Simultaneous detection of two lung cancer biomarkers using dual-color fluorescence quantum dots

Abstract

Quantum dots (QDs) have the potential to simplify the performance of multiplexed analysis. In this work, a novel protocol for performing a simultaneous dual-protein immunoassay, i.e. two lung cancer biomarkers, carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE), based on dual-color QDs, is described. First, two capture antibodies (both with biotin tags), two antigens and two detection antibodies were mixed together and the sandwich complexes were thus formed in the homogeneous solution, and then streptavidin coated polystyrene beads were directly added into the resultant system. Bead aggregation can be made self-limiting by controlling the shaker speed during the immunoassay. A distinct transition occurs between limited and complete aggregation as a function of the shaker speed during the immunoassay. Second, dual-color QDs with emission maxima at 525 and 655 nm were added after washing and reacted with the corresponding detection antibodies. Third, the bead-QD conjugates were dissociated in the dissociation buffer and then free QDs were directly used for the fluorescence detection of CEA and NSE. The results show that CEA and NSE could be sensitively determined with a common 96-well fluorescence plate reader and with equal detection limits down to the 1.0 ng mL(-1) level. Within the calibrated amount, the protocol had excellent precision within 0.53% for each target and was comparable in performance to commercial single-analyte ELISAs. Furthermore, the proposed method has been successfully applied to the determination of dual markers in real samples without cross-reaction, and a good correlation was achieved after comparison with the conventional assay for CEA and NSE in 25 human serum samples.

Multiplexing Determination of Cancer-Associated Biomarkers by Surface-Enhanced Raman Scattering Using Ordered Gold Nanohoneycomb Arrays

“…Through this method, the LOD of CEA and AFP was 1.67 and 1.56 ng/ml, respectively [36]. Li et al has used dual-color fluorescence quantum dots to detect CEA and NSE, both of the LODs were 1.0 ng/ml [47]. Cheng et al has designed field effect transistor-based biosensors to detecting CYFRA21-1 and NSE, the lowest detectable concentrations of CYFRA21-1 and NSE were 1 and 10 ng/ml, respectively [48].…”

Technological Development of Antibody Immobilization for Optical Immunoassays: Progress and Prospects

“…An avidin layer may be bound to a substrate surface that has been modified using aldehyde (Qi et al, 2006), 11-MUA on gold-coated magnetic nanoparticles (Torun et al, 2012), or EDC-activated polystyrene (PS) beads (Li et al, 2011a). However, the covalent attachment of avidin by carbodiimide may affect its binding activity (Vermette et al, 2003).…”

Synthetic (Inorganic) Nanoparticles Based Lung Cancer Diagnosis and Therapy

“…QDs may identify various potential biomarkers of lung cancer which include specific proteins, DNA, mRNA sequences, and circulating tumors. Dual-color QDs have been used successfully in performing a simultaneous dual-protein immunoassay of lung cancer-based biomarkers carcinoembryonic antigen (CEA) and neuron-specific enolase (NSE) in a single sample [51]. Akerman and his coworkers conjugated QDs with three different lung cancer cells targeting peptides such as CGFECVRQCPERC sequence (GFE), KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (F3), and CGNKRTRGC (LyP-1) [52].…”