Signal Amplification and Near-Infrared Translation of Enzymatic Reactions by Nanosensors

Enzymatic reactions are used to detect analytes in a range of biochemical methods such as enzyme-linked immunosorbent assays (ELISAs). To measure the presence of an analyte, they are conjugated to a recognition unit and convert a substrate into a (colored) product that is detectable by visible (VIS) light. Thus, the lowest enzymatic turnover that can be detected sets a limit on sensitivity. Here, we report that substrates and products of horseradish peroxidase (HRP) and β-galactosidase change the near-infrared (NIR, 800-2400 nm) fluorescence of (bio)polymer modified single-walled carbon nanotubes (SWCNTs). Therefore, SWCNTs translate a VIS signal into a beneficial NIR signal. Moreover, the affinity of the nanosensors cause a higher effective local concentration of the reactants for the optical measurement. This leads to a non-linear sensor-based signal amplification and translation (SENSAT). We find signal enhancement up to ≈ 120x for the HRP substrate p-phenylenediamine (PPD), which means that reactions below the limit of detection in the VIS can be followed in the NIR (≈ 1000 nm). The approach is also applicable to other substrates such as 3,3’-5,5’-tetramethylbenzidine (TMB) and direct observation of the HRP reaction. An adsorption-based theoretical model fits the observed signals and corroborates the sensor-based enhancement mechanism. This approach amplifies signals, translates them into the NIR and increases sensitivity of biochemical assays.