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.

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).

Ultrasensitive aptamer-functionalized Cu-MOF fluorescent nanozyme as an optical biosensor for detection of C-reactive protein.

In the present work, an aptasensing method based on integration of RNA on Cu-MOF was developed for detection of C-Reactive Protein (CRP). Cu-MOF showed stimulated fluorescence and mimetic peroxidase enzymatic activity at the time and can be used as dual-signal transduction. CRP binding RNA was used as a highly selective recognition element and immobilized on the Cu-MOF. The immobilized RNA can block the peroxidase activity and fluorescence of the signal traducer probe. Adding CRP to the RNA/Cu-MOF will release RNA from the surface of Cu-MOF and recover both the stimulated fluorescence and peroxidase activity. A biosensor was built for detection of CRP using the two modes of transduction, either colorimetry or fluorometry. A dynamic linear range was obtained from 0.1 to 50 ng mL -1with a limit of detection (LOD) as small as 40 pg mL -1was calculated in fluorescence mode and 240 pg mL -1 as LOD in colorimetry mode. The LODs are lower than the LOD of nephelometric techniques used in clinical practice and is comparable to the normal clinical cutoff value in high-sensitivity CRP assays (1 µg/mL). The aptasensor was successfully applied for detection of CRP in Covid-19 patients with spike recoveries between 84 and 102% and RSD from 0.94% to 2.05%.