Multiplexed detection of small analytes by structure-switching aptamer-based capillary electrophoresis.
Affinity probe capillary electrophoresis (APCE) assays, combining the separation power of CE with the specificity of interactions occurring between a target and a molecular recognition element (MRE), have become important analytical tools in many application fields. In this report, a rationalized strategy, derived from the structure-switching aptamer concept, is described for the design of a novel APCE mode dedicated to small molecule detection. Two assay configurations were reported. The first one, developed for the single-analyte determination, was based on the use of a cholesteryl-tagged aptamer (Chol-Apt) as the MRE and its fluorescein-labeled complementary strand (CS*) as the tracer (laser-induced fluorescence detection). Under micellar electrokinetic chromatography (MEKC) conditions, free CS* and the hybrid formed with Chol-Apt (duplex*) were efficiently separated (and then quantified) through the specific shift of the electrophoretic mobility of the cholesteryl-tagged species in the presence of a neutral micellar phase. When the target was introduced into the preincubated sample, the hybridized form was destabilized, resulting in a decrease in the duplex* peak area and a concomitant increase in the free CS* peak area. The second format, especially designed for multianalyte sensing, employed dually cholesteryl- and fluorescein-labeled complementary strands (Chol-CS*) of different lengths and unmodified aptamers (Apt). The size-dependent electrophoretic separation of different Chol-CS* forms from each other and from their corresponding duplexes* was also accomplished under MEKC conditions. The simultaneous detection of multiple analytes in a single capillary was performed by monitoring accurately each target-induced duplex-to-complex change. This method could expand significantly the potential of small solute APCE analysis in terms of simplicity, adaptability, generalizability, and high-throughput analysis capability.
Zhu
Zhenyu
Z
Departement de Pharmacochimie Moléculaire, UMR 5063 CNRS, ICMG FR 2607, Université Grenoble I, Campus universitaire, Saint-Martin d'Hères, France.
Ravelet
Corinne
C
Perrier
Sandrine
S
Guieu
Valérie
V
Roy
Béatrice
B
Perigaud
Christian
C
Peyrin
Eric
E
D000249:Adenosine Monophosphate / Q000032:analysis / Q000378:metabolism; D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D020374:Chromatography, Micellar Electrokinetic Capillary; D019075:Electrophoresis, Capillary / Q000379:methods*; D019793:Fluorescein / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D007834:Lasers; D008970:Molecular Weight; D009038:Motion; D009693:Nucleic Acid Hybridization; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D052157:Aptamers, Nucleotide; D005456:Fluorescent Dyes; D000249:Adenosine Monophosphate; D019793:Fluorescein