A signal-on ratiometric fluorometric heparin assay based on the direct interaction between amino-modified carbon dots and DNA.

Amino-modified carbon dots (C-dots) with positively charged surface were prepared. They display strong blue fluorescence and are shown to act as quenchers of the green fluorescence of FAM-labeled ssDNA such as the F-probe used in this work that was immobilized on the C-dots. On the addition of highly negatively charged heparin (Hep), it will interact with the C-dots and displace the F-probe from C-dots. Once the F-probe is displaced by Hep, its green fluorescence is restored. The intrinsic blue fluorescence of the C-dots remains stable after addition of Hep. Thus, a signal-on ratiometric fluorometric assay was developed for the ultra-sensitive detection of Hep. The underlying mechanisms of quenching and recovery are discussed. Under optimized conditions, the recovery of the ratiometric fluorescence of the system composed of C-dots and quenched F-probe is proportional to the Hep concentration in the range of 0.01-2.0 µg·mL-1 (= 0.00125-0.25 U·mL-1). The method was successfully applied to the determination of Hep in spiked serum samples. Graphical abstract Schematic of a signal-on ratiometric fluorometric method for the ultra-sensitive detection of heparin on the basis of the displacement and fluorescence enhancement of adsorbed FAM-labeled ssDNA from amino-modified carbon dots (C-dots) by heparin.

Ratiometric electrochemical immunoassay based on internal reference value for reproducible and sensitive detection of tumor marker.

A ratiometric assay in an electrochemical immunosensor for tumor marker, herein carcinoembryonic antigen (CEA) was chosen as a model analyte, was developed to improve simplicity and accuracy. The immunosensor was fabricated via the simple expedient way of using Polythionine-gold (PTh-Au) as electrode modified material to be an internal reference signal and K3[Fe(CN)6] in electrolyte as an indicator signal. When the CEA was fixed on the modified electrode via immunoreaction, only the indicator signal sensitively altered; by contrast, the internal reference signal of PTh-Au remained constant at a suitable pH of the electrolyte. The ratio between the alterations of the indicator signal of K3[Fe(CN)6] and the constant internal reference signal can be used to monitor the concentration of CEA reliably, reproducibly, and sensitively. The prepared ratiometric electrochemical immunosensor could detect CEA with good specificity within a wide linear range from 0.005ng/ml to 40ng/ml with a detection limit of 2.2pg/ml. Additionally, experimental results confirm that our proposed method is practical. Thus, this method can expand to recognize and test other protein markers.