Luminescent Screening Research Articles

The effects of PstR, a PadR family transcriptional regulatory factor, in Plesiomonas shigelloides are revealed by transcriptomics

BackgroundPlesiomonas shigelloides is a gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal diseases in humans. There have been reports of specific functional genes in the study of P. shigelloides, but there are also many unknown genes that may play a role in P. shigelloides pathogenesis as global regulatory proteins or virulence factors.ResultsIn this study, we found a transcriptional regulator of the PadR family in P. shigelloides and named it PstR (GenBank accession number: EON87311.1), which is present in various pathogenic bacteria but whose function has rarely been reported. RNA sequencing (RNA-Seq) was used to analyze the effects of PstR on P. shigelloides, and the results indicated that PstR regulates approximately 9.83% of the transcriptome, which includes impacts on motility, virulence, and physiological metabolism. RNA-seq results showed that PstR positively regulated the expression of the flagella gene cluster, which was also confirmed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Luminescence screening assay. Meanwhile, the ΔpstR mutant strains lacked flagella and were non-motile, as confirmed by motility assays and transmission electron microscopy (TEM). Additionally, PstR also positively regulates T3SS expression, which aids in P. shigelloides’ capacity to infect Caco-2 cells. Meanwhile, we also revealed that PstR negatively regulates fatty acid degradation and metabolism, as well as the regulatory relationship between PsrA, a regulator of fatty acid degradation and metabolism, and its downstream genes in P. shigelloides.ConclusionsOverall, we revealed the effects of PstR on motility, virulence, and physiological metabolism in P. shigelloides, which will serve as a foundation for future research into the intricate regulatory network of PstR in bacteria.

Transcriptome Analysis Reveals Cross-Talk between the Flagellar Transcriptional Hierarchy and Secretion System in Plesiomonas shigelloides.

Plesiomonas shigelloides, a Gram-negative bacillus, is the only member of the Enterobacteriaceae family able to produce polar and lateral flagella and cause gastrointestinal and extraintestinal illnesses in humans. The flagellar transcriptional hierarchy of P. shigelloides is currently unknown. In this study, we identified FlaK, FlaM, FliA, and FliAL as the four regulators responsible for polar and lateral flagellar regulation in P. shigelloides. To determine the flagellar transcription hierarchy of P. shigelloides, the transcriptomes of the WT and ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were carried out for comparison in this study. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and luminescence screening assays were used to validate the RNA-seq results, and the Electrophoretic Mobility Shift Assay (EMSA) results revealed that FlaK can directly bind to the promoters of fliK, fliE, flhA, and cheY, while the FlaM protein can bind directly to the promoters of flgO, flgT, and flgA. Meanwhile, we also observed type VI secretion system (T6SS) and type II secretion system 2 (T2SS-2) genes downregulated in the transcriptome profiles, and the killing assay revealed lower killing abilities for ΔflaK, ΔflaM, ΔfliA, and ΔfliAL compared to the WT, indicating that there was a cross-talk between the flagellar hierarchy system and bacterial secretion system. Invasion assays also showed that ΔflaK, ΔflaM, ΔfliA, and ΔfliAL were less effective in infecting Caco-2 cells than the WT. Additionally, we also found that the loss of flagellar regulators causes the differential expression of some of the physiological metabolic genes of P. shigelloides. Overall, this study aims to reveal the transcriptional hierarchy that controls flagellar gene expression in P. shigelloides, as well as the cross-talk between motility, virulence, and physiological and metabolic activity, laying the groundwork for future research into P. shigelloides' coordinated survival in the natural environment and the mechanisms that infect the host.

Reconstructing the Eemian to Middle Pleniglacial pedosedimentary evolution of the Baix loess–palaeosol sequence (Rhône Rift Valley, southern France) – basic chronostratigraphic framework and palaeosol characterisation

Abstract. Loess–palaeosol sequences (LPSs) are important archives of landscape evolution, recording alternating periods of geomorphic activity (dust deposition and slope processes) and landscape stability (soil formation). LPSs of the Rhône Rift Valley are located along a spatial climatic gradient from the mid-latitudes to the Mediterranean region. This position renders them extremely valuable archives for correlating LPSs in the European loess belt and the Mediterranean region. Despite this important function, Rhône Rift Valley LPSs have been little investigated. In this study, we aimed to narrow this knowledge gap, in a first step towards linking LPS-based mid-latitude and Mediterranean palaeoenvironmental reconstructions. We studied the ∼ 14 m thick Baix LPS located at the western edge of the Rhône Rift Valley near Valence. Here, we focus on the lower ∼ 7 m of the LPS, which comprises 12 of 19 soil horizons overall, and record regional palaeoenvironmental variations from the Eemian to the Middle Pleniglacial (∼ marine isotope stage (MIS) 5 to MIS 3). Our reconstruction is based on detailed field description, complemented by granulometry (10 cm vertical resolution), and micromorphological analyses. Luminescence screening of cut-out soil-sediment columns subsampled with ∼ 5 cm vertical resolution (126 subsamples over 7 m) provides a preliminary chronometry. The loess deposits of the Baix LPS are strongly calcareous and predominantly silty but also contain sandy and fine gravelly laminae typical of loess deposits subjected to slope-wash processes. From bottom to top, the investigated part shows three intensely pedogenised levels: (1) a basal sequence of reddish-brown Btg and Bt horizons of a thick Eemian Stagnic Luvisol (MIS 5e); (2) an early glacial yellowish-brown Bw horizon (MIS 5d-a); and (3) a yellowish-brown Bw horizon with large cone-shaped carbonate nodules in the associated underlying Bk horizon, interpreted as a truncated Middle Pleniglacial (MIS 3) Calcic Cambisol. The Baix LPS is in parts very similar to the Collias LPS ∼ 100 km further south but also exhibits differences, reflecting its position at the spatial climatic transition between the presently temperate and the Mediterranean zone.

Luminescence Screening of Copper and Gold Mixed Ligand Complexes with S-/P-Donor Atoms: An Advanced Multiweek Research Discovery Experiment for the Inorganic Chemistry Laboratory

An advanced research discovery laboratory course in inorganic chemistry has been developed to engage undergraduate students in authentic research experiences directly related to concepts and theory taught in the lecture portion of the course. The 12-week laboratory involves an initial 4 weeks of training experiments on inorganic/organometallic synthesis in air and under inert atmosphere, followed by an 8-week series of research discovery experiments designed to mirror research in a graduate- and/or industry-level inorganic chemistry laboratory. Students are first presented with a research problem related to a coinage metal (group 11: copper, silver, or gold). They are then asked to (1) design a synthetic route using peer-reviewed research articles from the literature with some modification as needed; (2) conduct their proposed synthetic reactions; and (3) characterize products to assess purity and determine the molecular structure, as well as screen their properties toward potential applications.

Determining the Dose-Response Curve of Exoelectrogens: A Microscale Microbial Fuel Cell Biosensor for Water Toxicity Monitoring.

Nowadays, the development of real-time water quality monitoring sensors is critical. However, traditional water monitoring technologies, such as enzyme-linked immunosorbent assay (ELISA), liquid chromatography, mass spectroscopy, luminescence screening, surface plasma resonance (SPR), and analysis of living bioindicators, are either time consuming or require expensive equipment and special laboratories. Because of the low cost, self-sustainability, direct current output and real-time response, microbial fuel cells (MFCs) have been implemented as biosensors for water toxicity monitoring. In this paper, we report a microscale MFC biosensor to study the dose–response curve of exoelectrogen to toxic compounds in water. The microscale MFC biosensor has an anode chamber volume of 200 μL, which requires less sample consumption for water toxicity monitoring compared with macroscale or mesoscale MFC biosensors. For the first time, the MFC biosensor is exposed to a large formaldehyde concentration range of more than 3 orders of magnitudes, from a low concentration of 1 × 10−6 g/L to a high concentration of 3 × 10−3 g/L in water, while prior studies investigated limited formaldehyde concentration ranges, such as a small concentration range of 1 × 10−4 g/L to 2 × 10−3 g/L or only one high concentration of 0.1 g/L. As a result, for the first time, a sigmoid dose–response relationship of normalized dose–response versus formaldehyde concentration in water is observed, in agreement with traditional toxicology dose–response curve obtained by other measurement techniques. The biosensor has potential applications in determining dose–response curves for toxic compounds and detecting toxic compounds in water.

Abstract 1117: Investigating PD-1/PD-L1 checkpoint inhibition as a treatment for platinum-resistant ovarian cancer

Abstract Ovarian cancer is the deadliest cancer of the female reproductive system, with the majority of patients experiencing relapse on first-line platinum-based chemotherapies. Identifying potential targets for additional second-line therapies is critical for treating platinum-resistant ovarian cancer. To that end, the evolution of platinum resistance in a cell-based model of ovarian cancer was monitored via characterization of proteomic changes in cancer-derived extracellular vesicles (EVs) after progressive rounds of cisplatin treatment. The content of cancer-derived EVs was analyzed via reverse phase protein array, mass spectrometry, and Western blotting to identify pathways that could be targeted by second-line therapies. Several therapeutically relevant markers were upregulated, including phospho-EGFR (Y1068), multidrug transporter ABCE1, and PD-L1. Monoclonal antibody-based therapies (mAbs) targeting PD-1/PD-L1 have been deployed successfully as second-line treatments for a variety of cancers, but their success has yet to be translated to ovarian cancer. Current mAb-based immunotherapies suffer from several pitfalls including poor tissue penetration, a long-standing issue in ovarian cancer treatment. To offer an alternative to mAb-based immunotherapies, we developed a small interfering peptide consisting of a portion of the PD-L1 interface sequence. This lead peptide underwent several rounds of in silico optimization via molecular dynamics and modeling simulations to identify modifications that could improve affinity for PD-1 or stability in serum. When efficacy was assessed in vitro via Amplified Luminescent Proximity Homogeneous Assay screening, the modifications to the lead peptide were not shown to improve efficacy. However, serum stability studies indicated the modifications dramatically improved peptide stability, with detectable levels still quantifiable via mass spectrometry after 48 hours. The stabilized peptide, titled MN1.4, was chosen for further efficacy testing in cell-based models. A cisplatin-treated daughter line from the OVCAR8 high grade serous ovarian carcinoma cell line was created to model the effects of clinical platinum-based chemotherapy treatments on the most common ovarian cancer subtype. OVCAR8 cells were co-cultured in the presence of stimulated Jurkat T cells to measure markers of T cell activation, including IL-2, via ELISA after treatment with MN1.4 in the presence or absence of EVs derived from cisplatin-treated cells. This model allows for examination of the potential sensitization effects of first-line cisplatin treatment on PD-1/PD-L1 immunotherapy, and how cancer-derived EVs from cisplatin-treated cells can abrogate this effect. We conclude that exosomal PD-L1 may impair responses to PD-1/PD-L1 immunotherapy and should be considered when assessing the viability of this treatment option for ovarian cancer patients. Citation Format: Rachel Carter, Marissa Howard, James Erickson, Paul Russo, Alessandra Luchini, Amanda Haymond, Fatah Kashanchi, Mikell Paige, Lance Liotta. Investigating PD-1/PD-L1 checkpoint inhibition as a treatment for platinum-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1117.

PDE-5 inhibitors in selected herbal supplements from the Ghanaian market for better erectile function as tested by a bioassay

Herbal supplements sold as ‘all natural’ on various markets in Accra (Ghana) and advertised as highly efficacious in treating erectile dysfunction (ED) were bought and analysed by a PDE-5 enzyme inhibition assay. The claimed efficacy of these products could be the result of inherent plant constituents, but also of intentionally added pharmaceuticals. Medically, ED is treated with potent inhibitors of the phosphodiesterase-5 (PDE-5) enzyme, as in the case of sildenafil. To test the efficacy of the Ghanaian supplements, extracts were made and tested using a PDE-Glo phosphodiesterase assay, a luminescent high-throughput screening (HTS) method.Results revealed that about 90% of the selected samples were able to inhibit PDE-5 activity to a high extent. Estimated concentrations in sildenafil equivalents ranged from traces to very high, with 25 samples (62.5%) pointing at daily doses higher than 25 mg sildenafil equivalents and 9 (22.5%) of these at doses higher than the maximal recommended daily intake of 100 mg sildenafil equivalents. Further investigations are needed to confirm if the observed effects are due to inherent plant constituents or merely the result of added synthetic PDE-5 enzyme inhibitors, especially because doses above 100 mg sildenafil equivalents per day may result in severe health risks.

Time-Resolved Luminescent High-Throughput Screening Platform for Lysosomotropic Compounds in Living Cells.

Lysosomes are membrane-bound organelles that regulate protein degradation and cellular organelle recycling. Homeostatic alteration by lysosomotropic compounds has been suggested as a potential approach for the treatment of cancer. However, because of the high false-negative rate resulting from strong fluorescent background noise, few luminescent high-throughput screening methods for lysosomotropic compounds have been developed for cancer therapy. Imidazole is a five-membered heterocycle that can act within the acidic interior of lysosomes. To develop an efficient lysosomotropic compound screening system, we introduced an imidazole group to iridium-based complexes and designed a long-lifetime lysosomal probe to monitor lysosomal activity in living cells. By integrating time-resolved emission spectroscopy (TRES) with the novel iridium-based lysosomal probe, a high-throughput screening platform capable of overcoming background fluorescent interference in living cells was developed for discovering lysosomotropic drugs. As a proof-of-concept, 400 FDA/EMA-approved drugs were screened using the TRES system, revealing five compounds as potential lysosomotropic agents. Significantly, the most promising potent lysosomotropic compound (mitoxantrone) identified in this work would have showed less activity if screened using a commercial lysosomal probe because of interference from the intrinsic fluorescence of mitoxantrone. We anticipate that this TRES-based high-throughput screening system could facilitate the development of more lysosomotropic drugs by avoiding false results arising from the intrinsic fluorescence of both bioactive compounds and/or the cell background.

Non-Destructive Luminescence-Based Screening Tool for Listeria monocytogenes Growth on Ham.

Listeria monocytogenes is a food-borne pathogen often associated with ready-to-eat (RTE) food products. Many antimicrobial compounds have been evaluated in RTE meats. However, the search for optimum antimicrobial treatments is ongoing. The present study developed a rapid, non-destructive preliminary screening tool for large-scale evaluation of antimicrobials utilizing a bioluminescent L. monocytogenes with a model meat system. Miniature hams were produced, surface treated with antimicrobials nisin (at 0–100 ppm) and potassium lactate sodium diacetate (at 0–3.5%) and inoculated with bioluminescent L. monocytogenes. A strong correlation (r = 0.91) was found between log scale relative light units (log RLU, ranging from 0.00 to 3.35) read directly from the ham surface and endpoint enumeration on selective agar (log colony forming units (CFU)/g, ranging from 4.7 to 8.3) when the hams were inoculated with 6 log CFU/g, treated with antimicrobials, and L. monocytogenes were allowed to grow over a 12 d refrigerated shelf life at 4 °C. Then, a threshold of 1 log RLU emitted from a ham surface was determined to separate antimicrobial treatments that allowed more than 2 log CFU/g growth of L. monocytogenes (from 6 log CFU/g inoculation to 8 log CFU/g after 12 d). The proposed threshold was utilized in a luminescent screening of antimicrobials with days-to-detect growth monitoring of luminescent L. monocytogenes. Significantly different (p < 0.05) plate counts were found in antimicrobial treated hams that had reached a 1 log RLU increase (8.1–8.5 log(CFU/g)) and the hams that did not reach the proposed light threshold (5.3–7.5 log(CFU/g)). This confirms the potential use of the proposed light threshold as a qualitative tool to screen antimicrobials with less than or greater than a 2 log CFU/g increase. This screening tool can be used to prioritize novel antimicrobials targeting L. monocytogenes, alone or in combination, for future validation.

Crystal engineering of an adenine-decavanadate molecular device towards label-free chemical sensing and biological screening.

Due to the inherent geometrical interdependencies of nucleic acid structures, the ability to engineer biosensors that rely on the specific interactions of these compounds is of considerable importance. Additionally, sensing or screening in a label-free fashion is a capability of these structures that can be readily achieved by exploiting the fluorescent component. In this work, the [AdH]6[V10O28].4(H2O) (1) supramolecular structure is introduced using adenine and decavanadate moieties that allow probing of selectivity to specific nucleic acid binding events by optical changes. The structure of (1) is an alternating organic-inorganic hybrid architecture of cationic adeninium (AdH+) ribbons and anionic decavanadate (DV)-water sheets. The luminescent screening and anticancer activity of compound (1) on the two human mammary carcinoma cell lines MDA-MB-231 and MCF7 were investigated using fluorescent microscopy and MTT assays, respectively. It was found that compound (1) is cell permeable with no toxicity below 12.5 µM concentration and moderate cytotoxicity at concentrations as high as 200 µM in human breast cancer cell lines, making it a useful tool to study the cell nucleus in real time.

Upconversion Luminescence Sandwich Assay For Detection of Influenza H7 Subtype.

With large anti-Stokes shifts and background-free signals, upconversion luminescent (UCL) screening assays have been a promising method to reduce the transmission of influenza epidemic, which can critically alleviate the disease burden and extra annual deaths. In this work, a luminescent resonance energy transfer sandwich assay is developed, which utilizes core-shell upconversion nanoparticles and gold nanoparticles as the donor and acceptor, respectively. The influenza H7 gene of H7N9 virus is used as the target for optimization of the assay. Importantly, the hybridization time of the assay is ≈40 min and the specificity test indicates the probes are specific toward the H7 target. The limit of detection of the system is ≈134 × 10-12 m (≈3.22 × 1010 molecules). Moreover, the assay is tested with the use of polymerase chain reaction validated samples from human isolates. The results are promising for implanting future on-site rapid influenza screening application.

Establishing a chronological framework for a late Quaternary seasonal swamp in the Australian ‘Top End’

Abstract Swamps in the seasonal tropics have good potential for the reconstruction of late Quaternary monsoonal dynamics. Their successful use, however, has often been compromised by chronological limitations introduced by a variety of depositional and post-depositional processes actively modifying the swamp deposits. We here present and discuss the results of a multiple dating approach at Table Top Swamp (TTS) in northern Australia (the ‘Top End’). Single-grain luminescence dating of quartz was successfully used to provide chronology in the lowermost core where insufficient organic material prevents the application of radiocarbon dating. In the uppermost, fine-grained and peaty section of the core, two different organic fractions (pollen concentrate and humins) were dated with AMS radiocarbon yielding significantly different chronologies. While this could point to the incorporation of younger pollen into the profile along seasonal dry cracks, older humins may also move up in the profile due to vertical mixing. Additional, spatially highly resolved measurements of the bulk OSL signal (Ln and Ln/Tn) combined with data on down-core variation in K, Th, and U concentration, grain size and moisture content were used to (i) guide the development of an age-depth relationship (i.e. age model) for the entire core based on three different data input scenarios, and (ii) test the applicability of novel luminescence screening techniques in seasonal swamp settings. Results suggest only minor differences among the applied models and scenarios, providing an overall reliable representation of the depositional history in the swamp. Even though all resulting age-depth models have relatively large uncertainties in the lower part of the core, there are significant changes in sedimentation rate over time, providing a chronological basis for a more detailed palaeoenvironmental analysis at TTS. The approach used may also be useful in developing age models in other complex environments, and has shown the importance of understanding carbon pathways as well as controls on luminescence signals when developing age models.

Regioselective Synthesis of 2-Substituted Indoles from Benzotriazoles and Alkynes by Photoinitiated Denitrogenation

Herein, we describe a photoinitiated and regioselective synthesis of 2-substituted indoles under mild reaction conditions. This biologically privileged scaffold was accessed in good yields from N-aroylbenzotriazoles, a quencher class previously identified using our mechanism-based luminescence screening, and terminal alkynes in the presence of a photocatalyst and blue light irradiation. This straightforward protocol displays a broad substrate scope and functional group tolerance. Furthermore, the mildness and robustness of the reaction were assessed by the application of an additive-based robustness screen. The determination of the reaction quantum yield and Stern–Volmer studies support the proposed photoinitiated radical chain mechanism.

Diverse Visible-Light-Promoted Functionalizations of Benzotriazoles Inspired by Mechanism-Based Luminescence Screening.

Three new visible-light-promoted functionalizations of benzotriazole substrates were discovered using a mechanism-based screening method. ortho-Thiolated, borylated, and alkylated N-arylbenzamide products were obtained under mild reaction conditions in a new denitrogenative synthetic approach to functionalized aniline derivatives. The functional group tolerance of the borylation reaction was further analyzed in the first application of an additive-based robustness screen in a photocatalytic transformation. All the functionalizations proceed via photocatalytically initiated chain mechanisms as indicated by determination of the reaction quantum yields and Stern-Volmer analyses.

ATP-Bioluminescence as a method to evaluated microbiological quality of UHT milk

New approaches are needed to quickly indicate possible contamination of UHT milk, among them the technique of ATP-Bioluminescence. Therefore, the aim of this study was to compare the results of culture methods with the results of ATP-Bioluminescence technique of 102 UHT whole milk samples incubated at 48, 72, and 168 hours. UHT milk samples were analyzed for the presence of mesophilic and psychrotrophic aerobic microorganisms using Plate Count Agar (PCA), Brain-Heart Infusion (BHI) media and PetrifilmTM Aerobic Count (AC) plates. The ATP-Bioluminescence technique was applied through the Microbial Luminescent Screening (MLS) system. Significant correlations were found between counts of aerobic mesophilic microorganisms on PCA, PetrifilmTM AC, BHI and results of ATP bioluminescence technique (P≤0.05). The ATP-Bioluminescence technique had higher correlation with counting method in PCA than BHI media. At lower pass/fail limits of Relative Light Units (60, 50, 45 and 40 RLU), the number of samples identified as positive increased and statistically agreed with aerobic mesophilic microorganism counts (P&gt;0.05). For the dairy industry, the ATP-Bioluminescence technique may become an important tool that assists the official methods to quickly monitor the microbiological quality of UHT milk though this will likely require a threshold below 150 RLU.

Toxicity of carbon-based nanomaterials against Escherichia coli depends on dispersion efficacy of their water suspensions

The relationships between surface wettability, dispersion efficacy in water suspensions and biotoxicity of nine carbon-based nanomaterials (CBN) samples represented by nanotubes, nanofibres and fullerenes are established. It is shown that presence of polar groups on the surface of similar in structure CBNs increases their hydrophilicity, reduces the particles size in water suspensions, and manifests itself by growth of the toxicity level in the luminescent screening assay based on Escherichia coli with the cloned luxCDABE-genes Photobacterium leiognathi. The artificial increase in individual CBN dispersion efficacy by primary suspension in the aprotic solvent dimethyl sulfoxide with the following transfer into an aqueous environment, also has led to growth of the registered biological activity. At the same time, the dispersion efficacy of morphologically diverse CBN is not the main cause of distinctions in their biotoxicity..

Terbium-sensitised luminescence screening method for fluoroquinolones in beef serum

Enrofloxacin and danofloxacin are the only fluoroquinolone antibiotics approved for use in cattle in the United States. Microbial screening methods commonly used for monitoring veterinary drug residues are not sensitive or selective for fluoroquinolones. In this work, a luminescence-based screening assay was developed to detect fluoroquinolones in beef serum. This approach takes advantage of the DNA-enhanced luminescence signal of a fluoroquinolone–Tb+3 complex. In this method, serum samples were extracted with acidified acetonitrile in the presence of magnesium sulfate. After centrifugation, evaporation of the supernatant was followed by dissolution of the residue in buffer and filtration. Addition of Tb+3 and DNA then allowed a reading of the luminescence signal. The technique was illustrated using enrofloxacin, and provided good recoveries (73–88%) at 25, 50 and 100 ng ml−1, with reasonable RSDs averaging at 11%. The LOD was 2.5 ng ml−1 based on the variability of response of control serum samples from 18 different steers. The method provided no false-positive or false-negative results while screening blind samples for enrofloxacin and was demonstrated to be quantitative over a range of 0–100 ng ml−1.

Luminescence Screening of Enrofloxacin and Ciprofloxacin Residues in Swine Liver after Dispersive Liquid–Liquid Microextraction Cleanup

A luminescence method was developed to screen residues of enrofloxacin (ENRO) and its metabolite, ciprofloxacin (CIPRO), in swine liver. Target analytes were extracted in acetonitrile/1.5% trifluoroacetic acid/NaCl, cleaned up by dispersive liquid-liquid microextraction (DLLME), and finally detected by terbium-sensitized luminescence (TSL) using a time-resolved luminescence photometer. CIPRO yielded slightly lower TSL response than ENRO, so a common threshold was derived from CIPRO and applied to both fluoroquinolones. Among 37 samples randomly spiked with CIPRO or ENRO up to 1 μg/g, all 19 samples spiked above the 500 ng/g tolerance were correctly screened as positive with no false negatives, but 3 of 18 samples spiked below 500 ng/g were classified as positive. This method minimized the use of chlorinated solvents and significantly improved sample throughput.

Luminescence Screening Assays for the Identification of Sensitizers for Lanthanides Based on the Controlled Formation of Ternary Lanthanide Complexes with DTPA–Bisamide Ligands

The formation of ternary complexes of lanthanide-diethylenetriamine pentaacetic acid (DTPA)-bisamide complexes with different aromatic acids and their application in luminescent screening assays are presented. The europium complexes of DTPA-bisethylamide (BEA), DTPA-bisbutylamide (BUA), DTPA-bis(2-norbornyl) (NBA), and DTPA-bis(1-adamantyl) (ADA) have been isolated and the sensitization of emission upon 1:1 formation with aromatic acids has been studied by luminescence spectroscopy. The ternary complexes show stronger luminescence with picolinate (PCA) rather than phthalate (PTA) or benzoate (BZA), with the latter forming 1:2 complexes. Isophthalate and dipicolinate sensitizers do not show a 1:1 formation of the ternary complexes. Electrospray mass spectra show characteristic peaks that confirm the formation of the ternary complexes, and NMR spectroscopic studies demonstrated a conformational locking effect upon formation of the ternary complex. It is shown that europium complexes with bulkier amide arms provide complexes with stronger luminescence enhancement that is clearly attributable to the bulkiness of the amide arms. In a luminescence screening experiment, different substitutions on BZA and PCA were examined. Sensitizers with a long alkyl chain show greater luminescent enhancements than the nonsubstituted acids, which can be attributed to an "umbrella" effect of the alkyl chain that protects the europium luminescent center from the quenching of secondary high-energy vibrations, in particular O-H from water molecules. The same effect is presented for the quinoline derivatives as sensitizers. Quinoline derivatives with salicylate binding units were identified as the best sensitizers when combined with EuADA, which is even bulkier than EuNBA, with HQ-3COO showing a 50-fold enhancement of emission upon formation of 1:1 complexes.

Time-Resolved Luminescence Screening of Antibiotics in Tissue Matrices without Centrifugation and Filtration: Spiked Recovery Studies

Analyses of chemical residues in animal tissue matrices require multistep sample preparation. To simplify this process, a methodology was developed that combines sorbent extraction and solid-matrix time-resolved luminescence (TRL); it was applied to tetracycline screening in milk. Reported here is an effort to extend its application to tissue matrices, illustrated by oxytetracycline (OTC) screening in catfish muscle. Extraction and enrichment are accomplished by immersing small C18 sorbent strips into tissue homogenates for 20 min, followed by a 3 min rinse in water and a 2 min dip in a reagent solution. After desiccation, TRL is measured directly on the sorbent surface. Tissue particulates no longer interfere via attenuation or scattering, rendering centrifugation and filtration unnecessary. The integrated TRL intensity shows a linear dependence on OTC concentration in the 0-8 microg/g range (R2 = 0.9992) with a 0.026 microg/g limit of detection. To screen OTC at 2 microg/g, the U.S. regulatory tolerance level, a threshold is established at x2-3sigma2, where x2 and sigma2 are the mean and standard deviation, respectively, of the TRL signals from 15 samples fortified at 2 microg/g. Among 45 blind samples randomly fortified at 0-4 microg/g, 41 were screened correctly and 4 negative samples were presumed positive. This method has the potential to improve throughput and save assay costs by eliminating acids, organic solvents, centrifugation, and filtration.