Real-time electrochemical monitoring of cellular H₂O₂ integrated with in situ selective cultivation of living cells based on dual functional protein microarrays at Au-TiO₂ surfaces

This paper demonstrates a novel strategy for site-selective cell adhesion and in situ cultivation of living cells, integrated with real-time monitoring of cellular small biomolecules based on dual functional protein microarrays. The protein microarrays have been produced on the superhydrophobic|philic Au-TiO₂ micropatterns, through further modification of l-cysteine (Cys) and followed by successive immobilization of a model protein, cytochrome c (cyt c). Experimental results have revealed that the created cyt c microarrays play dual functions: one is employed as a robust substrate for site-selective cell adhesion and in situ cultivation of living cells, because the protein microarrays exhibit high selectivity and bioaffinity toward cells, as well as long biostability under cell culture condition up to 7 days. Meanwhile, the cyt c microarrays can also serve as sensing elements for hydrogen peroxide (H ₂O₂) due to the inherent enzymatic activity of the heme center in cyt c. Direct electron transfer of cyt c has been enhanced at the Cys-modified Au-TiO₂ (Au-TiO₂/Cys) microarrays, and the electrochemical behavior can be tuned by varying the width and spacing of the microband arrays. Furthermore, cyt c is stably immobilized on the Au-TiO ₂/Cys microarrays and maintains its enzymatic activity after confined on the microarrays. Thus, the optimized cyt c microarrays show striking analytical performance for H₂O₂ determination, e.g., high sensitivity and selectivity, broad linear range from 10^-9 M to 10^-2 M, low detection limit down to 2 nM, and short response time within 5 s. As a result, the excellent analytical properties of the cyt c microarrays, as well as the characteristic of the protein microarrays themselves, including high selectivity, long biostability, and good bioaffinity, opens up a method for selective in situ cultivation of cells integrated with real-time detection of signaling biomolecules such as H₂O₂ released from living cells, which shows potential for physiological and pathological investigations.