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

• 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 2 mm 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 200 mM acetic acid and 2 M NaCI, and the eluted thrombin was successfully separated in an ionic buffer system of 20 mM 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.