Novel lectin-based quartz crystal microbalance biosensor for the identification of the pathogenic bacteria
|Gulnara Safina , Bengt Danielsson|
Lund University, Pure and Applied Biochemistry Department, Getingevägen 60, Lund SE-22100, Sweden
The development of the sensitive, rapid and simple methods for the detection of the pathogenic bacteria in the biological and food samples is the important problem which requires the special analytical approaches. The flow-through assay for screening of the pathogenic bacteria presence using lectin-based quartz crystal microbalance (QCM) biosensor has been proposed. The biosensor makes it possible to identify the bacteria presence using lectins immobilized on the surface of QCM crystal which bind specifically to the certain oligosaccharides present on the cell wall of the bacteria injected. For the obtaining of the biosensing part of the analytical device amine coupling procedure has been used. Firstly, the clean working surface of the crystal was treated with 11-mercaptoundecanoic acid (thiolization step) in order to obtain thiol self-assembled monolayers on its gold surface. Then the thiolized surface was activated on-line mode by sequential injections of the mixture of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N-hydroxisuccinimide (NHS). Then, the on-line immobilization of lectin with following blocking of the surface with ethanolamine hydrochoride to prevent non-specific binding was carried out. In this work we used different lectins as ligands – Concanavalin A (ConA), lectins from Ulex europeaus (UEA), Maackia amurensis (MAL), Lens culinaris (LCA), wheat germ agglutinin (WGA) – and different strains of the pathogenic bacteria Campylobacter jejuni and Helicobacter pylori as analytes. Formation of the complex [lectin-sugar] on the surface of the crystal after injection of the bacteria leads to the decreasing of the oscillation frequency of the crystal due to the increasing of its mass that is an analytical signal. The working conditions of the biosensor – flow rate, pH of the working buffer, the dilution of the bacterial cells, the regeneration solution – have been found and optimized. All the measurements were carried out in phosphate buffered saline (pH 7.4), the dilution of the cells was 1:50. Using the lectins having different specificity to various sugar residues makes it possible to distinguish and identify the different bacterial strains. It has been found that ConA revealed wider variety of the specificity to different bacteria observed. Lectin from UEA, vice versa, can bind only certain strains of Helicobacter pylori. Glycine with pH 2.5 was used as a regeneration solution which is able to remove the formed [lectin-sugar] complex from the crystal surface without significant damage of immobilized ligand to make it ready for the next experiment The most important advantage of proposed sensor is the ability to carry out the label-free assay, which makes it possible to analyze the bacteria directly in the sample. The assay is sensitive, much more rapid than the conventional bacteriological analysis (assay time is about 20-30 minutes) and simple which makes it as an alternative technique for the bacteria detection.
Presentation: Short communication at SMCBS'2007 International Workshop, by Gulnara Safina
See On-line Journal of SMCBS'2007 International Workshop
Submitted: 2007-08-31 18:38 Revised: 2009-06-07 00:44