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Label-Free Time-Gated Luminescent Detection Method for the Nucleotides with Varying Phosphate Content.

ABSTRACT: A new label-free molecular probe for luminescent nucleotide detection in neutral aqueous solution is presented. Phosphate-containing molecules, such as nucleotides possess vital role in cell metabolism, energy economy, and various signaling processes. Thus, the monitoring of nucleotide concentration and nucleotide related enzymatic reactions is of high importance. Two component lanthanide complex formed from Tb(III) ion carrier and light harvesting antenna, readily distinguishes nucleotides containing different number of phosphates and enable direct detection of enzymatic reactions converting nucleotide triphosphate (NTP) to nucleotide di/monophosphate or the opposite. Developed sensor enables the detection of enzymatic activity with a low nanomolar sensitivity, as highlighted with K-Ras and apyrase enzymes in their hydrolysis assays performed in a high throughput screening compatible 384-well plate format.

SUBMITTER: Kopra K

PROVIDER: S-EPMC6264117 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Label-Free Time-Gated Luminescent Detection Method for the Nucleotides with Varying Phosphate Content.

Kopra Kari K Seppälä Tanja T Rabara Dana D Abreu-Blanco Maria M Kulmala Sakari S Holderfield Matthew M Härmä Harri H

Sensors (Basel, Switzerland) 20181116 11

A new label-free molecular probe for luminescent nucleotide detection in neutral aqueous solution is presented. Phosphate-containing molecules, such as nucleotides possess vital role in cell metabolism, energy economy, and various signaling processes. Thus, the monitoring of nucleotide concentration and nucleotide related enzymatic reactions is of high importance. Two component lanthanide complex formed from Tb(III) ion carrier and light harvesting antenna, readily distinguishes nucleotides cont ...[more]

PMID: 30453509

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Project description: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 ?max = 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.

Dataset Information

Label-Free Time-Gated Luminescent Detection Method for the Nucleotides with Varying Phosphate Content.

Publications

Label-Free Time-Gated Luminescent Detection Method for the Nucleotides with Varying Phosphate Content.

Similar Datasets

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