Rapid Ultrasensitive and High-Throughput Bioburden Detection: Microfluidics and Instrumentation

Abstract

Contamination detection often requires lengthy culturing steps to detect low-level bioburden. To increase the rate of detection and decrease the limit of detection (LOD), a system featuring microfluidics and a multichannel fluorometer has been developed. The 8-channel fluorometer enables parallel testing of multiple samples with LOD as low as < 1 CFU/mL. This low-cost system utilizes the slope of fluorescence intensity that serves as the criterion for bioburden detection. The redox indicator dye resazurin is used to monitor the presence of viable cells, in this study and is reduced to resorufin with a high quantum yield at 585 nm. The sample under investigation is spiked with resazurin, loaded in a special-design microfluidic cassette and the rate of change is observed via the fluorometer. The method was validated using primary E. coli culture in comparison with a spectrophotometer which served as the gold standard. An optimized assay based on LB media was developed. The impact on the assay sensitivity based on incubation and filtration steps was also explored. The assay is shown to pick up inadvertent contamination from test tubes and pipette tips showing its applicability in real-world settings. The data analysis demonstrated a comparable performance of the multichannel fluorometer vis-a-vis the conventional plate reader. The multichannel system is shown to detect bioburden presence in as low as 20 seconds for bacterial concentrations ≥ 5 CFU/mL after 6 hours of incubation. Considering its portability, low cost, simplicity of operation and relevant assay sensitivity, the system is well-positioned to detect low-level bioburden in the laboratory, pharmaceutical and field settings.

A novel, low-cost microfluidic device with an integrated filter for rapid, ultrasensitive, and high-throughput bioburden detection

“…For this, the diluted samples from 1000 CFU/mL to 1 CFU/mL were prepared. After preparing different samples, they were incubated in shake flasks at 37 °C for 6 h as optimized in our previous publication 13 . In parallel, 200 µL of each sample was spread on an LB agar plate, and the plates were placed in an incubator at 37 °C overnight.…”

“…Sample preparation, filtration and fluorescence measurement procedure. The preparation of primary and secondary culture cells is done in the same way as our previous publications 13 . Briefly, a 50 mL primary culture was prepared using 200 µL of Bacillus cells, which was grown at 37 °C in a shaker at 150 rpm (Labline Instruments, Melrose Park, IL) overnight.…”

“…In recent years, resazurin has been used in cell viability measurements to measure the oxidation–reduction of a given sample. To detect bioburden rapidly and in a sensitive manner, our previous study 13 demonstrated the use of a USB-powered portable fluorometer for detecting viable cells in a given sample based on the detection of high quantum yield resorufin from resazurin. In this low-cost system, fluorescence intensity slope served as a criterion for detecting bioburden.…”

“…Bacterial trapping is done on the filter paper and detection is done in situ. After trapping the cells onto the filter paper, a resazurin-based assay is used as previously reported, and the fluorescence change due to the trapped cells (if any) is monitored using the previously developed multichannel fluorometer 13 . To the best of our knowledge, in this study, we have shown microwave-induced bonding to develop a microfluidic device with integrated filter papers for the first time for bioburden detection.…”