Miniaturization of an enclosed electrospinning process to enhance reproducibility in the fabrication of rapidly dissolving cell-based biosensors.

There is broad interest in producing electrospun films embedded with biological materials. It is well known that electrospinning requires careful control of the process conditions, especially the environmental conditions such as relative humidity (RH). Given that commercial electrospinning systems are expensive (> $10,000) and are typically too large to be used in standard biological safety cabinets (BSC), we designed and built a miniaturized electrospinning box (E-Box) that will fit inside a BSC, and the RH can be easily controlled using simple instrumentation (gas cylinder, regulator, needle valve, rotameter). It uses an inexpensive computerized numerical control machine to control the spinneret positioning and collector rotational speed-all the parts for the device (except the syringe pump and voltage supply) can be purchased for approximately $1000. We demonstrate the usefulness of our design in optimizing the production of Escherichia coli-embedded pullulan-trehalose films to be used as rapidly dissolving biosensors for environmental monitoring. At a fixed electrospinning recipe, we showed that decreasing the RH from approximately 48% to 22% resulted in the average fiber diameter increasing from 240 (± 11) nm to 314 (± 8) nm. We also demonstrate the usefulness of our design in performing sequential electrospinning experiments to evaluate process performance reproducibility. For example, from just 1 mL of a polymer solution, we produced 16 electrospun films (approximately 3 cm by 8 cm each)-from those films we hole-punched approximately 80 biosensor discs which were then used in subsequent experiments to determine the amount of two different biocides (Grotan BK and triclosan) in aqueous samples. The technique developed in this study is ideal for creating electrospun materials in high quantities that are highly reproducible through the precise control of RH.

Tuning the sensitivity of cell-based biosensors for the detection of biocides.

The presence of biocides in wastewater can negatively impact the efficiency of wastewater treatment processes, particularly the process of nitrification. In this paper, we describe the development of cell-based biosensors (CBBs) with tunable levels of sensitivity for rapidly detecting the presence and predicting the type and concentration of biocides. The CBB assay developed is performed by first exposing a panel of bacterial strains (E. coli, B. subtilis, B. cereus) to the sample being tested and to the control sample without biocide, and then adding a fluorescent dye (LIVE/DEAD BacLight). We then compare the fluorescence signals generated by the two samples, and the differences in the signals indicate the presence of a biocide, as previously reported in the literature. We found that the sensitivity of the CBB assay can be improved by 'tuning' the type/salinity of the buffer used to suspend the cells, and by changing the number of cells used in the assay. These changes improved the level of detection (LOD) of the biocide Cetyltrimethylammonium bromide (CTAB) from 10 ppm to 0.625 ppm and the biocide Grotan® BK from 500 ppm to 7.8 ppm. With the optimized conditions for each strain, we also establish that the combined response from the panel of bacterial strains can be used to predict the type and concentration of biocide sample tested. Additionally, we provide evidence that the CBB assay can be performed using a compact, commercially available fluorometer. Overall, the significance of this work will improve point-of-use testing and enable the discrimination between biocide-containing samples of similar toxicity and detection of lower toxicity samples, thereby improving the accuracy of the CBB assay.

Navigating Performance Standards for Face Mask Materials: A Custom-Built Apparatus for Measuring Particle Filtration Efficiency.

Public health agencies have recommended the community use of face masks to reduce the transmission of airborne diseases like COVID-19. Virus transmission is reduced when masks act as efficient filters, thus evaluating mask particle filtration efficiency (PFE) is essential. However, the high cost and long lead times associated with purchasing turn-key PFE systems or hiring certified laboratories hampers the testing of filter materials. There is a clear need for "custom" PFE test systems; however, the variety of standards that prescribe (medical) face mask PFE testing (e.g., ASTM International, NIOSH) vary widely in their protocols and clarity of guidelines. Herein, the development is described of an "in-house" PFE system and method for testing face masks in the context of current standards for medical masks. Pursuant to the ASTM International standards, the system uses an aerosol of latex spheres (0.1 µm nominal size) with particle concentrations upstream and downstream of the mask material measured using a laser particle analyzer. PFE measurements are obtained for a variety of common fabrics and medical masks. The approach described in this work conforms to the current standards for PFE testing while providing the flexibility to adapt to changing needs and filtration conditions.

A Rapid Assay to Assess Nitrification Inhibition Using a Panel of Bacterial Strains and Partial Least Squares Modeling.

As a proof of concept, a rapid assay consisting of a cell-based biosensor (CBB) panel of pure bacterial strains, a fluorescent dye, and partial least squares (PLS) modeling was developed to assess the nitrification inhibition potential of industrial wastewater (WW) samples. The current standard method used to assess the nitrification inhibition potential is the specific nitrification rate (SNR) batch test, which requires approximately 4 h to complete under the watch of an experienced operator. In this study, we exposed the CBB panel of seven bacterial strains (nitrifying and non-nitrifying) to 28 different industrial WW samples and then probed both the membrane integrity and cellular activity using a commercially available "live/dead" fluorescent dye. The CBB panel response acts as a surrogate measurement for the performance of nitrification. Of the seven strains, four (Nitrospira, Escherichia coli, Bacillus subtilis, Bacillus cereus) were identified via the modeling technique to be the most significant contributors for predicting the nitrification inhibition potential. The key outcome from this work is that the CBB panel fluorescence data (collected in approximately 10 min) can accurately predict the outcome of an SNR batch test (that takes 4 h) when performed with the same WW samples and has a strong potential to approximate the chemical composition of these WW samples using PLS modeling. Overall, this is a powerful technique that can be used for point-of-use detection of nitrification inhibition.

Optimization of microorganism preservation conditions for the development of an acute toxicity bioassay for biocides.

Biocides, also referred to as 'microbicides' or 'inhibitors', are widely used in industrial processes (e.g. utility water in cooling towers) to control and/or eliminate the growth of microorganisms. Because of their inherent toxicity, their presence in various sources (e.g. river sediments, potable water) can negatively affect ecosystems. Currently available biocide detection techniques are not suitable for 'point-of-use' applications since they are tedious, complicated, and often require experienced personnel to operate. To address this concern, we sought to develop a simple-to-use toxicity bioassay based on a model microorganism (E. coli) after short (<30 min) exposure to known biocides that can be stored at room temperature (preferably) or in the fridge. Based on recent work and our expertise in polymer-based preservation of biomolecules, we leveraged this knowledge to improve E. coli preservation for biocide detection purposes. A design-of-experiments strategy was used to evaluate 16 different preservation conditions from 5 process parameters (i.e. 25-1 fractional factorial). It was found that pullulan, a sugar-based polymer, improved E. coli culturability by an order of magnitude after three months of storage. Also, it was found that storing E. coli in the fridge in Milli-Q water was favorable for maintaining a high level of culturability. Finally, the toxicity of three common biocides (Cetyltrimethylammonium bromide (CTAB), ProClin™ 300, and Grotan® BK) was evaluated using a fluorescence-based assay across all 16 preservation conditions. The response of the preserved E. coli was biocide specific and at certain conditions did not vary during the entire three-month storage period.