Abstract
We validated a single-stranded, DNA aptamer-based, diagnostic method capable of detecting Lipocalin-2 (LCN2), a biomarker from clinically relevant hepatocellular carcinoma (HCC) patient serum, in the sandwich assay format. Nine aptamers (LCN2_apta1 to LCN2_apta9) for LCN2 were screened with SELEX processes, and a sandwich pair (LCN2_apta2 and LCN2_apta4) was finally chosen using surface plasmon resonance (SPR) and dot blotting analysis. The result of the proposed aptamer sandwich construction shows that LCN2 was sensitively detected in the concentration range of 2.5–500 ng mL−1 with a limit of detection of 0.6 ng mL−1. Quantitative measurement tests in HCC patients were run on straight serum and were compared with the performance of the conventional antibody-based ELISA kit. The aptamer sandwich assay demonstrated an excellent dynamic range for LCN2 at clinically relevant serum levels, covering sub-nanogram per mL concentrations. The new approach offers a simple and robust method for detecting serum biomarkers that have low and moderate abundance. It consists of functionalization, hybridization and signal read-out, and no dilution is required. The results of the study demonstrate the capability of the aptamer sandwich assay platform for diagnosing HCC and its potential applicability to the point-of-care testing (POCT) system.
Highlights
Hepatocellular carcinoma (HCC) is responsible for 5% of all deaths worldwide[1]
LCN2 (Lipocalin-2; neutrophil gelatinase-associated lipocalin (NGAL)), a 24 kDa secretory glycoprotein that was first identified in urine collected from mice with SV40-infected kidneys, is stored in human neutrophils[14,15]
LCN2 was actively explored as a potential biomarker for hepatocellular carcinoma (HCC)
Summary
Hepatocellular carcinoma (HCC) is responsible for 5% of all deaths worldwide[1]. Liver disease such as cirrhosis and HCC is the 5th most common cancer, after thyroid, stomach, colon and lung cancer, in Asian countries[2]. Most previous biomedical studies have focused primarily on measuring ultra-low or low abundance serum markers based on the enzyme-linked immunosorbent assay (ELISA) platform because it is a highly sensitive detection method, and the use of commercial antibodies is attractive[27]. This serum use is problematic due to the highly non-specific interaction and signal interference from the most abundant serum proteins, which generate a non-measurable signal. There is an urgent need to develop a diagnostic strategy that satisfies both the linearity in the range of sub-nanograms and high simplicity for clinical sample use[29]
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