Nanozyme and Stimulated Fluorescent Cu-Based Metal–Organic Frameworks (Cu-MOFs) Functionalized with Engineered Aptamers as a Molecular Recognition Element for Thrombin Detection in the Plasma of COVID-19 Patients

[Image: see text] An essential tool in the management and control of the COVID-19 pandemic is the development of a fast, selective, sensitive, and inexpensive COVID-19 biomarkers detection method. Herein, an ultrasensitive and label-free biosensing strategy was described for the colorimetric and fluorimetric detection of thrombin. A dual-mode aptasensing method based on integrating engineered ssDNA with a stimulated fluorescent enzyme-mimetic copper-based metal–organic framework (Cu-MOF) as a molecular recognition element for thrombin was investigated. Cu-MOFs displayed stimulated fluorescence and enzyme-mimetic peroxidase activities that oxidize the chromogenic colorless substance TMB to blue-colored oxTMB. The thrombin-based aptamer (ssDNA) can be immobilized on the Cu-MOF surface to form a functionalized composite, ssDNA/MOF, and quench the stimulated fluorescence emission and the enzymatic activity of the Cu-MOF. Later, addition of thrombin recovers the fluorescence and enzymatic activity of the MOF. Thus, a turn-on colorimetry/fluorimetry aptasensing probe was designed for the detection of thrombin. Based on colorimetric assay, 350 pM was recorded as the lower limit of detection (LOD), while based on the fluorescence mode, 110 fM was recorded as the LOD (when S/N = 3). The label-free aptasensing probe was used successfully for the detection of thrombin in COVID-19 patients with satisfactory recoveries, 95–98%. Since the detection time of our aptasensor is relatively rapid (45 min) and due to the low-cost precursors and easy-to-operate characteristics, we believe that it has great potential to be used in point-of-care testing (POCT).