Traditional Colorimetric Assay Research Articles

Antimicrobial activity of CuFe2O4 and CuFe2O4/ZnO: Squaring colorimetric and traditional microbiology assays with atomic force- and holotomography-microscopies

Intensive research efforts seek the discovery of efficient antimicrobial compounds to fight microbial resistance. Magnetic nanoparticles represent a promising option due to their multifunctional antimicrobial properties, as well as their versatility, stability, and the limited microbial resistance they generate. In this study, magnetic nanomaterials (MNMs) are synthesized using a microwave-activated solvothermal method. Characterization data indicate the crystalline nature (cubic spinel CuFe2O4 NMs), superparamagnetic behavior (MCF = 52 emu/g and MCF-ZnO = 29 emu/g) nanometric and regular size (spherical particles with a mean size of 7.4 ± 1.3 nm) of the MNMs. The antimicrobial activity of the as-prepared (naked) MNMs is moderate against bacteria, yeast, and fungal strains due to the aggregation of the MNMs in the exposure media. However, citrate-functionalized MNMs (CuFe2O4-Cit, CF-Cit) show enhanced antimicrobial activity, further increasing upon activation under a high-frequency magnetic field (magnetic hyperthermia). CF-Cit exerts stronger antibacterial activity towards S. aureus (MIC <25 μg/mL) than E. coli (MIC <50 μg/mL In addition, CuFe2O4-ZnO composites were synthesized and fully characterized. The MNMs under study exhibit efficient antifungal and anti-biofilm activity. Working towards the sustainable development of multifunctional (CuFe2O4 and CuFe2O4-ZnO) MNMs, their compatibility and bioactivity was evaluated using traditional assays and advanced microscopy techniques (Atomic Force Microscopy-AFM and Holotomographic Microscopy -HTM). Because of their chemical composition, the MNMs under study lixiviate transition metal ions to the exposure media, interfering with traditional colorimetric assays used to evaluate the bioactivity of the MNMs (antimicrobial, biocompatibility). Thus, AFM and HTM studies render complementary information during the interpretation of the bioactivity mechanisms exhibited by the MNMs.

An Electrochemical Activity Assay of Non-Electroactive Enzymes

Enzymatic tools serve important functions in all areas of human life. Research aimed at comparing the catalytic rates of functionally overlapping enzymes of interest (EOIs) is hampered by expensive and time-consuming growth and purification procedures. Assaying the performance of already purified EOIs requires further laborious optimization. These assays are typically colorimetric and employ a downstream signal producing enzyme such as NADH-coupled dehydrogenases, which complicate assay optimization with their own unique chemical and pH requirements. Electrocatalysts such as 2,2,6,6-tetramethylpiperidine 1-Oxyl (TEMPO) have much broader pH ranges and substrate scopes (TEMPO oxidizing aldehydes and non-tertiary alcohols) than signal enzymes and output their catalytic rates directly as current density. The field of electrochemistry is also making strides in enzyme-electrode immobilization, with several works now reporting methods to anchor enzymes onto electrode surfaces directly from cell lysate, avoiding lengthy column-purification procedures.This work proposes a hybrid electrolytic cell in which the catalytic rate of immobilized enzyme is quantified as current density produced by TEMPO-mediated oxidation of its products. The rate of production of carbon paper electrodes has been expedited and their functional area has been standardized through novel cutting and waxing procedures, demonstrating surface area coefficient of variation values up to an order of magnitude lower and production rates an order of magnitude higher than those of traditionally produced electrodes. Variation in electrode-loading of pyrene-anchored TEMPO has also been minimized, demonstrating current density variation identical to that of electrode functional area. Current work is investigating enzyme immobilization architectures employing electrode-immobilized DNA and Zinc-finger anchored enzyme to maximize enzyme loading, as well as enzyme-electrocatalyst reactivity to minimize background signal. In summary, we aim to develop an electrochemical assay of enzyme activity able to quantify the catalytic rates of a wide range of aldehyde and non-tertiary alcohol producing EOIs while minimizing the lengthy enzyme purification and assay optimization procedures of traditional colorimetric assays.

Thiohydantoins and hydantoins derived from amino acids as potent urease inhibitors: Inhibitory activity and ligand-target interactions

We report the investigation of hydantoins and thiohydantoins derived from L and d-amino acids as inhibitors against the Canavalia ensiformis urease (CEU). The biochemical in vitro assay against CEU revealed a promising inhibitory potential for most thiohydantoins with six of them showing %I higher than the reference inhibitor thiourea (56.5%). In addition, thiohydantoin derived from l-valine, 1b, as well as the hydantoin 2d, derived from l-methionine, were identified as the most potent inhibitors with %I = 90.5 and 85.9 respectively. Enzyme kinetic studies demonstrated a mixed and uncompetitive inhibition profile for these compounds with Ki values of 0.42 mM for 1b and 0.99 mM for 2d. These kinetic parameters, obtained from traditional colorimetric assay, were strictly related to the KD values measured spectroscopically by the Saturation Transfer Difference (STD) technique for the urease complex. STD was also used to evince the moieties of the ligands responsible for the binding with the enzyme. Molecular docking studies showed that the thiohydantoin and hydantoin rings can act as a pharmacophoric group due to their binding affinity by hydrogen bonding interactions with critical amino acid residues in the enzyme active and/or allosteric site. These findings agreed with the experimental alpha values, demonstrating that 1b has affinity by free enzyme, and 2d derivative, an uncompetitive inhibitor, has great binding affinity at the allosteric site. The results for the thiohydantoin 1a, derived from d-valine, demonstrated a drastic stereochemical influence on inhibition, kinetics, and binding parameters in comparison to its enantiomer 1b.

Generation of Dual functional Nanobody-Nanoluciferase Fusion and its potential in Bioluminescence Enzyme Immunoassay for trace Glypican-3 in Serum.

Glypican-3 (GPC3) is a serological biomarker for the diagnosis of Hepatocellular carcinoma (HCC), but it is a challenging task to develop a bioassay for determination of the trace GPC3 in serum. In this study, Bioluminescense immunoassay based on bifunctional nanobody-nanoluciferase fusion was developed with the ultra-sensitive feature to achieve this goal. First, nanobodies special against GPC-3 binder as biological recognition element were generated by immunization and phage display technology. Second, The best clone GPN2 was fused with nanoluciferase as a dual-functional immunoreagent to establish an ultra-sensitive bioluminescence enzyme immunoassay (BLEIA), which is 30 and 5 times more sensitive than the traditional colorimetric assay and fluorescent assay, respectively. The cross-reactivity analysis of BLEIA showed that there was no cross-reactivity with HCC related tumor markers AFP, CEA, CA19-9 and GPC1/GPC2. The limit of detection (LOD) of developed BLEIA was 1.5 ng/mL, which assured its application in the diagnosis of GPC3 in 94 serum samples. This study indicates that BLEIA based on nanobody-nanoluciferase fusion could be used as a useful tool for the diagnosis of HCC patients.

An Untargeted Metabolomic Approach for Microphytobenthic Biofilms in Intertidal Mudflats

Microphytobenthic (MPB) biofilms in intertidal muddy sediments play important ecological functions in coastal ecosystems. These biofilms are mainly composed of epipelic diatoms but also prokaryotes, with a dominance of bacteria, which excrete diverse extracellular polymeric substances (EPS) according to their environment. While numerous studies have investigated the main components of these EPS matrices via traditional colorimetric assays, their fine composition, notably in specialized metabolites, is still largely unknown. A better chemical characterization of these MPB biofilms is necessary, especially regarding the numerous functions their chemical components play for microorganisms (e.g. motility, cell protection, defense mechanisms, chemical communication), but also for coastal systems (e.g. primary production, sediment stabilization, larval settlement of some invertebrates with high economical value). An alternative approach to traditional analyses is the use of untargeted metabolomic techniques, which have not yet been applied to such MPB biofilms. The objectives of the present study were to (a) propose a protocol for metabolic fingerprinting by LC-MS and GC-MS for metabolites analysis in polar and non-polar fractions in MPB biofilms extracted from mudflat sediment and to (b) apply this protocol to a case study: the effect of light exposure on the metabolomic fingerprint of the MPB biofilm community. We compared three extraction methods using different mixes of solvents and selected a methanol/chloroform mix (1:1), which gave better results for both techniques and fractions. We then applied the selected protocol to our case study using a short-term light exposure experiment in aquaria (7 days). The present study is the first using a detailed untargeted metabolomic approach on MPB biofilms from mudflat sediment and will provide a solid baseline for further work in this area

Development of a novel reporter gene assay for platelet-derived growth factor-BB bioactivity

Platelet-derived growth factors (PDGFs) are involved in various physiological and pathological processes, making them important targets for drug development. However, current methods for measuring PDGF bioactivity do not meet the rapidly growing requirements of pharmaceutical research. Here, we describe a novel reporter gene assay (RGA) for PDGF-BB activity measurement. RGA was developed with engineered cells expressing a modified luciferase protein under the control of an SRE element. With PDGF-BB stimulation, cells produced stable dose-dependent signals with a correlation coefficient of R2 > 0.97 within several hours. The relative accuracy of the assay, represented by the relative bias for five independent samples with different bioactivity levels, was less than 4.67%. Variations in RGA caused by intra- and inter-assay factors were smaller than 10% and 15%, respectively. RGA not only yielded consistent results for estimating PDGF-BB activity, but also presented significantly lower variations than did the traditional colorimetric assay. Moreover, RGA could be completed within 2 days, showing a much higher efficiency than the WST method, which requires 4–5 days. Furthermore, RGA is suitable for other PDGF species besides PDGF-BB. Our results demonstrate that RGA could be a powerful tool for screening and identifying PDGF-related drug candidates in pharmaceutical applications.

Development and Validation of an Analytical Method for Total Polyphenols Quantification in Extra Virgin Olive Oils

Phenolic compounds in olive oil are considered interesting, both for their effect on human wellness and for their role in the shelf life of olive oils. Many methods have been suggested for quantitative analysis of phenolic compounds in extra virgin olive oil, which determine that results are not always comparable; therefore, it is necessary to have simple, inexpensive, robust, and validated methods for official routine analyses. In this work, solid phase extraction (SPE) was carried out to isolate polyphenols from olive oil and the traditional colorimetric assay (Folin-Ciocalteu method) was used to quantify them. SPE/colorimetric method was validated for total polyphenols official determinations in extra virgin oils. The proposed method was robust, precise, and accurate. Polyphenol recovery was excellent ≥ 95% in 100–500 ppm measuring range.

Self-Referenced Smartphone-Based Nanoplasmonic Imaging Platform for Colorimetric Biochemical Sensing.

Colorimetric sensors usually suffer due to errors from variation in light source intensity, the type of light source, the Bayer filter algorithm, and the sensitivity of the camera to incoming light. Here, we demonstrate a self-referenced portable smartphone-based plasmonic sensing platform integrated with an internal reference sample along with an image processing method to perform colorimetric sensing. Two sensing principles based on unique nanoplasmonics enabled phenomena from a nanostructured plasmonic sensor, named as nanoLCA (nano Lycurgus cup array), were demonstrated here for colorimetric biochemical sensing: liquid refractive index sensing and optical absorbance enhancement sensing. Refractive indices of colorless liquids were measured by simple smartphone imaging and color analysis. Optical absorbance enhancement in the colorimetric biochemical assay was achieved by matching the plasmon resonance wavelength with the chromophore's absorbance peak wavelength. Such a sensing mechanism improved the limit of detection (LoD) by 100 times in a microplate reader format. Compared with a traditional colorimetric assay such as urine testing strips, a smartphone plasmon enhanced colorimetric sensing system provided 30 times improvement in the LoD. The platform was applied for simulated urine testing to precisely identify the samples with higher protein concentration, which showed potential point-of-care and early detection of kidney disease with the smartphone plasmonic resonance sensing system.

High content screening as high quality assay for biological evaluation of photosensitizers in vitro.

A novel single step assay approach to screen a library of photdynamic therapy (PDT) compounds was developed. Utilizing high content analysis (HCA) technologies several robust cellular parameters were identified, which can be used to determine the phototoxic effects of porphyrin compounds which have been developed as potential anticancer agents directed against esophageal carcinoma. To demonstrate the proof of principle of this approach a small detailed study on five porphyrin based compounds was performed utilizing two relevant esophageal cancer cell lines (OE21 and SKGT-4). The measurable outputs from these early studies were then evaluated by performing a pilot screen using a set of 22 compounds. These data were evaluated and validated by performing comparative studies using a traditional colorimetric assay (MTT). The studies demonstrated that the HCS assay offers significant advantages over and above the currently used methods (directly related to the intracellular presence of the compounds by analysis of their integrated intensity and area within the cells). A high correlation was found between the high content screening (HCS) and MTT data. However, the HCS approach provides additional information that allows a better understanding of the behavior of these compounds when interacting at the cellular level. This is the first step towards an automated high-throughput screening of photosensitizer drug candidates and the beginnings of an integrated and comprehensive quantitative structure action relationship (QSAR) study for photosensitizer libraries.

Real-time cell-microelectronic sensing of nanoparticle-induced cytotoxic effects

We report a real-time cell analysis (RTCA) sensing method of 96 electronic microwells for profiling the cytotoxicity of nanoparticles on different cell lines. The method consists of 96 microwells embedded with microelectrodes (96x E-plate) to measure impedance changes of adherent cell lines. When the testing cells change in population, adhesion, and/or morphology, the impedance at the cell–electrode interface changes to provide real-time monitoring of overall cell status. To demonstrate this technique, we used three cell lines as sensing probes: two human lung carcinoma cell lines, A549 and SK-MES-1, and a normal mammalian cell line, CHO-K1. We tested two well-characterized nanoparticles: nano-titanium dioxide (nTiO2) and nano-silver (nAg). The three cell lines were separately seeded into 96x E-plates and treated with varying concentrations of nanoparticles (0.078–160μgmL−1). This method provides dynamic cell response profiles and temporal IC50 histograms, showing concentration-, time-, particle-, and cell-dependent cytotoxicity. The 24h and 48h IC50 values of nAg obtained using both the RTCA and the neutral red uptake (NRU) assays were in good agreement, validating the RTCA technique. The RTCA assay does not suffer interference from nTiO2, whereas the NRU assay cannot be used due to severe interference from nTiO2. A cytostatic response was observed in CHO-K1 cells after 24h exposure to 40μgmL−1 nTiO2, which was correlated with S-phase cell cycle arrest based on cell cycle analysis using flow cytometry. This suggests that the shapes of the response curves provide indicative information, directing further studies into the mode of action of the toxicant. Advantages of the RTCA technique over traditional colorimetric assays for screening the cytotoxicity of nanoparticles include minimizing interference, qualitative and quantitative cytotoxicity data, and the capability of real-time and high-throughput measurements.

Diversity in Production of Xylan-Degrading Enzymes Among Species Belonging to the Trichoderma Section Longibrachiatum

Xylan is an important part of plant biomass and represents a renewable raw material for biorefineries. Contrary to cellulose, the structure of hemicellulose is quite complex. Therefore, the biodegradation of xylan needs the cooperation of many enzymes. For industrial production of xylanase multienzyme complexes (cocktails) and selected monocomponent xylanases, different Trichoderma reesei mutants and recombinants are used. T. reesei QM 6a (wild-type parent of best existing mutants) was selected as a starting material in the 1960s when the modern in-depth analytical methods were not yet in use. Therefore, screening of fungi genetically close to T. reesei in biodegradation of xylan may have a scientific value. Fifteen different strains from Trichoderma section Longibrachiatum have been tested for extracellular xylan-degrading enzyme production on three carbon sources (wheat straw, corn fiber, and eucalyptus wood) in shake flask cultivation. The enzyme activities were evaluated by traditional colorimetric enzyme assays and by HPLC and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Degradation of xylan was studied on four different xylan-rich model substrates. T. reesei CPK 155, Trichoderma parareesei TUB F-2535, and Trichoderma gracile TUB F-2543 isolates were equally good or better in degradation of the wheat arabinoxylan (WAX) and corn fiber alcohol insoluble solids as hydolysis substrates than the well-known T. reesei QM 6a and RUT C30 strains. Though Trichoderma saturnisporum ATCC 18903 gave relatively low volumetric enzyme activities by traditional colorimetric assays, it could release quite large amount of hydrolysis products (mono- and oligosaccharides) from WAX. Therefore, these fungi may be potential candidates for further experiments. Enzyme production on wheat straw and corn fiber carbon sources was more effective than on eucalyptus wood.

Ferric and cupric reductase activities by iron‐limited cells of the green alga Chlorella kessleri: quantification via oxygen electrode

The colorimetric Fe2+ indicators bathophenanthroline disulfonic acid (BPDS) and 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine (FZ) are routinely used to assay for plasma membrane ferric reductase activity in iron-limited algal cells and also in roots from iron-limited plants. Ferric reductase assays using these colorimetric indicators must take into account the fact that Fe3+ chelators (e.g. ethylenediaminetetraacetic acid) can also in general bind Fe2+ and may therefore compete with the colorimetric Fe2+ indicators, leading to the potential for underestimation of the ferric reduction rate. Conversely, the presence of BPDS or FZ may also facilitate the reduction of Fe3+ chelates, potentially leading to overestimation of ferric reduction rates. Last, both BPDS and FZ have non-negligible affinities for Fe3+ in addition to their well-known affinities for Fe2+; this leads to potential difficulties in ascertaining whether free and/or chelated Fe3+ are potential substrates for the ferric reductase. Similar issues arise when assaying for cupric reductase activity using the colorimetric Cu+ indicator bathocuproinedisulfonic acid (BCDS). In this paper, we describe an oxygen-electrode-based assay (conducted in darkness) for both ferric and cupric reductase activities that does not use colorimetric indicators. Using this assay system, we show that the plasma membrane metal reductase activity of iron-limited cells of the green alga Chlorella kessleri reduced complexed Fe3+ (i.e. Fe3+ chelates) but did not reduce free (non-chelated) Fe3+, and also reduced free Cu2+ to Cu+, but did not reduce Cu2+ that was part of Cu2+ chelates. We suggest that the potential for reduction of free Fe3+ cannot be adequately assayed using colorimetric assays. As well, the BPDS-based assay system consistently yielded similar estimates of ferric reductase activity compared with the O2-electrode-based assays at relatively low Fe3+ concentration, but higher estimates at higher Fe3+ concentrations with chelators other than desferrioxamine mesylate. With respect to cupric reductase activity, the O2 electrode consistently provided much higher estimates; we suggest that this was as a result of Cu2+ chelation by BCDS leading to a large underestimation of the true cupric reduction rate. These results suggest that an O2-electrode-based metal reductase assay system has some specific advantages compared with the traditional colorimetric assay system, including especially the ability to discriminate between the reduction of free metal ions and chelated metal ions.

Development of microformat imaging microplate and membrane immunoenzyme assays of the herbicide atrazine

A microformat imaging technique in combination with registration of chemiluminescence by a charge-coupled device, CCD camera, were applied for the detection of the herbicide atrazine by two immunoenzyme assay formats – microplate and membrane assays. For the microplate (ELISA) case the detection limit was 95 pg mL−1, giving a 9.5-fold enhancement in comparison with a traditional colorimetric assay. The membrane dot blot assay allowed detection of up to 40 pg mL−1 of atrazine (4.5-fold enhancement). Due to the application of polyelectrolyte carriers for rapid reactants separation the membrane assay may be realized in 15 min. CCD-based registration of signals assures a high reproducibility of measurements; the variation coefficient is in the range 1–7%. The effectiveness of the assays developed for controlling triazine herbicides in mineral and tap water has been demonstrated.

Comparability and reliability of different techniques for the determination of phenolic compounds in virgin olive oil

AbstractPhenolic compounds represent a unique and functional part of the polar fraction of virgin olive oils. Many different approaches for the analysis of these compounds have been published, which has led to ambiguous results that are difficult to compare. In order to explain the controversial data reported in the literature, extraction techniques (solid‐phase extraction, SPE, and liquid‐liquid extraction, LLE), methods of analysis (HPLC and colorimetric assay) and quantification methods have been re‐investigated with genuine olive oil phenols. The optimized LLE system led to high recovery of the nine major olive oil phenolics (93%) and, in addition, was at least as good as the SPE technique in view of costs, solvent and sample consumption, and analysis time. SPE was shown to be problematic because of the selectivity towards the individual phenolics, particularly the aglycone‐type ones. The proposed LLE/HPLC method was compared with the traditional colorimetric assay (Folin‐Ciocalteu method) by analyzing 23 samples of virgin olive oils. A strong correlation between both methods has been found, suggesting that the colorimetric assay is reasonably valid for a rough prediction of the total phenolic content. In the literature, the level of phenolics is expressed in several different units (reference compound equivalents in case of colorimetric measurements and ppm in HPLC measurements). As these units can differ in orders of magnitude, it is necessary to convert the data to a common base before comparing or combining them.

Polynucleotide phosphorylase-based photometric assay for inorganic phosphate

Polynucleotide phosphorylase is a prokaryotic enzyme that catalyzes phosphorolysis of polynucleotides with release of nucleotide diphosphates. By taking advantage of this property, we developed a photometric assay for inorganic phosphate. In the presence of polyadenylic acid, phosphate is converted into adenosine 5 ′-diphosphate (ADP) by this enzyme. ADP then reacts with phospho enolpyruvate in a pyruvate kinase-catalyzed reaction, thus giving rise to adenosine 5 ′-triphosphate and pyruvate. Finally, pyruvate oxidizes reduced nicotinamide adenine dinucleotide (NADH) through the action of l-lactate dehydrogenase, with concomitant decrease in absorbance at 340 nm. As expected, in this detection system 1 mol of NADH was oxidized per mole of phosphate. The assay showed an excellent reproducibility, as the standard deviations never exceeded 5%. It also was shown to be unaffected by several compounds that are regarded as major interferents of the traditional colorimetric assays. Absence of interference was also demonstrated when determining phosphate content in different biological samples, such as human serum and perchloric acid extracts from Escherichia coli, yeast, and bovine liver. An E. coli strain overexpressing His-tagged polynucleotide phosphorylase developed in our laboratories allowed quick and straightforward purification of enzyme, making the assay feasible and convenient. Since all other reagents required are inexpensive, the assay represents a cheaper alternative to commercially available phosphate assay kits.

Design and Use of Background-Reduced 27Al NMR Probes for the Study of Dilute Samples from the Environment

Development of methods for the detection and measurement of aluminum (Al) is crucial for our understanding of Al(III) chemistry and toxicity in natural waters, soil solutions, and environmental samples. Traditional colorimetric assays, by their very nature, alter solution Al(III) chemistry, potentially biasing measurements. Methods based on 27Al NMR spectroscopy have the advantage of being nondestructive and of not altering the chemistry of the solution. Standard commercial NMR probes and sample tubes, unfortunately, are constructed from aluminum-containing components. These materials give substantial background signal, which is detected as a large, broad hump, overwhelming signals from dilute samples. We describe here the construction of two novel NMR probes and a sample container built from a variety of materials with low Al content. The designs feature the use of transversely mounted solenoid coils with aluminum-free sample holders. The sample container features a second chamber which can be filled with an external reference solution. These novel 27Al NMR probes are being used for the NMR spectroscopic investigation and quantitation of natural, dilute (10−6 M) Al(III) samples from the environment.

Assays for Serodiagnosis of Prostate Cancer

In this study, five serologic assays for prostate cancer were compared: immunoenzymometric assay for prostate-specific antigen (PSA-E), immunoradiometric assay for prostate-specific antigen (PSA-R), immunoenzymometric assay for prostatic acid phosphatase (PAP-E), immunoradiometric assay for prostatic acid phosphatase (PAP-R), and a traditional colorimetric assay for prostatic acid phosphatase (PAP-C). Precision, linearity, and analytical sensitivity for all the assays were good. There was excellent correlation between the two PSA assays (r=0.973), good correlation between the two immunologic tests for PAP (r=0.785), and poor correlation with the colorimetric test for PAP (r=0.041). Sera from 200 healthy male subjects were assayed, and reference intervals and decision values were established. Sera from 519 patients were assayed in a random-blind manner. The results were correlated with diagnoses which were established histologically. Samples included 189 patients with prostate cancer, 252 with benign prostatic hyperplasia, and 78 with miscellaneous genitourinary pathologies. Using the selected decision values, the diagnostic efficiencies for the assays were established as: PSA-E, 67.2%; PSA-R, 66.9%; PAP-E, 68.2%; PAP-R, 58.5%; and PAP-C, 42.9%. We eliminated from the study 94 patients who were receiving cancer therapy, so the recalculated diagnostic efficiencies were: PSA-E, 81.4%; PSA-R, 81.4%; PAPE, 81.2%; PAP-R, 74.2%; and PAPC, 57.9%.

A simple and sensitive in vivo luciferase assay for tRNA-mediated nonsense suppression

We present a rapid assay for tRNA suppression in living Escherichia coli. An amber, ochre, or opal nonsense mutation in a cloned luxB gene from the bacterium Vibrio harveyi was suppressed. Because luciferase (Lux) activity depends completely on the appearance of the full-length luxB gene product, the amount of light produced was proportional to tRNA-mediated nonsense suppression in the cell. This luminometric assay was notably quicker, easier, and more sensitive than a traditional colorimetric assay employing beta-galactosidase. Assays required only one addition to a growing culture and were complete within 1 min. Light output was directly proportional to the amount of bacterial luciferase in a sample over a range of greater than or equal to 40,000-fold. Fewer than 100 cells were required for detection of Lux with ordinary instrumentation; assays were 80-fold more sensitive than simultaneous beta-galactosidase measurements. Assayed cells survived and could be recovered as colony formers. The beta-galactosidase colorimetric assay and the luciferase assay were similarly reproducible. Light from colonies expressing Lux was visible to the dark-adapted eye and useful for screening. A rapid assay that does not depend on the formation of permanent transformants can be based on electroporation followed by luminometry.

Separation and quantitation of serum beta-carotene and other carotenoids by high performance liquid chromatography.

We describe a specific assay for serum provitamin A (alpha- and beta-carotene) by high pressure liquid chromatography (HPLC). The system separates alpha- and beta-carotene in 7.4 min using a C18 muBondapak, 10-micron particle size column with a mobile phase of acetonitrile-chloroform 92:8 at 2 ml per min and a 462 nm detector. The HPLC assay had a recovery of 94.8% of added beta-carotene and, at a serum concentration of 215.2 micrograms/L, had within-run and between-run precisions of 3.1% and 3.6%, respectively. In 65 subjects, the HPLC-determined provitamin A (alpha- and beta-carotene) value was 343 +/- 166 micrograms/L and averaged 23.4 +/- 7.9% (range 9-43%) of the values obtained by a traditional colorimetric assay for total serum "carotenes." Although total serum carotenes showed no relationship to serum vitamin A (r = -0.048; P = 0.78), HPLC-determined alpha- and beta-carotene was significantly inversely correlated (r = -0.357; P = 0.05).