino acid can be measured in the presence of 1000-fold excess of the l isomer. The column can also be monitored at lower sensitivity, using an ultraviolet detector operating at or near the absorption maximum of 222 nm. Chromatographic data are presented on the resolution of 17 amino acid pairs.

Fluorometric flow injection determination of aqueous peroxides at nanomolar level using membrane reactors

A flow injection system based on the p-hydroxyphenylacetate-peroxide-peroxidase reaction allows the simultaneous determination of H/sub 2/O/sub 2/ and CH/sub 3/HO/sub 2/ at 50 samples/h with an LOD of 0.1 /sup +/g/L (3 nM) H/sub 2/O/sub 2/. A pressurized porous PTFE membrane reactor introduces the enzyme, and the pH of the flow stream is altered by introducing NH/sub 3/ through a nonporous cation exchange membrane reactor. Excellent reproducibility, precision, and sensitivity are achieved with the membrane reactors and an inexpensive filter fluorometer with a monochromatic excitation source. Low levels of residual peroxide in water are ubiquitous; such levels return soon after the H/sub 2/O/sub 2/ is catalytically removed.

String Collectors for Spray Pattern Analysis

ABSTRACT TRING, as a collector to sample liquid spray patterns, and a unique filter fluorometer door, designed to enhance the string fluorescence, were evaluated. String and paper tape collectors were compared for treatment combinations involving two nozzle types, four application rates and three dye concentrations. The fluorometric response from the string collector was equal to or better than that from the paper tape. The spray application rate was highly correlated with collector fluorescence. Optimum dye concentrations for the string and paper tape collectors were determined to be 100 and 300 ppm, respectively.