Organic Cyanide Decorated SERS Active Nanopipettes for Quantitative Detection of Hemeproteins and Fe3+ in Single Cells

It is challenging to develop a robust nanoprobe for real-time operational and accurate detection of heavy metals in single cells. Fe-CN coordination chemistry has been well studied to determine the structural characteristics of hemeproteins by different techniques. However, the frequently used cyanide ligands are inorganic molecules that release cyanide anion under particular conditions and cause cyanide poisoning. In the present study, organic cyanide (4-mercaptobenzonitrile, MBN) was utilized for the first time in developing a facile nanoprobe based on surface-enhanced Raman scattering (SERS) for quantitative detection of hemeproteins (oxy-Hb) and trivalent iron (Fe³⁺) ions. The nanoprobe prepared by coating the glass capillary tip (100 nm) with a thin gold film, which enables highly localized study in living cell system. The cyanide stretching vibration in MBN was highly sensitive and selective to Fe³⁺ and oxy-Hb with excellent binding affinity (K_d 0.4 pM and 0.1 nM, respectively). The high sensitivity of the nanoprobe to analyte (Fe³⁺) was attributed to the two adsorption conformations (-SH and -CN) of MBN to the gold surface. Therefore, MBN showed an exceptional dual-peak (2126 and 2225 cm^-1) behavior. Furthermore, the special Raman peaks of cyanide in 2100-2300 cm^-1 (silent region of SERS spectra) are distinguishable from other biomolecules characteristic peaks. The selective detection of Fe³⁺ in both free and protein-bound states in aqueous solution is achieved with 0.1 pM and 0.08 μM levels of detection limits, respectively. Furthermore, practical applicability of fabricated nanoprobe was validated by detection of free Fe³⁺ in pretreated living HeLa cells by direct insertion of a SERS active nanoprobe. Regarding the appropriate precision, good reproducibility (relative standard deviation, RSD 7.2-7.6%), and recyclability (retain good Raman intensity even after three renewing cycles) of the method, the developed sensing strategy on a nanopipette has potential benefits for label-free, qualitative and quantitative recognition of heavy metal ions within nanoliter volumes.