Sample Cuvette Research Articles

Fluorescence Lifetime Microplate Reader for Structural Biology: High Performance and High-Throughput in the Same Tool

Microplate readers are widely considered as designed and intended for high-throughput (HT) applications such as drug discovery, whereas “research-grade” (RG) fluorescence spectrometers are the tools of choice when quality and diversity of data are the prime considerations. However, a definite trend of the past decade is the commercial availability of microplate readers that come equipped with emission and excitation monochromators (even double monochromators) very similar to those found on research-grade spectrometers. The flashlamp sources used in most microplate readers are very similar or even identical in design to the flashlamp sources of the research-grade spectrometers. Microplate readers that employ fluorescence lifetime as a primary readout represent another possible form of convergence between the high-throughput assay and research. An interesting question is to what degree the biggest difference between these two worlds is now simply the sample format, i.e., cuvette vs. microplate. It seems reasonable to assume that the cuvette with its straightforward implementation of right angle excitation-emission geometry offers significant advantages over the epi-illumination geometry and uncontained sample imposed by a microplate when it comes to data quality. We have implemented a prototype microplate reader equipped with a variety of pulsed laser sources for measurement of fluorescence spectra, fluorescence lifetimes, and anisotropy. The subject of this poster is benchmarking its performance relative to cuvette format. The plate reader employs direct waveform recording as an alternative to TCSPC; studies to compare the speed, accuracy, and precision of the two lifetime approaches are presented along with several examples of titration curves for rapid determination of binding affinities via time-resolved FRET.

High pressure sample cell for total internal reflection fluorescence spectroscopy at pressures up to 2500 bar

Total internal reflection fluorescence (TIRF) spectroscopy is a surface sensitive technique that is widely used to characterize the structure and dynamics of molecules at planar liquid-solid interfaces. In particular, biomolecular systems, such as protein adsorbates and lipid membranes can easily be studied by TIRF spectroscopy. Applying pressure to molecular systems offers access to all kinds of volume changes occurring during assembly of molecules, phase transitions, and chemical reactions. So far, most of these volume changes have been characterized in bulk solution, only. Here, we describe the design and performance of a high pressure sample cell that allows for TIRF spectroscopy under high pressures up to 2500 bar (2.5 × 10(8) Pa), in order to expand the understanding of volume effects from the bulk phase to liquid-solid interfaces. The new sample cell is based on a cylindrical body made of Nimonic 90 alloy and incorporates a pressure transmitting sample cuvette. This cuvette is composed of a fused silica prism and a flexible rubber gasket. It contains the sample solution and ensures a complete separation of the sample from the liquid pressure medium. The sample solution is in contact with the inner wall of the prism forming the interface under study, where fluorescent molecules are immobilized. In this way, the new high pressure TIRF sample cell is very useful for studying any biomolecular layer that can be deposited at a planar water-silica interface. As examples, high pressure TIRF data of adsorbed lysozyme and two phospholipid membranes are presented.

Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Δ7-24, 25-dihydrolanosterol

Sterol 14α-demethylase (CYP51) that catalyzes the removal of the 14α-methyl group from the sterol nucleus is an essential enzyme in sterol biosynthesis, a primary target for clinical and agricultural antifungal azoles and an emerging target for antitrypanosomal chemotherapy. Here, we present the crystal structure of Trypanosoma (T) brucei CYP51 in complex with the substrate analog 14α-methylenecyclopropyl-Δ7-24,25-dihydrolanosterol (MCP). This sterol binds tightly to all protozoan CYP51s and acts as a competitive inhibitor of F105-containing (plant-like) T. brucei and Leishmania (L) infantum orthologs, but it has a much stronger, mechanism-based inhibitory effect on I105-containing (animal/fungi-like) T. cruzi CYP51. Depicting substrate orientation in the conserved CYP51 binding cavity, the complex specifies the roles of the contact amino acid residues and sheds new light on CYP51 substrate specificity. It also provides an explanation for the effect of MCP on T. cruzi CYP51. Comparison with the ligand-free and azole-bound structures supports the notion of structural rigidity as the characteristic feature of the CYP51 substrate binding cavity, confirming the enzyme as an excellent candidate for structure-directed design of new drugs, including mechanism-based substrate analog inhibitors.

Tryptamine-Gallic Acid Hybrid Prevents Non-steroidal Anti-inflammatory Drug-induced Gastropathy

We have investigated the gastroprotective effect of SEGA (3a), a newly synthesized tryptamine-gallic acid hybrid molecule against non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy with mechanistic details. SEGA (3a) prevents indomethacin (NSAID)-induced mitochondrial oxidative stress (MOS) and dysfunctions in gastric mucosal cells, which play a pathogenic role in inducing gastropathy. SEGA (3a) offers this mitoprotective effect by scavenging of mitochondrial superoxide anion (O(2)(·-)) and intramitochondrial free iron released as a result of MOS. SEGA (3a) in vivo blocks indomethacin-mediated MOS, as is evident from the inhibition of indomethacin-induced mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. SEGA (3a) corrects indomethacin-mediated mitochondrial dysfunction in vivo by restoring defective electron transport chain function, collapse of transmembrane potential, and loss of dehydrogenase activity. SEGA (3a) not only corrects mitochondrial dysfunction but also inhibits the activation of the mitochondrial pathway of apoptosis by indomethacin. SEGA (3a) inhibits indomethacin-induced down-regulation of bcl-2 and up-regulation of bax genes in gastric mucosa. SEGA (3a) also inhibits indometacin-induced activation of caspase-9 and caspase-3 in gastric mucosa. Besides the gastroprotective effect against NSAID, SEGA (3a) also expedites the healing of already damaged gastric mucosa. Radiolabeled ((99m)Tc-labeled SEGA (3a)) tracer studies confirm that SEGA (3a) enters into mitochondria of gastric mucosal cell in vivo, and it is quite stable in serum. Thus, SEGA (3a) bears an immense potential to be a novel gastroprotective agent against NSAID-induced gastropathy.

Substrate Preferences and Catalytic Parameters Determined by Structural Characteristics of Sterol 14α-Demethylase (CYP51) from Leishmania infantum

Leishmaniasis is a major health problem that affects populations of ∼90 countries worldwide, with no vaccine and only a few moderately effective drugs. Here we report the structure/function characterization of sterol 14α-demethylase (CYP51) from Leishmania infantum. The enzyme catalyzes removal of the 14α-methyl group from sterol precursors. The reaction is essential for membrane biogenesis and therefore has great potential to become a target for antileishmanial chemotherapy. Although L. infantum CYP51 prefers C4-monomethylated sterol substrates such as C4-norlanosterol and obtusifoliol (V(max) of ∼10 and 8 min(-1), respectively), it is also found to 14α-demethylate C4-dimethylated lanosterol (V(max) = 0.9 min(-1)) and C4-desmethylated 14α-methylzymosterol (V(max) = 1.9 min(-1)). Binding parameters with six sterols were tested, with K(d) values ranging from 0.25 to 1.4 μM. Thus, L. infantum CYP51 is the first example of a plant-like sterol 14α-demethylase, where requirements toward the composition of the C4 atom substituents are not strict, indicative of possible branching in the postsqualene portion of sterol biosynthesis in the parasite. Comparative analysis of three CYP51 substrate binding cavities (Trypanosoma brucei, Trypanosoma cruzi, and L. infantum) suggests that substrate preferences of plant- and fungal-like protozoan CYP51s largely depend on the differences in the enzyme active site topology. These minor structural differences are also likely to underlie CYP51 catalytic rates and drug susceptibility and can be used to design potent and specific inhibitors.

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Thiol-disulfide Redox Dependence of Heme Binding and Heme Ligand Switching in Nuclear Hormone Receptor Rev-erbβ

Rev-erbβ is a heme-binding nuclear hormone receptor that represses a broad spectrum of target genes involved in regulating metabolism, the circadian cycle, and proinflammatory responses. Here, we demonstrate that a thiol-disulfide redox switch controls the interaction between heme and the ligand-binding domain of Rev-erbβ. The reduced dithiol state of Rev-erbβ binds heme 5-fold more tightly than the oxidized disulfide state. By means of site-directed mutagenesis and by UV-visible and EPR spectroscopy, we also show that the ferric heme of reduced (dithiol) Rev-erbβ can undergo a redox-triggered switch from imidazole/thiol ligation (via His-568 and Cys-384, based on a prior crystal structure) to His/neutral residue ligation upon oxidation to the disulfide form. On the other hand, we find that change in the redox state of iron has no effect on heme binding to the ligand-binding domain of the protein. The low dissociation constant for the complex between Fe(3+)- or Fe(2+)-heme and the reduced dithiol state of the protein (K(d) = ∼ 20 nM) is in the range of the intracellular free heme concentration. We also determined that the Fe(2+)-heme bound to the ligand-binding domain of Rev-erbβ has high affinity for CO (K(d) = 60 nM), which replaces one of the internal ligands when bound. We suggest that this thiol-disulfide redox switch is one mechanism by which oxidative stress is linked to circadian and/or metabolic imbalance. Heme dissociation from Rev-erbβ has been shown to derepress the expression of target genes in response to changes in intracellular redox conditions. We propose that oxidative stress leads to oxidation of cysteine(s), thus releasing heme from Rev-erbβ and altering its transcriptional activity.

Cost-effective and compact wide-field fluorescent imaging on a cell-phone

We demonstrate wide-field fluorescent and darkfield imaging on a cell-phone with compact, light-weight and cost-effective optical components that are mechanically attached to the existing camera unit of the cell-phone. For this purpose, we used battery powered light-emitting diodes (LEDs) to pump the sample of interest from the side using butt-coupling, where the pump light was guided within the sample cuvette to uniformly excite the specimen. The fluorescent emission from the sample was then imaged using an additional lens that was positioned right in front of the existing lens of the cell-phone camera. Because the excitation occurs through guided waves that propagate perpendicular to our detection path, an inexpensive plastic colour filter was sufficient to create the dark-field background required for fluorescent imaging, without the need for a thin-film interference filter. We validate the performance of this platform by imaging various fluorescent micro-objects in 2 colours (i.e., red and green) over a large field-of-view (FOV) of ∼81 mm(2) with a raw spatial resolution of ∼20 μm. With additional digital processing of the captured cell-phone images, through the use of compressive sampling theory, we demonstrate ∼2 fold improvement in our resolving power, achieving ∼10 μm resolution without a trade-off in our FOV. Further, we also demonstrate darkfield imaging of non-fluorescent specimen using the same interface, where this time the scattered light from the objects is detected without the use of any filters. The capability of imaging a wide FOV would be exceedingly important to probe large sample volumes (e.g., >0.1 mL) of e.g., blood, urine, sputum or water, and for this end we also demonstrate fluorescent imaging of labeled white-blood cells from whole blood samples, as well as water-borne pathogenic protozoan parasites such as Giardia Lamblia cysts. Weighing only ∼28 g (∼1 ounce), this compact and cost-effective fluorescent imaging platform attached to a cell-phone could be quite useful especially for resource-limited settings, and might provide an important tool for wide-field imaging and quantification of various lab-on-a-chip assays developed for global health applications, such as monitoring of HIV+ patients for CD4 counts or viral load measurements.

Monomeric Rhodopsin Is Sufficient for Normal Rhodopsin Kinase (GRK1) Phosphorylation and Arrestin-1 Binding

G-protein-coupled receptor (GPCR) oligomerization has been observed in a wide variety of experimental contexts, but the functional significance of this phenomenon at different stages of the life cycle of class A GPCRs remains to be elucidated. Rhodopsin (Rh), a prototypical class A GPCR of visual transduction, is also capable of forming dimers and higher order oligomers. The recent demonstration that Rh monomer is sufficient to activate its cognate G protein, transducin, prompted us to test whether the same monomeric state is sufficient for rhodopsin phosphorylation and arrestin-1 binding. Here we show that monomeric active rhodopsin is phosphorylated by rhodopsin kinase (GRK1) as efficiently as rhodopsin in the native disc membrane. Monomeric phosphorylated light-activated Rh (P-Rh*) in nanodiscs binds arrestin-1 essentially as well as P-Rh* in native disc membranes. We also measured the affinity of arrestin-1 for P-Rh* in nanodiscs using a fluorescence-based assay and found that arrestin-1 interacts with monomeric P-Rh* with low nanomolar affinity and 1:1 stoichiometry, as previously determined in native disc membranes. Thus, similar to transducin activation, rhodopsin phosphorylation by GRK1 and high affinity arrestin-1 binding only requires a rhodopsin monomer.

Teaching, Learning, and Using Spectroscopy with Commercial, Off-the-Shelf Technology

The astonishing propagation of sophisticated electronics across the globe has attracted the attention of many for developing disease diagnoses, water purity measurements, and security applications using cellular telephones and the complementary metal oxide semiconductor (CMOS) cameras with which many are equipped. This Focal Point article builds on the theme of using technology already in the hands of students/consumers to teach spectrophotometry and, by extension, to suggest how one might inexpensively develop an instrumental analysis curriculum at extraordinarily low cost. A grating spectrophotometer using a white light-emitting diode (LED) light source, plastic sample cuvette, holographic transmission grating, and any camera that produces JPG files as output is described. Optical characteristics are explained and a sample working curve provided. The raw data for that curve are available as Supplementary Material on-line. The behavior of the "Cell Phone Spectrometer" is inadequate for routine analysis but outstanding for teaching the workings of optical instrumentation components and systems.

Direct determination of bromine in plastics by electrothermal vaporization/inductively coupled plasma mass spectrometry using a tungsten boat furnace vaporizer and an exchangeable sample cuvette system

A tungsten boat furnace vaporization inductively coupled plasma mass spectrometry (TBF/ICP-MS) method has been applied to the direct determination of bromine in plastic samples. In the pretreatment, the plastic sample is spread over a small sample cuvette made of tungsten by treating it with a strongly basic organic solution, e.g., octanol or diisobutyl ketone in the presence of potassium hydroxide. The cuvette is placed on a tungsten boat furnace, with which the electrothermal vaporizer is equipped. At the vaporization step, a widely spread thin layer of the sample facilitates its efficient evaporation and introduction into an ICP mass spectrometer. The most remarkable feature is that all the bromine species in plastic samples are decomposed to form a thermally stable inorganic salt during the pretreatment procedure. Therefore, the bromine content in plastic samples can be measured by a calibration curve method constructed with an aqueous standard solution of potassium bromate(V). The detection limit (3sigma) was estimated to be 0.77 pg of bromine, which corresponds to a concentration of 0.31 ng g(-1) of bromine in plastic samples when a sample amount taken of 2.5 mg is studied. The relative standard deviation was calculated to be 2.2%. Analytical results of some plastic samples, which contained both inorganic bromide salts and also organic bromine species, are given.

Pulsed laser excited photothermal lens spectrometry of cadmium sulfoselenide doped silica glasses

Experimental results for photothermal lens measurements are compared to finite elemental analysis models for commercial colored glass filters. Finite elemental analysis software is used to model the photothermal effect by simulating the coupling of heat both within the sample and out to the surroundings. Modeling shows that heat transfer between the glass surface and the air coupling fluid has a significant effect on the predicted time dependent photothermal lens signals. For comparison with experimental signals, a simple equation based on the finite element analysis result is proposed for accounting for the variance of experimental data where this type of heat coupling situation occurs. The colored glass filters are found to have positive thermo-optical coefficient. Finite element analysis modeling results are also used to correlate experimental measurements of different sample geometries. In particular, the glass samples are compared to ethanol solutions of iron (II) dicyclopentadiene in a sample cuvette even though heat transfer is different for these two samples.

STUDIES ON A FOUR-ARM POLYSTYRENE GEL BY LASER LIGHT SCATTERING

Atom transfer radical polymerization(ATRP)was used to synthesize narrowly distributed 4-arm-polystyrene star chains(4-PS-Br).Then the bromine end functional groups were further modified to thiol groups(4-PS-SH).Oxidization of each two thiol end groups leads to a disulfide bond(—S—S—),which enables us to slowly crosslink the PS star chains in THF solutions to form polymer clusters,then gels.Static laser light scattering(SLS)shows that in both semidilute solutions and gels the time-average scattered intensity changes little with the sample position,where the sample position is arbitrarily chosen by rotating and lifting the sample cuvette,indicating this chemical gel is homogeneous and speckle-free.Dynamic laser light scattering(DLS)shows that first there is only one fast decay mode of the time-time intensity correlation function in the original semidilute solution.Then the slow modes of the correlation function appear and the contributions of the slow modes increase at the early oxidation stage,indicating the formation of large cross-linked clusters.With oxidation time increase the contribution of the slow mode decreases,finally disappears.The correlation functions of the chemical gel are nearly the same as those of the original semidilute solution.The normalized intermediate scattering function can fully relax to zero,indicating that the gel has no frozen-in static component,and the speckles of other polymer gels originate from large voids inside,not cross-linked chains(clusters).Both SLS and DLS results indicate that this polystyrene gel is a homogeneous chemical gel.

Pulsed Laser Excited Photothermal Lens Spectrometry of CdSxSe1—x Doped Silica Glasses

Experimental results for photothermal lens measurements are compared to finite elemental analysis models for commercial colored glass filters. Finite elemental analysis software is used to model the photothermal effect by simulating the coupling of heat both within the sample and out to the surroundings. Modeling shows that heat transfer between the glass surface and the air coupling fluid has a significant effect on the predicted time-dependent photothermal lens signals. For comparison with experimental signals, a simple equation based on the finite element analysis result is proposed for accounting for the variance of experimental data where this type of heat coupling situation occurs. The colored glass filters are found to have positive thermo-optical coefficients. The net positive dn/dT of CdSxSe(1-x) doped glass filters is considered to be the consequence of counteracting factors: optical nonlinearity, stress-induced birefringence, and the structural network of glass. Finite element analysis modeling results are also used to correlate experimental measurements of different sample geometries. In particular, the glass samples are compared to ethanol solutions of iron (II) dicylopentadiene in a sample cuvette even though heat transfer is different for these two samples.

The Puzzle of Ligand Binding to Corynebacterium ammoniagenes FAD Synthetase

In bacteria, riboflavin phosphorylation and subsequent conversion of FMN into FAD are carried out by FAD synthetase, a single bifunctional enzyme. Both reactions require ATP and Mg(2+). The N-terminal domain of FAD synthetase appears to be responsible for the adenylyltransferase activity, whereas the C-terminal domain would be in charge of the kinase activity. Binding to Corynebacterium ammoniagenes FAD synthetase of its products and substrates, as well as of several analogues, is analyzed. Binding parameters for adenine nucleotides to each one of the two adenine nucleotide sites are reported. In addition, it is demonstrated for the first time that the enzyme presents two independent flavin sites, each one related with one of the enzymatic activities. The binding parameters of flavins to these sites are also provided. The presence of Mg(2+) and of both adenine nucleotides and flavins cooperatively modulates the interaction parameters for the other ligands. Our data also suggest that during its double catalytic cycle FAD synthetase must suffer conformational changes induced by adenine nucleotide-Mg(2+) or flavin binding. They might include not only rearrangement of the different protein loops but also alternative conformations between domains.

Biosynthesis of the Sesquiterpene Antibiotic Albaflavenone in Streptomyces coelicolor A3(2)

Cytochrome P450 170A1 (CYP170A1) is encoded by the sco5223 gene of the Gram-positive, soil-dwelling bacterium Streptomyces coelicolor A3(2) as part of a two-gene cluster with the sco5222 gene. The SCO5222 protein is a sesquiterpene synthase that catalyzes the cyclization of farnesyl diphosphate to the novel tricyclic hydrocarbon, epi-isozizaene (Lin, X., Hopson, R., and Cane, D. E. (2006) J. Am. Chem. Soc. 128, 6022-6023). The presence of CYP170A1 (sco5223) suggested that epiisozizaene might be further oxidized by the transcriptionally coupled P450. We have now established that purified CYP170A1 carries out two sequential allylic oxidations to convert epi-isozizaene to an epimeric mixture of albaflavenols and thence to the sesquiterpene antibiotic albaflavenone. Gas chromatography/mass spectrometry analysis of S. coelicolor culture extracts established the presence of albaflavenone in the wild-type strain, along with its precursors epi-isozizaene and the albaflavenols. Disruption of the CYP170A1 gene abolished biosynthesis of both albaflavenone and the albaflavenols, but not epi-isozizaene. The combined results establish for the first time the presence of albaflavenone in S. coelicolor and clearly demonstrate that the biosynthesis of this antibiotic involves the coupled action of epi-isozizaene synthase and CYP170A1.

Separate vaporisation of boric acid and inorganic boron from tungsten sample cuvette–tungsten boat furnace followed by the detection of boron species by inductively coupled plasma mass spectrometry and atomic emission spectrometry (ICP-MS and ICP-AES)

Utilising extremely different vaporisation properties of boron compounds, the determination procedures of volatile boric acid and total boron using tungsten boat furnace (TBF) ICP-MS and TBF-ICP-AES have been investigated. For the determination of volatile boric acid by TBF-ICP-MS, tetramethylammonium hydroxide (TMAH, Me 4NOH) was used as a chemical modifier to retain it during drying and ashing stages. As for the total boron, not only non-volatile inorganic boron such as boron nitride (BN), boron carbide (B 4C), etc. but also boric acid (B(OH) 3) was decomposed by a furnace-fusion digestion with NaOH to produce sodium salt of boron, a suitable species for the electrothermal vaporisation (ETV) procedure. The proposed method was applied to the analysis of various standard reference materials. The analytical results for various biological and steel samples are described.

Determination of the particulate absorption of microalgae using a point‐source integrating‐cavity absorption meter: verification with a photometric technique, improvements for pigment bleaching, and correction for chlorophyll fluorescence

The determination of the spectral light absorption coefficient of planktonic algae in seawater is crucial for many applications, including optical remote sensing. Common techniques are adversely affected by light‐scattering effects, or by low particle concentrations in situ. Measurements with a point‐source integrating‐cavity absorption meter (PSICAM) can overcome both difficulties. In our study, we assessed the performance of a PSICAM for measuring the absorption coefficients of microalgae by a comparison with state‐of‐the‐art techniques. The PSICAM was evaluated by comparative measurements of diluted algal cultures with a photometric setup, where the sample cuvette is placed in the center of an integrating sphere (CIS). The accuracy of both the CIS and the PSICAM technique was calibrated against a commercial spectrophotometer by measurements of dense algal cultures. The remaining sources of error for the PSICAM technique were the bleaching procedure and chlorophyll fluorescence. We propose an improved bleaching technique and introduce a correction procedure for the fluorescence effects. Applying both, the overall differences between PSICAM and CIS measurements were less than ± 1%. We further demonstrate that the accuracy of the PSICAM is at least as good as that of a commercial spectrophotometer, but its precision should be two to three orders of magnitude higher. Due to its high sensitivity and accuracy, and to the fact that sources of error can be identified and accounted for, the PSICAM has a great potential to significantly improve the determination of absorption spectra of particulate matter in natural seawater.

Uncoupling the Enzymatic and Autoprocessing Activities of Helicobacter pylori γ-Glutamyltranspeptidase

Gamma-glutamyltranspeptidase (gammaGT), a member of the N-terminal nucleophile hydrolase superfamily, initiates extracellular glutathione reclamation by cleaving the gamma-glutamyl amide bond of the tripeptide. This protein is translated as an inactive proenzyme that undergoes autoprocessing to become an active enzyme. The resultant N terminus of the cleaved proenzyme serves as a nucleophile in amide bond hydrolysis. Helicobacter pylori gamma-glutamyltranspeptidase (HpGT) was selected as a model system to study the mechanistic details of autoprocessing and amide bond hydrolysis. In contrast to previously reported gammaGT, large quantities of HpGT were expressed solubly in the inactive precursor form. The 60-kDa proenzyme was kinetically competent to form the mature 40- and 20-kDa subunits and exhibited maximal autoprocessing activity at neutral pH. The activated enzyme hydrolyzed the gamma-glutamyl amide bond of several substrates with comparable rates, but exhibited limited transpeptidase activity relative to mammalian gammaGT. As with autoprocessing, maximal enzymatic activity was observed at neutral pH, with hydrolysis of the acyl-enzyme intermediate as the rate-limiting step. Coexpression of the 20- and 40-kDa subunits of HpGT uncoupled autoprocessing from enzymatic activity and resulted in a fully active heterotetramer with kinetic constants similar to those of the wild-type enzyme. The specific contributions of a conserved threonine residue (Thr380) to autoprocessing and hydrolase activities were examined by mutagenesis using both the standard and coexpression systems. The results of these studies indicate that the gamma-methyl group of Thr380 orients the hydroxyl group of this conserved residue, which is required for both the processing and hydrolase reactions.

Versatile portable fluorometer for time-resolved luminescence analysis

A robust, filter-based portable fluorometer was designed, prototyped, and tested for time-resolved luminescence (TRL) analysis. Its flexible optical design allows interchangeable configurations to support three measurement modes: liquid-phase TRL using a sample cuvette, solid-matrix TRL using a sorbent strip, and evanescent-field TRL using a quartz-rod waveguide. A xenon flashlamp is used as the light source and a photomultiplier tube (PMT) as the photodetector. A gating technique was implemented to overcome PMT saturation by the intense xenon lamp flash, therefore higher gains can be set to measure weak luminescence signals. The TRL signal is digitized at a 4μs time resolution and a 12bit amplitude resolution. Individual flashes were monitored by a photodiode and its current was integrated to compensate for source light fluctuation. Using tetracycline as a model analyte, a 0.025ppb limit of detection (LOD) with a typical 2% relative standard deviation, and a 3 orders of magnitude (0.5–300ppb) linear dynamic range (r2=0.9996) were achieved.