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Rapid detection of heparin-binding protein (HBP) is essential for timely intervention in sepsis cases. Current detection techniques are usually antibody-based immunological methods, which have certain problems, such as complexity and slow detection, and fall short in meeting the urgency of clinical needs. The application of an aptamer can address these concerns well. In this study, HBP-specific DNA aptamers were screened first. Among which, Apt-01, Apt−02, and Apt−13 had a high affinity for HBP, exhibiting impressive KD values of 3.42, 1.44, and 1.04 nmol/L, respectively. Then, the aptamer of HBP and its partially complementary primer probe were combined to form double-stranded DNA (dsDNA) and synthesize a circular DNA template. The template is complementary to the primer probe, but due to the presence of dsDNA, ExoIII cleaves C2-13 as an RCA primer probe, rendering the template unable to recognize the primer probe and preventing the RCA reaction from proceeding. When the target is present, it competes with the adapter for recognition and releases C2-13, exposing its 3′ end. After initiating the RCA at room temperature and reacting with SYBR GreenII at 37 °C for 20 min, fluorescence changes can be observed and quantitatively analyzed at a 530 nm wavelength, achieving quantitative biological analysis. Apt-01 was used to develop a fluorescent biosensor for HBP detection, which exhibited a good linear range (0.01 nmol/L to 10 nmol/L) and detection limit (0.0056 nmol/L). This advancement holds the potential to lay a solid groundwork for pioneering sensitive and specific methods for HBP detection and to significantly enhance the diagnostic processes for sepsis. [ABSTRACT FROM AUTHOR]

Abstract Golgi protein 73 (GP73) is a potential hepatocellular carcinoma serum marker with better sensitivity and specificity. In this study, a highly sensitive electrochemical immunosensor which combines the highly selective proximity ligation assay (PLA) and highly efficient enzyme-powered recycling amplification has been developed for GP73 determination. PLA is triggered by affinity binding of two labelled antibody-DNA (P1-RAb and P2-MAb) to target protein, resulting in increased specificity. The formed immunocomplex hybrids with DNA2 to realize the release of methylene blue labelled mononucleotides fragment with the help of exonuclease III, which acts as the power of recycling amplification. The fragment can diffuse easily to the nafion modified indium tin oxide electrode surface and a strong electrochemical signal can be detected. Under the optimized conditions, the enhanced electrochemical intensity has a linear relationship with the concentration of GP73 in the range of 0.3 pg mL−1 to 6.0 ng mL−1 with the detection limit of 0.10 pg mL−1. The developed immunosensor has been applied to detect the GP73 in the clinical serum samples with satisfactory results. Graphical abstract Image 1 Highlights • A highly sensitive electrochemical immunosensor for the detection of GP73 was developed. • The enzyme-powered recycling amplification exhibited the high amplification efficiency. • The proximity ligation assay was applied for GP73 quantification with highly selectivity. • The nafion modified ITO electrodes can significantly enhance the amount of negative charge on the surface of ITO electrode. [ABSTRACT FROM AUTHOR]