Browsing by Author "Ge, Xudong"
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Item Bioburden detection on surface and water samples in a rapid, ultra-sensitive and high-throughput manner(Wiley, 2024-03-17) Hasan, Md Sadique; Sundberg, Chad Alan; Gilotte, Elias; Ge, Xudong; Kostov, Yordan; Rao, GovindBioburden 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.Item Improving the recombinant human erythropoietin glycosylationusing microsome supplementation in CHO cell-free system(Wiley, 2018) Gurramkonda, Chandrasekhar; Rao, Aniruddha; Borhani, Shayan; Pilli, Manohar; Deldari, Sevda; Ge, Xudong; Pezeshk, Niloufar; Tolosi, Michael; Kostov, Yordon; Tolosa, Leah; Frey, Douglas D.; Rao, GovindCell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the HO cell-free system. However, many complex proteins are still difficult to express using this method. To investigate the current bottlenecks in cell-free glycoprotein production, we chose erythropoietin (40% glycosylated), an essential endogenous hormone which stimulates the development of red blood cells. Here, we report the production of recombinant erythropoietin (EPO) using CHO cell-free system. Using this method, EPO was expressed and purified with a twofold increase in yield when the cell-free reaction was supplemented with CHO microsomes. The protein was purified to near homogeneity using an ion-metal affinity column. We were able to analyze the expressed and purified products (glycosylated cell-free EPO runs at 25–28 kDa, and unglycosylated protein runs at 20 kDa on an SDS–PAGE), identifying the presence of glycan moieties by PNGase shift assay. The purified protein was predicted to have ∼2,300 IU in vitro activity. Additionally, we tested the presence and absence of sugars on the cell-free EPO using a lectin-based assay system. The results obtained in this study indicate that microsomes augmented in vitro production of the glycoprotein is useful for the rapid production of single doses of a therapeutic glycoprotein drug and to rapidly screen glycoprotein constructs in the development of these types of drugs. CFPS is useful for implementing a lectin-based method for rapid screening and detection of glycan moieties, which is a critical quality attribute in the industrial production of therapeutic glycoproteins.Item Microwave induced thermally assisted solvent-based bonding of biodegradable thermoplastics: an eco-friendly rapid approach for fabrication of microfluidic devices and analyte detection(Nature, 2022-09-27) Hasan, Md Sadique; Borhani, Shayan; Ramamurthy, Sai Sathish; Andar, Abhay; Ge, Xudong; Choa, Fow-Sen; Kostov, Yordan; Rao, GovindThere 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.Item Minimally invasive technique for measuring transdermal glucose with a fluorescent biosensor(Springer Berlin Heidelberg, 2018-08-17) Brown, Sheniqua; Zambrana, Paige N.; Ge, Xudong; Bagdure, Dayanand; Stinchcomb, Audra L.; Rao, Govind; Tolosa, LeahThere is a need for blood glucose monitoring techniques that eliminate the painful and invasive nature of current methods, while maintaining the reliability and accuracy of established medical technology. This research aims to ultimately address these shortcomings in critically ill pediatric patients. Presented in this work is an alternative, minimally invasive technique that uses microneedles (MN) for the collection of transdermal glucose (TG). Due to their comparable skin properties, diffusion studies were performed on full thickness Yucatan miniature pig skin mounted to an in-line diffusion flow cell and on different skin sites of human subjects. Collected TG samples were measured with a L255C mutant of the E. coli glucose-binding protein (GBP) with an attached fluorescent probe. The binding constant (Kd = 0.67 μM) revealed the micromolar sensitivity and high selectivity of the his-tagged GBP biosensor for glucose, making it suitable for TG measurements. In both the animal and human models, skin permeability and TG diffusion across the skin increased with MN application. For intact and MN-treated human skin, a significant positive linear correlation (r > 0.95, p < 0.01) existed between TG and BG. The micromolar sensitivity of GBP minimized the volume required for interstitial fluid glucose analysis allowing MN application time (30 s) to be shortened compared to other studies. This time reduction can help in eliminating skin irritation issues and improving practical use of the technique by caregivers in the hospital. In addition, the his-tagged optical biosensor used in this work can be immobilized and used with a portable sensing fluorometer device at the point of care (POC) making this minimally invasive technology more ideal for use in the pediatric intensive care unit.Item Minimally invasive technique for measuring transdermal glucose with a fluorescent biosensor(Springer Nature Switzerland AG., 2018-08-31) Brown, Sheniqua; Zambrana, Paige N.; Ge, Xudong; Bagdure, Dayanand; Stinchcomb, Audra L.; Rao, Govind; Tolosa, LeahThere is a need for blood glucose monitoring techniques that eliminate the painful and invasive nature of current methods, while maintaining the reliability and accuracy of established medical technology. This research aims to ultimately address these shortcomings in critically ill pediatric patients. Presented in this work is an alternative, minimally invasive technique that uses microneedles (MN) for the collection of transdermal glucose (TG). Due to their comparable skin properties, diffusion studies were performed on full thickness Yucatan miniature pig skin mounted to an in-line diffusion flow cell and on different skin sites of human subjects. Collected TG samples were measured with a L255C mutant of the E. coli glucose-binding protein (GBP) with an attached fluorescent probe. The binding constant (Kd = 0.67 μM) revealed the micromolar sensitivity and high selectivity of the his-tagged GBP biosensor for glucose, making it suitable for TG measurements. In both the animal and human models, skin permeability and TG diffusion across the skin increased with MN application. For intact and MN-treated human skin, a significant positive linear correlation (r > 0.95, p < 0.01) existed between TG and BG. The micromolar sensitivity of GBP minimized the volume required for interstitial fluid glucose analysis allowing MN application time (30 s) to be shortened compared to other studies. This time reduction can help in eliminating skin irritation issues and improving practical use of the technique by caregivers in the hospital. In addition, the his-tagged optical biosensor used in this work can be immobilized and used with a portable sensing fluorometer device at the point of care (POC) making this minimally invasive technology more ideal for use in the pediatric intensive care unit.Item Rapid Bacterial Detection and Identification of Bacterial Strains Using Machine Learning Methods Integrated With a Portable Multichannel Fluorometer(IEEE, 2023-08-09) Hasan, Md Sadique; Sundberg, Chad; Hasan, Hasibul; Kostov, Yordan; Ge, Xudong; Choa, Fow-Sen; Rao, GovindRapid and sensitive bioburden detection is of paramount importance in different applications including public health, and food and water safety. To overcome the traditional limitations of bacterial detection i.e., lengthy culture time, and complicated procedure, a low-cost, portable multichannel fluorometer coupled with machine learning (ML) has been implemented in this study. Five different strains of bacterial samples were tested along with the negative control for time-series fluorescence data collection and analysis. We applied different conventional unsupervised and supervised machine learning techniques with extracted features followed by preprocessing of the data. Initially, machine learning algorithms were applied for the qualitative detection of bacteria by binary classification followed by regression analysis to predict the level of contamination for E. coli. The multiclass classification was used to identify gram-positive, and gram-negative bacterial strains and differentiate all the bacterial strains tested. Our results show that around 97.9% accuracy can be achieved for bacterial contamination detection for as low as 1 CFU/mL while 92.1% accuracy can be achieved for differentiating the gram-positive and gram-negative strains. Additionally, with 1 minute of data, high accuracy is obtained for detecting bioburden, proving the multichannel fluorometer’s rapid detection capability. The multichannel fluorometer integrated with ML analytics is capable of automating data analysis and determining accurate and rapid bacterial detection on-site with the prediction of bioburden levels and differentiating bacterial strains and the protocol can be applied to the biosensors with a similar data type.Item Rapid recombinant protein expression in cell-free extracts from human blood(Nature, 2018-06-22) Burgenson, David; Gurramkonda, Chandrasekhar; Pilli, Manohar; Ge, Xudong; Andar, Abhay; Kostov, Yordan; Tolosa, Leah; Rao, GovindSeveral groups have recently reported on the utility of cell-free expression systems to make therapeutic proteins, most of them employing CHO or E. coli cell-free extracts. Here, we propose an alternative that uses human blood derived leukocyte cell extracts for the expression of recombinant proteins. We demonstrate expression of nano luciferase (Nluc), Granulocyte-colony stimulating factor (G-CSF) and Erythropoietin (EPO) in cell-free leukocyte extracts within two hours. Human blood is readily available from donors and blood banks and leukocyte rich fractions are easy to obtain. The method described here demonstrates the ability to rapidly express recombinant proteins from human cell extracts that could provide the research community with a facile technology to make their target protein. Eventually, we envision that any recombinant protein can be produced from patient-supplied leukocytes, which can then be injected back into the patient. This approach could lead to an alternative model for personalized medicines and vaccines.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.