Aptamer affinity chromatography for rapid assay of adenosine in microdialysis samples collected in vivo

• Published in: Journal of Chromatography A 2003-07, Vol.1005 (1), p.123-130
• Main Authors: Deng, Qing, Watson, Christopher J., Kennedy, Robert T.
• Format: Article
• Language: English
• Subjects: Adenosine; Adenosine - analysis; Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; Cations, Divalent; Chelating Agents - chemistry; Chromatography, Affinity - methods; Chromatography, High Pressure Liquid - methods; DNA Primers; Edetic Acid - chemistry; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Magnesium - chemistry; Microdialysis; Nucleic acids; Nucleic bases, nucleotides; Oligonucleotides; Osmolar Concentration; Somatosensory Cortex - chemistry
• ISSN: 0021-9673
• DOI: 10.1016/S0021-9673(03)00812-4

An anti-adenosine aptamer was evaluated as a stationary phase in packed capillary liquid chromatography. Using an aqueous mobile phase containing 20 m M Mg 2+, adenosine was strongly retained on the column. A gradient of increasing Ni 2+ (to 18 m M), which is presumed to complex with nitrogen atoms in adenosine involved in binding to the aptamer, eluted adenosine in a narrow zone. Up to 6 μl of 1.2 μ M adenosine could be injected onto the 150-μm I.D.×7 cm long column without loss of adenosine. With UV absorbance detection, the detection limit was 30 n M or 120 fmol (4 μl injected). Samples could be repetitively injected with 4.6% relative standard deviation in peak area. Columns were stable to at least 200 injections. The adenosine assay, which required no sample preparation, was used on microdialysis samples collected from the somatosensory cortex of chloral hydrate anesthetized rats. Total analysis times were short enough that dialysate samples could be injected every 5 min. Basal dialysate concentrations of adenosine stabilized at 87±10 n M ( n=5) with the probe operated at 0.6 μl/min.