Abstract
In the present work, a pair of molecularly imprinted polymers (MIPs) targeting distinct peptide targets were packed into trap columns and combined for automated duplex analysis of two low abundant small cell lung cancer biomarkers (neuron-specific enolase [NSE] and progastrin-releasing peptide [ProGRP]). Optimization of the on-line molecularly imprinted solid-phase extraction (MISPE) protocol ensured that the MIPs had the necessary affinity and selectivity towards their respective signature peptide targets – NLLGLIEAK (ProGRP) and ELPLYR (NSE) – in serum. Two duplex formats were evaluated: a physical mixture of the two MIPs (1:1 w/w ratio) inside a single trap column, and two separate MIP trap columns connected in series. Both duplex formats enabled the extraction of the peptides from serum. However, the trap columns in series gave superior extraction efficiency (85.8±3.8% and 49.1±6.7% for NLLGLIEAK and ELPLYR, respectively). The optimized protocol showed satisfactory intraday (RSD≤23.4 %) and interday (RSD≤14.6%) precision. Duplex analysis of NSE and ProGRP spiked into digested human serum was linear (R2≥0.98) over the disease range (0.3-30 nM). The estimated limit of detection (LOD) and limit of quantification (LOQ) were 0.11 nM and 0.37 nM, respectively, for NSE, and 0.06 nM and 0.2 nM, respectively, for ProGRP. Both biomarkers were determined at clinically relevant levels. To the best of our knowledge, the present work is the first report of an automated MIP duplex biomarker analysis. It represents a proof of concept for clinically viable duplex analysis of low abundant biomarkers present in human serum or other biofluids.
Highlights
The analysis of clinically relevant protein biomarkers has played an indispensable role in medicine for decades
As the primary function of molecularly imprinted polymers (MIPs) in SPE is the capture of the target analyte, considerations such as binding site heterogeneity and mass transfer kinetics are of less relevance in the optimisation of the loading mobile phase
The present work demonstrates a successful proof of concept for automated duplex analysis via the use of two MIP trap columns connected in series targeting the signature peptides of the Small cell lung cancer (SCLC) biomarkers NSE and progastrin-releasing peptide (ProGRP)
Summary
The analysis of clinically relevant protein biomarkers has played an indispensable role in medicine for decades. Cormack), trinegha enolase (NSE) [3] These biomarkers are analyzed via two singleplex assays; this approach can be laborious, expensive, and require larger quantities of patient sample compared to a potential multiplex assay [4]. Over the last 20 years, there have been considerable developments in multiplex immunoassays, including assays brought to the market [5]. This is due to the considerable advantages of multiplex assays, including reduced labour and consumable costs, lower sample volumes, lower turnaround times, and improved disease monitoring [5]. The development of novel multiplex technologies will contribute to improvements in patient care and clinical outcomes
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