On selectivity and sensitivity of synthetic multifunctional pores as enzyme sensors: Discrimination between ATP and ADP and comparison with biological pores

Abstract

This report delineates scope and limitation of the selectivity of synthetic multifunctional pores as enzyme sensors using glycolytic enzymes as example (G. Das, P. Talukdar, and S. Matile, Science, 2002, Vol. 298, pp. 1600-1602). Unproblematic detectability of hexokinase and phosphofructokinase demonstrates that the selectivity of synthetic multifunctional pore (SMPs) sensors suffices to sense ATP in mixed analytes containing ADP, whereas detection of the isomerization of glucose 6-phosphate into fructose 6-phosphate by phosphoglucose isomerase is not possible with confidence. The sensitivity of SMP sensors is sufficient for end-point detection of one picomole poly-L-glutamate hydrolyzed by papain in unoptimized assay format; the sensitivity of melittin as representative biological pore of similar charge and aggregation number to detect the same reaction is more than four orders of magnitude inferior.

Interface Engineering of Synthetic Pores: Towards Hypersensitive Biosensors

“…ATP/ADP discrimination (ATP = adenosine 5'-triphosphate, ADP = adenosine 5'-diphosphate) provides an ideal example because it is important for the fluorometric detection of many enzymes (e.g., kinases) and the sensing of their substrates. [1,12,13,23,25,36] Under standard conditions, blockage of pore 1 by ATP occurred with IC 50 = 44.2 mm and was clearly better than blockage by ADP (Table 1, entries 5 and 6). The high discrimination factor of D = 6.1 suggested, together with relatively poor IC 50 values, that nucleotide recognition is not affected by stoichiometric binding.…”

“…The discrimination factor D = 3.0 found for the detection of amplified a-ketoglutarate with nanomolar sensitivity (IC 50 = 400 nm) is sufficient for sensing applications. [1,36] To confirm the validity of the interpretation of this breakthrough, control dilution experiments without stoichiometric binding were made. ATP/ADP discrimination (ATP = adenosine 5'-triphosphate, ADP = adenosine 5'-diphosphate) provides an ideal example because it is important for the fluorometric detection of many enzymes (e.g., kinases) and the sensing of their substrates.…”

“…In pore 1, which is introduced in this report, the rigid-rod b barrel of the classical [32][33][34][35][36] pores 2 and 3 is elongated with four of Tomichs hydrophilic tetralysine (K 4 ) anchors (Figure 1). [29] Rigid-rod b barrels in general consist of p-octiphenyl staves and b-sheet hoops.…”

“…The synthesis of the protected LRLHL peptide 26 has been reported previously. [34][35][36] Chemoselective hydrogenolysis of the terminal benzyl ester of rod 5 liberated the carboxylic acid at one rod terminus without deprotection of those along the rigid-rod scaffold. The deprotected carboxylic acid at one end of rod 27 was coupled with the N terminus of the Z-protected K 4 anchor 25.…”

“…[34][35][36] The cationic K 4 anchors were selected because they were best in an extensive optimization with readily accessible peptidic anion channels. [29] Moreover, cationic anchors were preferred over anionic or neutral anchors because repulsion from the cationic interior of pore 1 was considered as essential to prevent backfolding of the anchor into the pore.…”