<strong>MONITORING OF MICROORGANISMS WITH FUNCTIONALIZED MICRO BEADS ON A DIFFUSOMETRIC PLATFORM</strong>

Abstract

Sepsis is a fatal infectious disease claiming thousands of lives every year. Antimicrobial susceptibility testing (AST) plays a pivotal role in the success of treatments. However, the turnaround time for the outcome of conventional AST usually requires over 24 hours, resulting in high patient mortality1. Moreover, antibiotics abuse can also incubate the booming of superbugs. A reliable and efficient drug screen becomes increasingly important to date to save lives in a timely fashion. To this end, a technique combining optical diffusometry and bead-based immunoassays is developed herein to achieve a rapid quantification of target microorganisms. For rapid ASTs, a variety of techniques, such as image analysis2, dielectrophoretic force3, and Raman-enhancing spectrum4, have been developed in the recent years. Nevertheless, complicated procedures and equipments impede their prevalence in clinics. Lately, bead-based immunoassays are emerging because of high flexibility. Taking advantaging of the concept, Kinnunen et al.5 used magnetic bead rotation integrating immunoassays to monitor growth of bacteria. However, a sophisticated driving magnetic source was required. Unlike the abovementioned techniques, Brownian motion is a self-driven phenomenon. According to the Stokes-Einstein relation, particle diffusivity is a function of particle size6. When the particle size is increased from conjugated target analytes, the particle diffusivity will decrease. A proof of concept was firstly carried out in a pilot study conducted by Gorti et al.6 in detecting the M13 virus. In this work, they explored the detection of virus by measuring the change of particle diffusivity. The successful attempt then enabled the potential to quantify low-abundance microorganisms in the same manner. In this study, an optical diffusometric platform was used to monitor two microorganisms, P. aeruginosa and S. aureus. The platform was composed of an inverted epifluorescent microscope (IX71), a digital camera (Flea®3, Point Grey), a microchip and a computer. Bead-based immunoassays were used to detect desired bacteria suspended in physiological fluids (Fig. 1). Quantitative analysis of Brownian motion was realized by the spatially cross-correlation algorithm. Two amine-modified color particles (2-μm, Sigma) were respectively conjugated with anti-P. aeruginosa IgG and anti-S. aureus IgG and then incubated with P. aeruginosa and S. aureus bacteria for an hour. The bacteria were firmly attached to the functionalized particles. The limit of detection could achieve 100 CFU/mL. Two bacteria were also measured at different concentrations in a mixed sample by using triple color particles composed of equal amounts of anti-P. aeruginosa Ab- (orange), anti-S. aureus Ab- (red), and anti-TNF-α (green) Ab-functionalized particles (Fig. 2). The experimental result agrees with the theoretical prediction (Fig. 3). When the bacterium-conjugated particles were further mixed with gentamicin in the TSB solution at 37 °C for 2 h, particle images were recorded every 20 min with a 10× objective to monitor the bacterial activity. Results indicated that effective concentrations of the antibiotic, gentamicin, on the co-cultured bacteria could be explicitly distinguished from their temporal diffusivity changes (Fig. 4). This study provides valuable information to timely treatments against polymicrobial diseases in the near future.