Browsing by Author "Tolosa, Michael"
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Item Rapid ultra-sensitive and high-throughput bioburden detection: Microfluidics and instrumentation(ACS, 2022-06-06) Hasan, Md Sadique; Marsafari, Monireh; Tolosa, Michael; Andar, Abhay; Ramamurthy, Sai Sathish; Ge, Xudong; Kostov, Yordan; Rao, GovindContamination 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.Item Real‐time dissolved carbon dioxide monitoring I: Application of a novel in situ sensor for CO2 monitoring and control(Wiley Online Library, 2019-12-16) Chopda, Viki R.; Holzberg, Timothy; Ge, Xudong; Folio, Brandon; Tolosa, Michael; Kostov, Yordan; Tolosa, Leah; Rao, GovindDissolved carbon dioxide (dCO₂) is a well‐known critical parameter in bioprocesses due to its significant impact on cell metabolism and on product quality attributes.Processes run at small‐scale faces many challenges due to limited options for modular sensors for online monitoring and control. Traditional sensors are bulky, costly, and invasive in nature and do not fit in small‐scale systems. In this study, we present the implementation of a novel, rate‐based technique for real‐time monitoring of dCO₂ in bioprocesses. A silicone sampling probe that allows the diffusion of CO₂ through its wall was inserted inside a shake flask/bioreactor and then flushed with air to remove the CO₂ that had diffused into the probe from the culture broth (sensor was calibrated using air as zero‐point calibration). The gas inside the probe was then allowed to recirculate through gas‐impermeable tubing to a CO₂ monitor. We have shown that by measuring the initial diffusion rate of CO₂ into the sampling probe we were able to determine the partial pressure of the dCO₂ in the culture. This technique can be readily automated, and measurements can be made in minutes.Demonstration experiments conducted with baker’s yeast and Yarrowia lipolytica yeast cells in both shake flasks and mini bioreactors showed that it can monitor dCO₂ in real‐time. Using the proposed sensor, we successfully implemented a dCO₂‐based control scheme, which resulted in significant improvement in process performance.Item Real‐time dissolved carbon dioxide monitoring II: Surface aeration intensification for efficient CO2 removal in shake flasks and mini‐bioreactors leads to superior growth and recombinant protein yields(Wiley Online Library, 2019-12-13) Chopda, Viki R.; Holzberg, Timothy; Ge, Xudong; Folio, Brandon; Wong, Lynn; Tolosa, Michael; Kostov, Yordan; Tolosa, Leah; Rao, GovindMass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O₂ (DO) and dissolved CO₂ (dCO₂) is of paramount importance. At bioreactor level these parameters can be monitored online and can be controlled by sparging air/oxygen or stirrer speed. However, traditional small‐scale systems such as shake flasks lack real time monitoring and also employ only surface aeration with additional diffusion limitations imposed by the culture plug. Here we present implementation of intensifying surface aeration by sparging air in the headspace of the reaction vessel and real‐time monitoring of DO and dCO₂ in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO₂ at low level, which significantly improved not only biomass growth but also protein yield. We expect that implementing such controlled smart shake flasks can minimize the process development gap which currently exists in shake flask level and bioreactor level results.