Quantifying picomoles of analyte from less than 100 live bacteria: A novel method with a buffering hydrogel as an electrochemical cell

Abstract

Microenvironmental changes in the chemical surrounding of bacterial cells might have a profound impact on the ecology of biofilms. However, quantifying total amount of picomoles of analyte from a miniscule number of bacteria is an analytical challenge. Here we provide a novel microliter volume hydrogel based electrochemical cell platform suitable of coulometrically measuring hydrogen peroxide (H2O2) produced by less than 100 cells of Streptococcus sanguinis, a relevant member of the healthy oral microbiome. A morpholine moiety was incorporated into the polymer structure of the hydrogel to create a controlled microenvironment at biological pH. We calculated the buffering capacity of this hydrogel as 0.257 ± 0.135 molHNO3molMEA×ΔpHover the pH range of 7.2–6.2 by using a novel method designed for buffering hydrogels. The H2O2 sensors coated in microliter volume of buffering hydrogel showed no change in sensitivity within the pH range of 7.0–3.0, allowing for H2O2 measurements of S. sanguinis independent of any acid they produce. The novel platform was able to measure down to 22.7 ± 3.5 pmol H2O2 produced by less than 100 bacterial cells, which would otherwise not be attainable in large solution-based assays. These findings indicate that this is a suitable platform for quantifying metabolites from sub-milligram biological samples and may even be suitable for direct analysis of raw biofilms samples with little to no sample pretreatment.