Abstract

2,6-Pyridinedicarboxylic acid (PDC) and its derivatives are introduced as a new sensitizer system for enzyme-amplified lanthanide luminescence (EALL), a detection scheme for bioassays, which combines enzymatic amplification with time-resolved luminescence measurements of lanthanide chelates. Various PDC esters have been synthesized as esterase substrates that are cleaved to PDC in the presence of the enzyme. PDC forms luminescent complexes with Tb(III) or Eu(III), and the evaluation of the reaction is used for the selective and sensitive detection of esterases. For an esterase from hog liver a limit of detection of 10⁻³ u/mL (equivalent to 10⁻⁹ mol/L) and a limit of quantification of 3 × 10⁻³ u/mL (equivalent to 3 × 10⁻⁹ mol/L) could be achieved. As a second model reaction, xanthine oxidase (XOD) catalyzes the oxidation of 2,6-pyridinedicarboxaldehyde to PDC. Here, the limit of detection was 3 × 10⁻³ u/mL and the limit of quantification 10⁻² u/mL for XOD from microorganisms. Major advantage of the tridentate PDC ligand is the possibility to perform all steps of the assay within or close to the physiological pH range, while the established EALL schemes based on bidentate salicylates or bisphenols have to be carried out at strongly alkaline pH to ensure sufficient complexation with the lanthanides.

Keywords: Lanthanides; Luminescence; Enzymes; Ligands; Energy transfer

Article Outline

1. Experimental

1.1. Chemicals

1.2. Instrumentation

1.3. Synthesis of dipropyl-2,6-pyridinedicarboxylate, diisopropyl-2,6-pyridinedicarboxylate, dihexyl-2,6-pyridinedicarboxylate and dibenzyl-2,6-pyridinedicarboxylate

1.3.1. Dipropyl-2,6-pyridinedicarboxylate (PDCPE)

1.3.2. Diisopropyl-2,6-pyridinedicarboxylate (PDCIPE)

1.3.3. Dihexyl-2,6-pyridinedicarboxylate (PDCHE)

1.3.4. Dibenzyl-2,6-pyridinedicarboxylate (PDCBAE)

1.4. Preparation of the solutions

1.5. Optimization of the PDC/lanthanide(III) system

1.5.1. Optimization of the lanthanide ion concentration

1.5.2. Addition of Cs(I) to investigate the heavy atom effect

1.5.3. Addition of Y(III) ions to investigate the co-fluorescence effect

1.5.4. Optimization of the parameters for time-resolved measurements

1.5.5. Calibration for 2,6-pyridinedicarboxylic acid with lanthanide(III)

1.5.6. Calibration for the lanthanide(III) with 2,6-pyridinedicarboxylic acid

1.6. Optimization of the PDC/lanthanide(III)/esterase system

1.6.1. Optimization of the pH

1.6.2. Esterase determination with time-resolved fluorescence

1.6.3. Investigation on the reactivity of esterases from other sources

1.7. Optimization of the PDC/lanthanide(III)/XOD system

1.7.1. Optimization of the pH

1.7.2. XOD determination with time-resolved fluorescence

2. Results and discussion

3. Conclusions

Acknowledgements

References