Electrical detection of germination of viable model Bacillus anthracis spores in microfluidic biochips

Abstract

In this paper, we present a new impedance-based method to detect viable spores by electrically detecting their germination in real time within microfluidic biochips. We used Bacillus anthracis Sterne spores as the model organism. During germination, the spores release polar and ionic chemicals, such as dipicolinic acid (DPA), calcium ions, phosphate ions, and amino acids, which correspondingly increase the electrical conductivity of the medium in which the spores are suspended. We first present macro-scale measurements demonstrating that the germination of spores can be electrically detected at a concentration of 10⁹ spores ml⁻¹ in sample volumes of 5 ml, by monitoring changes in the solution conductivity. Germination was induced by introducing an optimized germinant solution consisting of 10 mM L-alanine and 2 mM inosine. We then translated these results to a micro-fluidic biochip, which was a three-layer device: one layer of polydimethylsiloxane (PDMS) with valves, a second layer of PDMS with micro-fluidic channels and chambers, and the third layer with metal electrodes deposited on a pyrex substrate. Dielectrophoresis (DEP) was used to trap and concentrate the spores at the electrodes with greater than 90% efficiency, at a solution flow rate of 0.2 μl min⁻¹ with concentration factors between 107–109 spores ml⁻¹, from sample volumes of 1–5 μl. The spores were captured by DEP in deionized water within 1 min (total volume used ranged from 0.02 μl to 0.2 μl), and then germinant solution was introduced to the flow stream. The detection sensitivity was demonstrated to be as low as about a hundred spores in 0.1 nl, which is equivalent to a macroscale detection limit of approximately 10⁹ spores ml⁻¹. We believe that this is the first demonstration of this application in microfluidic and BioMEMS devices.