Europium chelate-labeled lateral flow assay for rapid and multiple detection of β-lactam antibiotics by the penicillin-binding protein

A lateral flow assay for rapid and multiple detection of β-lactam antibiotics by the penicillin-binding protein was developed.

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.

A Nanoparticle-Lectin Immunoassay Improves Discrimination of Serum CA125 from Malignant and Benign Sources

BACKGROUND Measurement of serum cancer antigen 125 (CA125) is the standard approach for epithelial ovarian cancer (EOC) diagnostics and follow-up. However, the clinical specificity is not optimal because increased values are also detected in healthy controls and in benign diseases. CA125 is known to be differentially glycosylated in EOC, potentially offering a way to construct CA125 assays with improved cancer specificity. Our goal was to identify carbohydrate-reactive lectins for discriminating between CA125 originating from EOC and noncancerous sources. METHODS CA125 from the OVCAR-3 cancer cell line, placental homogenate, and ascites fluid from patients with cirrhosis were captured on anti-CA125 antibody immobilized on microtitration wells. A panel of lectins, each coated onto fluorescent europium-chelate–doped 97-nm nanoparticles (Eu+3-NPs), was tested for detection of the immobilized CA125. Serum samples from high-grade serous EOC or patients with endometriosis and healthy controls were analyzed. RESULTS By using macrophage galactose-type lectin (MGL)-coated Eu+3-NPs, an analytically sensitive CA125 assay (CA125MGL) was achieved that specifically recognized the CA125 isoform produced by EOC, whereas the recognition of CA125 from nonmalignant conditions was reduced. Serum CA125MGL measurement better discriminated patients with EOC from endometriosis compared to conventional immunoassay. The discrimination was particularly improved for marginally increased CA125 values and for earlier detection of EOC progression. CONCLUSIONS The new CA125MGL assay concept could help reduce the false-positive rates of conventional CA125 immunoassays. The improved analytical specificity of this test approach is dependent on a discriminating lectin immobilized in large numbers on Eu+3-NPs, providing both an avidity effect and signal amplification.

A FRET based pH probe with a broad working range applicable to referenced ratiometric dual wavelength and luminescence lifetime read out

A FRET system composed of a europium chelate and carboxynaphthofluorescein enables ratiometric pH sensing with an exceptionally broad dynamic range.