Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic Staphylococcus aureus.

We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, Staphylococcus aureus (S. aureus) was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific S. aureus was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to S. aureus via in situ hybridisation to differentiate S. aureus from a closely related but less pathogenic Staphylococcus species (S. epidermidis). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the S. aureus. After excitation (365 nm, Chelate λmax = 335 nm), the long-lived (Eu3+) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological samples. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained S. aureus with mean signal intensity 3.5 times higher than the threshold level of signal from S. epidermidis (with SNR 8 times higher). A positive control probe (EUB338-BHHTEGST-Eu3+) has mean signal intensity for S. aureus and S. epidermidis equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has great potential for highly sensitive homogeneous in situ hybridisation detection of the vast range of intracellular DNA targets.

Sensitive Time-Gated Immunoluminescence Detection of Prostate Cancer Cells Using a TEGylated Europium Ligand.

We describe the application of a synthetically developed tetradentate ß-diketonate-europium chelate with high quantum yield (39%), for sensitive immunodetection of prostate cancer cells (DU145). MIL38 antibody, a mouse monoclonal antibody against Glypican 1, conjugated directly to the chelate via lysine residues, resulted in soluble (hydrophilic) and stable immunoconjugates. Indirect labeling of the antibody by a europium chelated secondary polyclonal antibody and a streptavidin/biotin pair was also performed. All of these bright luminescent conjugates were used to stain DU145 cells, a prostate cancer cell line, using time gated luminescence microscopy for imaging, and their performances were compared to conventional FITC labeling. For all prepared conjugates, the europium chelate in conjunction with a gated autosynchronous luminescence detector (GALD) completely suppressed the cellular autofluorescence background to allow capture of vivid, high contrast images of immune-stained cancer cells.

A Novel Universal Detection Agent for Time-Gated Luminescence Bioimaging.

Luminescent lanthanide chelates have been used to label antibodies in time-gated luminescence (TGL) bioimaging. However, it is a challenging task to label directly an antibody with lanthanide-binding ligands and achieve control of the target ligand/protein ratios whilst ensuring that affinity and avidity of the antibody remain uncompromised. We report the development of a new indirect detection reagent to label antibodies with detectable luminescence that circumvents this problem by labelling available lysine residues in the linker portion of the recombinant fusion protein Linker-Protein G (LPG). Succinimide-activated lanthanide chelating ligands were attached to lysine residues in LPG and Protein G (without Linker) and the resulting Luminescence-Activating (LA-) conjugates were compared for total incorporation and conjugation efficiency. A higher and more efficient incorporation of ligands at three different molar ratios was observed for LPG and this effect was attributed to the presence of eight readily available lysine residues in the linker region of LPG. These Luminescence-Activating (LA-) complexes were subsequently shown to impart luminescence (upon formation of europium(III) complexes) to cell-specific antibodies within seconds and without the need for any complicated bioconjugation procedures. The potential of this technology was demonstrated by direct labelling of Giardia cysts and Cryptosporidium oocysts in TGL bioimaging.

A novel biocompatible europium ligand for sensitive time-gated immunodetection.

We describe the synthesis of a novel hydrophilic derivative of a tetradentate ß-diketone europium ligand that was used to prepare an immunoconjugate probe against Giardia lamblia cysts. We used a Gated Autosynchronous Luminescence Detector (GALD) to obtain high quality delayed luminescence images of cells 30-fold faster than ever previously reported.

Express fluorescence in situ hybridization methods for the detection of Staphylococcus aureus.

BACKGROUND: As a proof-of-concept, the feasibility of detecting Staphylococcus aureus faster than previous whole-cell fluorescent in situ hybridization (FISH) methods was tested. METHODS: Isolates of Staphylococcus were treated with three rapid slide-based FISH protocols and DNA probes. Protocols were shortened by optimizing, combining or omitting steps. RESULTS: All FISH protocols detected S. aureus and not the phenotypically similar Staphylococcus epidermidis. The express FISH assay was completed in 24 minutes. The one-step FISH assay with NaCl and the one-step with phosphate buffered saline (PBS) assay took 19 minutes, but yielded a weaker signal. CONCLUSIONS: The exploratory study identified S. aureus two to three times faster than previous methods. Additional testing in a clinical laboratory scenario (for example with positive blood-culture bottles) is warranted.

Optimization of a two-step permeabilization fluorescence in situ hybridization (FISH) assay for the detection of Staphylococcus aureus.

BACKGROUND: Aspects of the fluorescence in situ hybridization (FISH) method for the detection of clinically important bacteria, such as Staphylococcus aureus, Staphylococcus epidermidis, and Escherichia coli, were investigated for optimization. METHODS: Various approaches to optimizing the FISH procedure were taken and different methods were compared. To save time, hybridization and washing buffers were prepared beforehand and stored at -20 °C and mixed to their final formamide and NaCl concentrations just before use. The use of 50-ml tubes for hybridization incubation reduced drying out, reagent wastage, and reaction times. RESULTS: A two-step permeabilization FISH assay was developed that used phosphate-buffered saline as a buffer for lysostaphin. It could detect bacteria with DNA probes conjugated to fluorophores with a higher signal intensity and the less expensive biotinylated DNA probes with minimal cell lysis in 1 hr. CONCLUSIONS: The two-step assay might be used when the FISH signal is weak, bacterial numbers are low or if there is a need to use other reporter molecules.

Detection of Staphylococcus aureus with a fluorescence in situ hybridization that does not require lysostaphin.

To detect with whole-cell fluorescence in situ hybridization (FISH), Staphylococcus aureus is typically permeabilized with lysozyme and lysostaphin. We tested whether it was feasible to detect S. aureus and differentiate it from Staphylococcus epidermidis with lysozyme-only permeabilization. We compared lysozyme permeabilization to S. aureus permeabilized with lysozyme in combination with lysostaphin. It was determined that S. aureus treated with agarose, methanol, and lysozyme could be detected with FISH. The 1 hr protocol is a useful alternative to conventional FISH.

Time-gated luminescence microscopy.

Autofluorescent algal samples were spiked with europium beads for analysis on a novel all-solid-state, time-gated luminescence (TGL) microscope. Pulsed UV excitation (365 nm) was provided by a high-power UV-LED source fitted to an Olympus BX51 microscope. An "Impactron" electron multiplying charge-coupled-device (CCD) camera acquired images in delayed luminescence mode. Second, we evaluated sensitivity of the instrument by acquiring images of immunofluorescently labeled Giardia cysts with a single-exposure period of 3 ms. The camera was triggered 3 micros after the LED had extinguished to yield a 14-fold increase in signal-to-noise ratio within a single 33 ms capture cycle. This novel instrument could be switched instantly from prompt epifluorescence mode to TGL mode for suppression of short-lived fluorescence.