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

Abstract

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.

Lanthanide-Complex-Enhanced Bioorthogonal Branched DNA Amplification

“…In comparison to the “double Z” RNAscope method, LEBODA offers two distinct advantages, including (1) the utilization of the Click Chemistry strategy to enhance the density and stability of the bridge probes and (2) the integration of high-density lanthanide complexes in each bridge probe (Table ). This innovative approach to signal enhancement effectively overcomes the concentration-quenching challenge often encountered with conventional fluorophores, as the lanthanide complex possesses a large Stokes shift that allows for high-density labeling. − These advancements make LEBODA a promising technique for nucleic acid in situ detection, offering improved sensitivity and signal amplification compared to those of existing methods.…”

Long-lifetime green-emitting Tb

“…The abovementioned long decay times can most favourably be used to discriminate the emission signal from other dyes, and the highly problematic autofluorescence in matrices of biological origin in particular (by time-gated luminescence). [21][22][23] The feasibility of using long-lived red-emitting microbe stains based on Eu diketonate complexes has recently been evaluated. [4] Furthermore, the narrow-line emissions of Tb 3+ , as well as most other rare earth ions of interest, can be of extreme value where filters have to be used to separate excitation and emission.…”

Lifetime Multiplexing with Lanthanide Complexes for Luminescence In Situ Hybridisation

“…Long‐lived luminescence probes with lifetimes in the microsecond‐to‐millisecond range, together with time‐resolved luminescence detection, offer high‐contrast detection of targets without confounding background from biological autofluorescence and residual excitation light. This concept, first proposed for immunoassays in the mid‐1970s, [1] has been widely demonstrated for accurate detection and quantification of proteins, [2–3] nucleic acids, [4] molecular biomarkers, [5–6] and pathogens [7–9] . The technique has been successfully implemented in various analytical platforms for testing liquid samples, [10] solid substrates, [11] cultured cells, [12–13] extracted tissues, [14] and small animals in vivo [15–17] .…”