Bioburden detection on surface and water samples in a rapid, ultra-sensitive and high-throughput manner.

Bioburden detection is crucial for food, water, and biopharmaceutical applications as it can directly impact public health. The objective of this study is to develop and validate an assay and protocol for detecting bioburden on solid surfaces, as well as in water, with high sensitivity and accuracy in a rapid manner. Henceforth, a resazurin-based assay optimized for detecting bioburden has been integrated with a previously developed portable multichannel fluorometer. The microbes were isolated from solid surfaces in different laboratory settings by swabbing technique, and stream water was collected for contamination analysis. Based on the results, the assay and protocol can successfully detect bioburden as low as 20 CFU/cm2 and 10 CFU/mL present in both surface and water samples, respectively.

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

Rapid and accurate bioburden detection has become increasingly necessary for food, health, pharmaceutical and environmental applications. To detect bioburden accurately, and in a highly sensitive manner, we have fabricated a novel microfluidic device with an integrated filter to trap the cells. Bioburden is detected on the filter paper in situ using the redox reaction of fluorescent label resorufin and a portable multichannel fluorometer is used for fluorescence measurement. The microfluidic device was fabricated in a facile, low-cost, and rapid way with microwave-induced thermally assisted bonding. To characterize the bonding quality of the microfluidic cassettes, different tests were performed, and the filter paper material and size were optimized. Primary Bacillus subtilis culture bacterial samples were filtered through the device to validate and investigate the performance parameters. Our results show that a limit of detection (LOD) of 0.037 CFU/mL can be achieved through this microfluidic device whereas the LOD in a normal microfluidic cassette in the fluorometer and the golden standard spectrophotometer are 0.378 and 0.128 CFU/mL respectively. The results depict that three to ten times LOD improvement is possible through this microfluidic cassette and more sensitive detection is possible depending on the volume filtered within a rapid 3 min. This novel microfluidic device along with the fluorometer can be used as a rapid portable tool for highly sensitive, accurate and high-throughput bacterial detection for different applications.

Microwave induced thermally assisted solvent-based bonding of biodegradable thermoplastics: an eco-friendly rapid approach for fabrication of microfluidic devices and analyte detection.

There is an increasing interest in low-cost, facile and versatile thermoplastic bonding for microfluidic applications that can be easily transitioned from laboratory prototyping to industrial manufacturing. In addition, owing to the surge in the usage of thermoplastic microfluidics and its adverse effect on the environment, it is prudent to source alternative materials that are biodegradable, providing a sustainable, green approach. To address the problems, here we introduce an environment friendly, low-cost and safe welding technology used in the fabrication of microcassettes from biodegradable cellulose acetate (CA) thermoplastics. The thermally assisted solvent based bonding of the thermoplastics was accomplished in a domestic microwave oven with the aid of a polyether ether ketone (PEEK) vise. To characterize the quality of the bonding, our in-house technique was compared with a conventional thermally assisted solvent bonding configuration using a heat press machine and tested under different conditions. Our microwave induced bonding of CA presents three times reduced bonding time with higher bonding strength, good reliability and does not necessitate the use of cumbersome instrumentation. Finally, we demonstrate an electrophoresis application and vitamin C detection accomplished using this biodegradable microcassette presenting comparable results with traditional techniques, illustrating the potential of this fabrication technique in different microfluidic applications.

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

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 eight-channel fluorometer enables parallel testing of multiple samples with the 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 and loaded in a special-design microfluidic cassette, and the rate of change is observed via the fluorometer. The method was validated using primary Escherichia coli culture in comparison with a spectrophotometer which served as the gold standard. An optimized assay based on Luria-Bertani medium 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 s for bacterial concentrations ≥5 cfu/mL after 6 h 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.

Wood Microfluidics.

As nonbiodegradable plastics continue to pollute our land and oceans, countries are starting to ban the use of single-use plastics. In this paper, we demonstrated the fabrication of wood-based microfluidic devices and their adaptability for single-use, point-of-care (POC) applications. These devices are made from easily sourced renewable materials for fabrication while exhibiting all the advantages of plastic devices without the problem of nonbiodegradable waste and cost. To build these wood devices, we utilized laser engraving and traditional mechanical methods and have adapted specific surface coatings to counter the wicking effect of wood. To demonstrate their versatility, wood microfluidic devices were adapted for (i) surface plasmon coupled enhancement (SPCE) of fluorescence for detection of proteins, (ii) T-/Y-geometry microfluidic channel mixers, and (iii) devices for rapid detection of microbial contamination. These provide proof of concept for the use of wooden platforms for POC applications. In this study, we measured the fluorescence intensities of recombinant green fluorescent protein (GFP) standards (ranging from 1.5-25 ng/µL) and 6XHis-G-CSF (ranging from 0.1-100 ng/µL) expressed in cell-free translation systems. All tested devices perform as well as or better than their plastic counterparts.