A one-step sandwich method is described for detecting proteins with magnetic nanospheres (MNs) and fluorescent nanospheres
(FNs). Thrombin is selected as a model analyte to validate the method. Two DNA aptamers (Apt 29 and Apt 15 targeting two
different exosites of thrombin) are chosen as recognition elements to modify MNs and FNs. The superparamagnetic MN-Apt 29
conjugate is used to separate and concentrate thrombin. The FN-Apt 15 conjugate encapsulates hundreds of fluorescent quantum
dots and is used as reporter to provide a stable signal. Magnetic capture and fluorescence identification are performed simultaneously to form a sandwich complex (MN-Apt 29-thrombin-FN-Apt 15) for fluorescence determination (at excitation/emission
wavelengths of 380/622 nm). The method is convenient, time saving, and gives a strong signal (compared to the two-step method
where capture and identification are performed in two steps). The one-step method presented here is completed within 30 min and
has a 3.5 ng·mL−1 (97 pM) detection limit. The method is reproducible, has an intra-assay variability of 1.5%, and an inter-assay
variability of 4.9%. Other serum proteins (HSA, CEA, PSA, and AFP) do not interfere. The method was also applied to analyze
serum samples. Almost the same fluorescence intensity was measured when analyzing 1% serum samples (compared to buffer
samples).

• Aptamer-functionalized magnetic and fluorescent nanospheres for one-step sensitive detection of thrombin