Isothermal amplification (IA) is a nucleic acid amplification technology (NAAT) that has contributed significantly to the healthcare system. The combination of NAAT with a suitable detection platform resulted in higher sensitivity, specificity, and rapid disease diagnosis. Traditional NAAT, such as polymerase chain reaction (PCR), is widely applied in the general healthcare system but is rarely accessed in resource-limited hospitals. Some IA methods provide a rapid, sensitive, specific, and simple method for disease diagnosis. However, not all IA techniques have been regularly used in clinical applications because different biomarkers and sample types affect either the enzyme in the IA system or sample preparation. This review focuses on the application of some IA techniques that have been applied in the medical field and have the potential for use at points of care.

In this study, we developed magnetic graphene oxide composites by chemically attaching Fe₃O₄ nanoparticles to graphene oxide nanosheets. Characterization techniques, including Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), Raman spectroscopy, thermogravimetric analysis (TGA), X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and transmission electron microscopy (TEM), confirmed the successful synthesis of Fe₃O₄@GO composites with desirable properties. The resulting composites exhibited superparamagnetic behavior, solubility, and compatibility for efficient miRNA separation. Using miR-29a as a model, we demonstrated the effective binding of miR-29a to the magnetic graphene oxide (GO) composites at an optimal concentration of 1.5 mg/mL, followed by a simple separation using magnetic forces. Additionally, the addition of 5.0 M urea enhanced the miRNA recovery. These findings highlight the potential use of our magnetic graphene oxide composites for the efficient separation and recovery of miR-29a, suggesting their broad applicability in various miRNA-based studies. Further exploration can focus on investigating endogenous miRNAs with aberrant expression patterns, contributing to the advancements in precision medicine.

Rolling Circle Amplification (RCA) is a robust isothermal nucleic acid amplification technique widely used in molecular diagnostics. In this study, we combine RCA with solid-state nanopore sensing to monitor the amplification process at the single-molecule level using miR-21 as a model biomarker. This label-free platform enables detailed analysis of amplification kinetics and structural transitions over time. Changes in translocation dwell time and current blockage were evaluated across RCA incubation periods (30 min, 1 h, 2 h), revealing time-dependent increases consistent with the generation of longer and more complex DNA concatemers. These findings were validated by Urea-PAGE and atomic force microscopy (AFM), while Mfold-based secondary structure predictions further supported the evolution of more stable and folded configurations. Additionally, a custom-developed signal extraction application facilitated reproducible event classification and visualization. Overall, this integrated approach provides new insights into RCA behavior and highlights the potential of nanopore-based sensing for the development of sensitive, structure-resolved diagnostic tools.

Human serum albumin (HSA) is a key biomarker for early detection of kidney disorders such as microalbuminuria. However, its structural complexity and susceptibility to degradation in urine challenge current diagnostic assays, particularly immunoturbidimetric methods that depend on epitope stability. In this study, we expressed and characterized full-length recombinant HSA (rHSA) and its proteolytic fragment F8 (residues 496–585) in both Escherichia coli and rice-based systems to explore their suitability for biosensor validation and diagnostic use. Comparative analysis via SDS-PAGE, LC–MS/MS, and dynamic light scattering revealed differences in solubility and aggregation profiles between expression systems, with rice-derived rHSA showing greater resistance to proteolytic degradation. The F8 fragment retained structural integrity and remained detectable in both native and protease-treated samples. Using a previously developed Cy5-labeled ssDNA aptamer-based fluorescence aptasensor, we evaluated binding to rHSA and F8 across a clinically relevant concentration range (0.37–1500 μg/mL). Sigmoidal fluorescence recovery curves with R2 > 0.99 were observed for all recombinant proteins. Notably, the aptasensor successfully detected both targets even under conditions mimicking nonfresh urine, highlighting its diagnostic robustness. These findings establish recombinant F8 as a stable, diagnostically relevant biomarker candidate and confirm the practical utility of aptamer-based platforms for albumin detection in diverse testing scenarios.