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

摘要

This paper demonstrates a novel strategy for site-selectivencell adhesion and in situ cultivation of living cells,nintegrated with real-time monitoring of cellular smallnbiomolecules based on dual functional protein microarrays.nThe protein microarrays have been produced on thensuperhydrophobic|philic Au-TiO2 micropatterns, throughnfurther modification of L-cysteine (Cys) and followednby successive immobilization of a model protein,ncytochrome c (cyt c). Experimental results have revealednthat the created cyt c microarrays play dualnfunctions: one is employed as a robust substrate fornsite-selective cell adhesion and in situ cultivation ofnliving cells, because the protein microarrays exhibitnhigh selectivity and bioaffinity toward cells, as well asnlong biostability under cell culture condition up to 7ndays. Meanwhile, the cyt c microarrays can also servenas sensing elements for hydrogen peroxide (H2O2) duento the inherent enzymatic activity of the heme centernin cyt c. Direct electron transfer of cyt c has beennenhanced at the Cys-modified Au-TiO2 (Au-TiO2/Cys)nmicroarrays, and the electrochemical behavior can bentuned by varying the width and spacing of the microbandnarrays. Furthermore, cyt c is stably immobilizednon the Au-TiO2/Cys microarrays and maintainsnits enzymatic activity after confined on thenmicroarrays. Thus, the optimized cyt c microarraysnshow striking analytical performance for H2O2 determination,ne.g., high sensitivity and selectivity, broadnlinear range from 10-9 M to 10-2 M, low detection limitndown to 2 nM, and short response time within 5 s. Asna result, the excellent analytical properties of the cyt cnmicroarrays, as well as the characteristic of the proteinnmicroarrays themselves, including high selectivity, longnbiostability, and good bioaffinity, opens up a methodnfor selective in situ cultivation of cells integrated withnreal-time detection of signaling biomolecules such asnH2O2 released from living cells, which shows potentialnfor physiological and pathological investigations.