Fluorescent measurement of affinity binding between thrombin and its aptamers using on-chip affinity monoliths

• Published in: Journal of Chromatography A Vol. 1291; pp. 92 - 96
• Main Authors: Gao, Changlu, Sun, Xiuhua, Woolley, Adam T.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 24-05-2013; Elsevier
• Subjects: acetic acid; Acetic Acid - chemistry; Adsorption; Affinity; Analytical, structural and metabolic biochemistry; Antifouling; Aptamer; Aptamers, Nucleotide - chemistry; Aptamers, Nucleotide - metabolism; Binding; binding capacity; Biological and medical sciences; chromatography; Elution; Enzymes and enzyme inhibitors; Fluorescence; Fluorescent; Fundamental and applied biological sciences. Psychology; green fluorescent protein; human serum albumin; Humans; Hydrolases; hydrophilicity; Microfluidic; Microfluidic Analytical Techniques - instrumentation; Microfluidic Analytical Techniques - methods; Microfluidics; Monolith; oligonucleotides; sodium bicarbonate; Sodium Chloride - chemistry; Spectrometry, Fluorescence - methods; Surface Properties; Thrombin; Thrombin - chemistry; Thrombin - metabolism; Fluorescent; Aptamer; Monolith; Microfluidic; Chromatographic retention; Thrombin; Surface structure; Affinity adsorbent; Characterization; Crosslinked copolymer; Separation method; Affinity chromatography; Microequipment; Fluid mechanics; Scanning electron microscopy; Fluorescence detector; Serine endopeptidases; Enzyme; Monolithic column; System on a chip; Real time; Peptidases; Glycidyl methacrylate copolymer; Morphology; Preparation; Hydrolases; Miniaturization; Microfluidics
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2013.03.063

•The bioaffinity binding between thrombin and aptamers was fluorescently monitored.•PEG-monolith greatly decreased nonspecific adsorption.•Macroporous structure of PEG-monolith allows high efficiency separation.•Binding and separation procedures can be performed on the microdevice.
A microfluidic chip with integrated 2mm long monoliths incorporated with poly(ethylene glycol) (PEG) groups was developed for thrombin–aptamer interaction study. The non-G quartet forming oligonucleotide coated monoliths was compared to a 15 mer thrombin-binding aptamer, in which affinity binding and elution processes were real-time monitored fluorescently. The results showed that the fluorescence intensity of aptamer stationary phase is approximately 10 times higher than that of the control column, which is probably due to the successful suppression of nonspecific adsorption between thrombin and aptamers/monoliths by using PEG-monolith. The experiment was repeated using human serum albumin (HSA) and green fluorescence protein (GFP) as interferences, it was double confirmed that thrombin was selectively retained by PEG-monolith. An elution efficiency of 75% was achieved with an elute of 200mM acetic acid and 2M NaCI, and the eluted thrombin was successfully separated in an ionic buffer system of 20mM NaHCO3 (pH 9.5) with 3% PEG. The hydrophilic and antifouling properties of PEG-monolith greatly decrease nonspecific adsorption and enhance detection sensitivity, which provided an alternative method to perform on-chip fluorescent measurement of bioaffinity binding.