Advanced biomarker detection methods: development of peroxidase mimics and microfluidic paper-based platforms

In the development of analytical techniques for biomarker detection, two advanced approaches have emerged, demonstrating significant improvements in sensitivity, reproducibility, and practicality. Firstly, nitrogen-doped carbon dots/Ni-MnFe-layered double hydroxides (N-CDs/Ni-MnFe-LDHs) were demonstrated as superior peroxidase mimics for immunoassays, offering a promising alternative to traditional natural peroxidase. This nanomaterial efficiently catalyzed the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2), resulting in an intense blue product. Employing low-density lipoprotein cholesterol (LDL-C) as a model target, the method achieved an ultra-low detection limit of 0.0051 mg/dL and a linear range of 0.0625-0.750 mg/dL, outperforming natural enzyme-based assays in sensitivity.
Subsequently, a fluorometric aptasensor LDL-C detection was developed based on the aptamer-enhanced peroxidase-mimicking activity of gold nanoparticles (AuNPs). Functionalized with LDL-C-specific thiolated aptamer, the AuNPs exhibited increased peroxidase-like activity, catalyzing the oxidation of o-phenylenediamine dihydrochloride (OPD) into a yellow-fluorescent product. The sensor demonstrated a linear response between smartphone-recorded signal intensity and analyte concentration within the range of 0.05-1 mg/dL, achieving a detection limit of 0.0230 mg/dL. This method showed agreement with clinical laboratory results and presented a deployable prototype for point-of-care testing applications.
In the second approach, the paper-based analytical device (PAD) platform has been refined using an in-house computer-controlled X-Y knife and pen plotter, overcoming traditional fabrication limitations. Laminated PAD (LPAD) was developed to increase mechanical strength and reduce sample evaporation during analysis. This LPAD was used to simultaneously determine glucose and total cholesterol in whole blood, employing the LF1 membrane to separate plasma from blood cells for enzymatic reactions selectively. The analytical results were consistent with those obtained using clinical laboratory methods, with detection limits of 0.16 mM for glucose and 0.57 mM for total cholesterol. The LPAD maintained stability in color intensity after 60 days of storage at 4C, offering a low-cost, high-performance option for chemical sensing devices.