A Paper-Based Colorimetric Sensor for Tumor Marker CA-125 Using Aptamer-Enhanced Ni–MnFe Layered Double Hydroxide Nanozymes

Cancer antigen 125 (CA-125) is a glycoprotein associated with cholangiocarcinoma, making it a valuable biomarker for diagnosis. However, conventional detection methods have limitations due to their reliance on antibodies for specificity and natural enzymes as labels for signal amplification. Both are costly and unstable under extreme pH conditions, and enzymes exhibit a low catalytic efficiency. Addressing these limitations could enhance the diagnostic accuracy of CA-125 detection in cholangiocarcinoma diagnosis. In this work, Ni-MnFe-layered double hydroxides (Ni-MnFe-LDHs) conjugated with an aptamer (Ni-MnFe-LDHs@aptamer) are demonstrated as a superior peroxidase mimic to enhance the sensitivity for the colorimetric detection of CA-125 on a paper-based analytical device (PAD) platform. Ni-MnFe-LDHs exhibited efficient peroxidase-like activity, catalyzing the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) into an intense blue product in the presence of hydrogen peroxide (H2O2). Upon modification with a CA 125-specific aptamer, Ni-MnFe-LDHs became more dispersed due to electrostatic repulsion, exposing more active sites and generating more hydroxyl radicals (·OH). Moreover, π–π stacking and hydrogen bond interactions between the aptamer and TMB increased the substrate affinity of Ni-MnFe-LDHs, thereby enhancing their catalytic performance. This resulted in a darker blue signal in the absence of CA-125. When CA-125 was present in the sample, it was captured by the aptamer on Ni-MnFe-LDHs, resulting in a decreased blue color signal. Using CA-125 as the target analyte, a linear relationship between the scanner-recorded signal intensity and analyte concentration was observed in the range of 10–25 U/mL. The results obtained from real human sample application using the developed method were consistent with those from a clinical laboratory. This method can be easily implemented and holds great potential as a prototype for various diagnostic applications, especially for detecting biomarkers in serum or plasma samples. It offers significant benefits for point-of-care testing.