Facilitating serum determination of neuron specific enolase at clinically relevant levels by coupling on-line...

The identification and quantification of biomarkers is essential for the diagnosis, treatment, and long-term monitoring of many human diseases. In the present work, macromolecular synthetic receptors with pre-determined affinity and selectivity for the signature peptide of a prognostically significant small cell lung cancer (SCLC) biomarker - neuron-specific enolase (NSE) - were prepared in a porous polymer microsphere format using a template-directed synthesis strategy performed under precipitation polymerization conditions. The polymer microspheres were packed into short trap columns and then exploited as molecularly selective sorbents in a fully automated, on-line molecularly imprinted solid-phase extraction (MISPE) protocol. The on-line MISPE protocol was optimised with respect to the composition of the loading mobile phase, the flow rate, and the extraction time. The molecularly imprinted polymers (MIPs) showed high affinity and useful selectivity for the peptide target - the hexapeptide ELPLYR - compared to non-imprinted control polymers. The MIPs were able to retain the biomarker on-column for extraction times of up to 20 min, and the on-line MISPE method enabled complete recovery of the biomarker over the linear range 10-100 ng mL^-1 when the biomarker was present in spiked ammonium bicarbonate solution (R² = 0.994). For extractions of ELPLYR from very complex biological matrices, the recoveries of ELPLYR from reversed-phase SPE (RP-SPE)-treated and untreated digested human serum were 100.8 ± 6.2% and 61.6 ± 1.9%, respectively. Extractions of ELPLYR from spiked untreated digested serum were linear in the range of 7.5-375 ng mL^-1 (R² = 0.99). The limit of detection (LOD) and limit of quantification (LOQ) for the biomarker in digested serum were estimated to be 1.8 ng mL^-1 and 6.0 ng mL^-1, respectively, which is below the median reference level of NSE in humans (8.6 ng mL^-1). This work sets in place the basis for a new diagnostic tool for SCLC that is sensitive, robust, automated, and antibody-free, and which works very well with complex human plasma samples.