Mass transfer is known to play a critical role in bioprocess performance and henceforth monitorin... more Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O 2 (DO) and dissolved CO 2 (dCO 2) 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 dCO 2 in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO 2 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.
There is a need for blood glucose monitoring techniques that eliminate the painful and invasive n... more There 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 (K = 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 hum...
Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable pr... more Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (µCol) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. µCols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any HPLC system. µCols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared to current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and µCol validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was employed to validate the experimental performance of the µCols. In this study, µCols were used for improving the purification achieved for granulocyte-colony stimulating factor (GCSF) expressed using a cell-free CHO in-vitro translation (IVT) system and were compared to a conventional 1 mL IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin and purification time for the μCols compared to conventional columns for similar protein yields. This article is protected by copyright. All rights reserved.
Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant thera... more Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the CHO 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 2-fold 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 puri...
The public reporting burden for this collection of information is estimated to average 1 hour per... more The public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing the burden, to the Department of Defense, Executive Services and Communications Directorate (0704-0188). Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number.
Develop a high-performance pCO 2 sensor that is affordable enough to be deployed in geat numbers ... more Develop a high-performance pCO 2 sensor that is affordable enough to be deployed in geat numbers to autonomously monitor the overall patterns of CO 2 emissions and ocean acidification.
Electrodes based on graphite, graphene, and carbon nanomaterials have been used in the anode cham... more Electrodes based on graphite, graphene, and carbon nanomaterials have been used in the anode chamber of microbial fuel cells (MFCs). Carbon quantum dots (C-dots) are a class of versatile nanomaterials hitherto not reported in MFCs. C-dots previously synthesized from coconut husk were reported to possess hydroxyl and carboxyl functional groups on their surface. The presence of these functional groups on a carbon matrix conferred on the C-dots the ability to conduct and transfer electrons. Formation of silver nanoparticles from silver nitrate upon addition of C-dots confirmed their reducing ability. DREAM assay using a mixed microbial culture containing C-dots showed a 172% increase in electron transfer activity and thus confirmed the involvement of C-dots in supplementing redox activity of a microbial culture. Addition of C-dots as a suspension in the anode chamber of an MFC resulted in a 22.5% enhancement in maximum power density. C-dots showed better performance as electron shuttle...
Methods in molecular biology (Clifton, N.J.), 2017
A portable kinetics fluorometer is developed to detect viable cells which may be contaminating va... more A portable kinetics fluorometer is developed to detect viable cells which may be contaminating various samples. The portable device acts as a single-excitation, single-emission photometer that continuously measures fluorescence intensity of an indicator dye and plots it. The slope of the plot depends on the number of colony forming units per milliliter. The device uses resazurin as the indicator dye. Viable cells reduce resazurin to resorufin, which is more fluorescent. Photodiode is used to detect fluorescence change. The photodiode generated current proportional to the intensity of the light that reached it, and an op-amp is used in a transimpedance differential configuration to ensure amplification of the photodiode's signal. A microfluidic chip is designed specifically for the device. It acts as a fully enclosed cuvette, which enhances the resazurin reduction rate. In tests, the E. coli-containing media are injected into the microfluidic chip and the device is able to detect...
Current glucose monitoring techniques for neonates rely heavily on blood glucose monitors which r... more Current glucose monitoring techniques for neonates rely heavily on blood glucose monitors which require intermittent blood collection through skin-penetrating pricks on the heel or fingers. This procedure is painful and often not clinically conducive, which presents a need for a noninvasive method for monitoring glucose in neonates. Our motivation for this study was to develop an in vitro method for measuring passive diffusion of glucose in premature neonatal skin using a porcine skin model. Such a model will allow us to initially test new devices for noninvasive glucose monitoring without having to do in vivo testing of newborns. The in vitro model is demonstrated by comparing uncompromised and tape-stripped skin in an in-line flow-through diffusion apparatus with glucose concentrations that mimic the hypo-, normo-, and hyper-glycemic conditions in the neonate (2.0, 5.0, and 20 mM, respectively). Transepidermal water loss (TEWL) of the tape-stripped skin was approximately 20 g m(-2...
Trichoplusia ni larvae were infected with baculoviruses containing genes for the expression of ul... more Trichoplusia ni larvae were infected with baculoviruses containing genes for the expression of ultraviolet optimized green fluorescent protein (GFPuv) and several product proteins. A GFP-specific optical probe was used to both excite the green fluorescent protein (ex = 385 nm), and subsequently monitor fluorescence emission (em = 514 nm) from outside the infected larvae. The probe's photodetector was connected to a voltmeter, which was used to quantify the amount of GFPuv expressed in infected larvae. Voltage readings were significantly higher for infected vs. uninfected larvae and, by Western analysis, linear with the amount of GFPuv produced. In addition, the probe sensitivity and range were sufficient to delineate infection efficiency and recombinant protein production for model proteins, chloramphenicol acetyltransferase and human interleukin-2. This work represents a critical step in developing an automated process for the production of recombinant proteins in insect larvae.
Single-wall carbon nanotubes (CNT) are one of the most attractive engineered nanomaterials due to... more Single-wall carbon nanotubes (CNT) are one of the most attractive engineered nanomaterials due to their unique electrical, mechanical and thermal properties, and potential use in a variety of commercial products. Due to their small size, CNT could become easily airborne and reach the various environmental compartments and eventually the food chain and humans. However, the environmental fate processes and health
Mass transfer is known to play a critical role in bioprocess performance and henceforth monitorin... more Mass transfer is known to play a critical role in bioprocess performance and henceforth monitoring dissolved O 2 (DO) and dissolved CO 2 (dCO 2) 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 dCO 2 in the bioprocesses to evaluate the impact of intensified surface aeration. We observed that sparging air in the headspace allowed us to keep dCO 2 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.
There is a need for blood glucose monitoring techniques that eliminate the painful and invasive n... more There 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 (K = 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 hum...
Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable pr... more Biopharmaceutical separations require tremendous amounts of optimization to achieve acceptable product purity. Typically, large volumes of reagents and biological materials are needed for testing different parameters, thus adding to the expense of biopharmaceutical process development. This study demonstrates a versatile and customizable microscale column (µCol) for biopharmaceutical separations using immobilized metal affinity chromatography (IMAC) as an example application to identify key parameters. µCols have excellent precision, efficiency, and reproducibility, can accommodate any affinity, ion-exchange or size-exclusion-based resin and are compatible with any HPLC system. µCols reduce reagent amounts, provide comparable purification performance and high-throughput, and are easy to automate compared to current conventional resin columns. We provide a detailed description of the fabrication methods, resin packing methods, and µCol validation experiments using a conventional HPLC system. Finite element modeling using COMSOL Multiphysics was employed to validate the experimental performance of the µCols. In this study, µCols were used for improving the purification achieved for granulocyte-colony stimulating factor (GCSF) expressed using a cell-free CHO in-vitro translation (IVT) system and were compared to a conventional 1 mL IMAC column. Experimental data revealed comparable purity with a 10-fold reduction in the amount of buffer, resin and purification time for the μCols compared to conventional columns for similar protein yields. This article is protected by copyright. All rights reserved.
Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant thera... more Cell-Free Protein Synthesis (CFPS) offers many advantages for the production of recombinant therapeutic proteins using the CHO 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 2-fold 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 puri...
The public reporting burden for this collection of information is estimated to average 1 hour per... more The public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing the burden, to the Department of Defense, Executive Services and Communications Directorate (0704-0188). Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number.
Develop a high-performance pCO 2 sensor that is affordable enough to be deployed in geat numbers ... more Develop a high-performance pCO 2 sensor that is affordable enough to be deployed in geat numbers to autonomously monitor the overall patterns of CO 2 emissions and ocean acidification.
Electrodes based on graphite, graphene, and carbon nanomaterials have been used in the anode cham... more Electrodes based on graphite, graphene, and carbon nanomaterials have been used in the anode chamber of microbial fuel cells (MFCs). Carbon quantum dots (C-dots) are a class of versatile nanomaterials hitherto not reported in MFCs. C-dots previously synthesized from coconut husk were reported to possess hydroxyl and carboxyl functional groups on their surface. The presence of these functional groups on a carbon matrix conferred on the C-dots the ability to conduct and transfer electrons. Formation of silver nanoparticles from silver nitrate upon addition of C-dots confirmed their reducing ability. DREAM assay using a mixed microbial culture containing C-dots showed a 172% increase in electron transfer activity and thus confirmed the involvement of C-dots in supplementing redox activity of a microbial culture. Addition of C-dots as a suspension in the anode chamber of an MFC resulted in a 22.5% enhancement in maximum power density. C-dots showed better performance as electron shuttle...
Methods in molecular biology (Clifton, N.J.), 2017
A portable kinetics fluorometer is developed to detect viable cells which may be contaminating va... more A portable kinetics fluorometer is developed to detect viable cells which may be contaminating various samples. The portable device acts as a single-excitation, single-emission photometer that continuously measures fluorescence intensity of an indicator dye and plots it. The slope of the plot depends on the number of colony forming units per milliliter. The device uses resazurin as the indicator dye. Viable cells reduce resazurin to resorufin, which is more fluorescent. Photodiode is used to detect fluorescence change. The photodiode generated current proportional to the intensity of the light that reached it, and an op-amp is used in a transimpedance differential configuration to ensure amplification of the photodiode's signal. A microfluidic chip is designed specifically for the device. It acts as a fully enclosed cuvette, which enhances the resazurin reduction rate. In tests, the E. coli-containing media are injected into the microfluidic chip and the device is able to detect...
Current glucose monitoring techniques for neonates rely heavily on blood glucose monitors which r... more Current glucose monitoring techniques for neonates rely heavily on blood glucose monitors which require intermittent blood collection through skin-penetrating pricks on the heel or fingers. This procedure is painful and often not clinically conducive, which presents a need for a noninvasive method for monitoring glucose in neonates. Our motivation for this study was to develop an in vitro method for measuring passive diffusion of glucose in premature neonatal skin using a porcine skin model. Such a model will allow us to initially test new devices for noninvasive glucose monitoring without having to do in vivo testing of newborns. The in vitro model is demonstrated by comparing uncompromised and tape-stripped skin in an in-line flow-through diffusion apparatus with glucose concentrations that mimic the hypo-, normo-, and hyper-glycemic conditions in the neonate (2.0, 5.0, and 20 mM, respectively). Transepidermal water loss (TEWL) of the tape-stripped skin was approximately 20 g m(-2...
Trichoplusia ni larvae were infected with baculoviruses containing genes for the expression of ul... more Trichoplusia ni larvae were infected with baculoviruses containing genes for the expression of ultraviolet optimized green fluorescent protein (GFPuv) and several product proteins. A GFP-specific optical probe was used to both excite the green fluorescent protein (ex = 385 nm), and subsequently monitor fluorescence emission (em = 514 nm) from outside the infected larvae. The probe's photodetector was connected to a voltmeter, which was used to quantify the amount of GFPuv expressed in infected larvae. Voltage readings were significantly higher for infected vs. uninfected larvae and, by Western analysis, linear with the amount of GFPuv produced. In addition, the probe sensitivity and range were sufficient to delineate infection efficiency and recombinant protein production for model proteins, chloramphenicol acetyltransferase and human interleukin-2. This work represents a critical step in developing an automated process for the production of recombinant proteins in insect larvae.
Single-wall carbon nanotubes (CNT) are one of the most attractive engineered nanomaterials due to... more Single-wall carbon nanotubes (CNT) are one of the most attractive engineered nanomaterials due to their unique electrical, mechanical and thermal properties, and potential use in a variety of commercial products. Due to their small size, CNT could become easily airborne and reach the various environmental compartments and eventually the food chain and humans. However, the environmental fate processes and health
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Papers by Govind Rao