Interfacial Electrochemical Electron Transfer Processes in Bacterial Biofilm Environments on Au(111)

Abstract

We have studied Streptococcus mutans (S. mutans) biofilm growth and growth inhibition on Au(111)-surfaces using atomic force microscopy (AFM) and interfacial electrochemistry of a number of redox probe molecules. AFM of the biofilm growth and growth inhibition on both mica and Au(111)-surfaces was followed by sampling at given times, drying the samples naturally, and imaging. The electrochemical investigations were based on single-crystal Au(111)-electrode surfaces to exclude polycrystallinity as a cause of inhomogeneous voltammetric broadening on the biofilm covered electrode surfaces. The redox couples were chosen for their positive ([Ru(NH(3))(6)](3+/2+), [Co(terpy)(2)](3+/2+), terpy = 2,2',2''-terpyridine) or negative ([Fe(CN)(6)](3-/4-), [IrCl(6)](3-/4-)) electrostatic charge. [Co(NH(3))(6)](3+/2+) and [Co(phen)(3)](3+/2+) (phen = 1,10-phenanthroline) were other inhibition factors investigated. The positively and negatively charged redox probe couples displayed antagonistic inhibition and voltammetric patterns. [Ru(NH(3))(6)](3+/2+) and the homologous compound [Co(NH(3))(6)](3+/2+) were the only probe compounds to effect growth inhibition. On the other hand, cyclic voltammetry (CV) of both [Ru(NH(3))(6)](3+/2+) (positively charged, biofilm growth inhibitor) and [Co(terpy)(2)](3+/2+) (positively charged, no biofilm growth inhibition) displayed fully reversible CV on biofilm covered electrodes, almost indistinguishable from CV at bare Au(111)-electrode surfaces. In comparison, CVs of [Fe(CN)(6)](3-/4-) and [IrCl(6)](3-/4-) (both negatively charged and no growth inhibition) were distorted from planar diffusion behavior on bare Au(111)-electrode surfaces toward spherical diffusion behavior on S. mutans biofilm covered Au(111)-electrode surfaces. DNAase treatment of the biofilm covered Au(111)-electrode surface partly restores planar diffusion CV of [Fe(CN)(6)](3-/4-) and [IrCl(6)](3-/4-). This is reflected in a decrease of the growth rate and the appearance of molecular-scale structures near the bacterial edges as imaged by AFM after DNAase treatment. A rationale for the different voltammetric behavior of positively and negatively charged redox probe molecules based on electrostatic properties of the local surface environment is offered.

Investigation of Streptococcus mutans biofilm growth on modified Au(111)-surfaces using AFM and electrochemistry

“…These cells do not form biofilms but are distributed individually over the alkanthiol SAM surface. This pattern is in contrast to bare Au(1 1 1)-or mica surfaces [21]. Individually scattered degraded cells are seldom found on the fully developed biofilms on modified Au(1 1 1)-surface, say after 24 hour growth.…”

“…S. mutans was stored in Brain Heart Infusion broth (BHI) medium at À80°C and incubated in BHI broth shaken at 37°C overnight [21].…”

“…S mutans is highly acidogenic, producing short-chain carboxylic acids which dissolve even hard tissues such as dental enamel and dentine [18], ultimately leading to dental decay. A significant number of studies have focused on S .mutans using AFM [18][19][20][21][22][23][24][25][26], to address adhesion forces between bacterial biofilms and different dental materials such as dental enamel and dentine. High-resolution images of different S. mutans strains have also been obtained.…”

“…In a recent study we reported a combined AFM/voltammetric approach, targeted on S. mutans, and focused on electrostatic interactions between the negatively charged bacteria and both the electrode surface, and positively and negatively charged electroactive probe molecules, with the following observations [21]:…”

Microscopic imaging ellipsometry of submicron-scale bacterial cells

“…S. mutans was incubated in BHI medium shaken at 37 ºC overnight. Another solution consists of 7.4 g BHI, 20 g sucrose, and 200 ml Millipore water was prepared to grow bacterial cells on Au film [19]. A substrate consists of SiO 2 300 nm/ Ti 40 nm/ Au 50 nm was cleaned by sonication in acetone and isopropanol solutions for 30 and 20 minutes respectively.…”