Xudong Ge

High-stability non-invasive autoclavable naked optical CO2 sensor.

The fabrication and characterization of a high-stability non-invasive autoclavable naked optical CO(2) sensor is described in this report. The sensor was made by using 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) as the fluorescence dye and cetyltrimethylammonium hydroxide (CTMAOH) as the phase transfer agent (the base). A highly hydrophobic two-component silicone film was used as the polymer matrix, which overcame some of the limitations of the existing plastic type CO(2) sensors, such as dye leaching and cross-sensitivity to ions. To improve the stability of the sensor, several affecting factors were investigated. Experimental results showed that sufficient base and a small amount of water in the sensing film were critical factors that affected the stability of the sensor. Although the sensor was more stable when kept in water, the function of the sensor could recover when the sensor kept in air was transferred into water. The sensor has a lifetime of several months. The detection limit of the sensing film was about 0.03%. The average response and recovery times were 0.66 and 1.94 min, respectively. It had no cross-sensitivity to salt concentrations in the range of 0-0.2 M and to pH in the range of 5.6-8.0, so it can be used in processes with changing ion concentration and pH. It was sterilizable and could be autoclaved many times without losing its sensitivity. The applicability of the sensor in real application was successfully tested in the fermentation of Escherichia coli.

On the Possibility of Real-Time Monitoring of Glucose in Cell Culture by Microdialysis Using a Fluorescent Glucose Binding Protein Sensor

Although glucose sensors with millimolar sensitivity are still the norm, there is now a developing interest in glucose sensors with micromolar sensitivity for applications in minimally invasive sampling techniques such as fast microdialysis and extraction of interstitial fluid by iontophoresis and laser poration. In this regard, the glucose binding protein (GBP) with a binding constant for glucose in the micromolar range is of particular relevance. GBP is one of the soluble binding proteins found in the periplasmic space of Gram‐negative bacteria. Because of its hinge‐like tertiary structure, glucose binding induces a large conformational change, which can be used for glucose sensing by attaching a polarity sensitive fluorescent probe to a site on the protein that is allosterically responsive to glucose binding. Correspondingly, the resulting optical biosensor has micromolar sensitivity to glucose. Because binding is reversible, the biosensor is reusable and can be stored at 4 °C for 6 months without losing its sensitivity. In this paper, we show the feasibility of using the GBP biosensor to monitor glucose in microdialysis. The effect of perfusion rate, bulk glucose concentration and temperature on microdialysis efficiency was determined. Additionally, the glucose concentrations in mammalian cell culture were monitored to demonstrate the applicability of this sensor in complex and dynamic processes over a period of time. As the sensor is sensitive to micromolar glucose, high dialysis efficiency is not required when the bulk glucose concentration is within the millimolar physiological range. Thus, a perfusion rate of 10 μL/min or faster can be used, resulting in delay times of 1 min or less.

Comparing the performance of the optical glucose assay based on glucose binding protein with high-performance anion-exchange chromatography with pulsed electrochemical detection: efforts to design a low-cost point-of-care glucose sensor.

Background: The glucose binding protein (GBP) is one of many soluble binding proteins found in the periplasmic space of gram-negative bacteria. These proteins are responsible for chemotactic responses and active transport of chemical species across the membrane. Upon ligand binding, binding proteins undergo a large conformational change, which is the basis for converting these proteins into optical biosensors. Methods: The GBP biosensor was prepared by attaching a polarity-sensitive fluorescent probe to a single cysteine mutation at a site on the protein that is allosterically responsive to glucose binding. The fluorescence response of the resulting sensor was validated against high-performance anion-exchange chromatography (HPAEC) with pulsed electrochemical detection. Finally, a simple fluorescence reader was built using a lifetime-assisted ratiometric technique. Results: The GBP assay has a linear range of quantification of 0.100–2.00 μM and a sensitivity of 0.164 μM−1 under the specified experimental conditions. The comparison between GBP and HPAEC readings for nine blind samples indicates that there is no statistical difference between the analytical results of the two methods at the 95% confidence level. Although the methods of fluorescence detection are based on different principles, the response of the homemade device to glucose concentrations was comparable to the response of the larger and more expensive tabletop fluorescence spectrophotometer. Conclusions: A glucose binding protein labeled with a polarity-sensitive probe can be used for measuring micromolar amounts of glucose. Using a lifetime-assisted ratiometric technique, a low-cost GBP-based micromolar glucose monitor could be built.

Study on low-cost calibration-free pH sensing with disposable optical sensors

As labor costs become more expensive, less labor-intensive disposable devices have become more ubiquitous. Similarly, the disposable optical pH sensor developed in our lab could provide a convenient yet cost-effective way for pH sensing in processes that require stringent pH control. This optical pH sensor is prepared in uniform individual lots of 100-200 sensors per lot. Calibration is accomplished on a few randomly selected sensors out of each lot. We show that all others in the same lot can then be used directly without requiring individual calibration. In this paper, a calibration model is derived to include all the factors that affect the signal of the disposable sensor. Experimental results show that the derived calibration model fits the experimental data. The readings of 28 randomly selected disposable sensors with 4 sensors from each of the 7 lots show an error less than 0.1 pH units in the useful sensing range of the sensor. The calibration model indicates that if further improvement on precision is desired, more uniform porous material and more advanced coating techniques will be required. When it comes to the effects of the varying coasters, house-made low-cost fluorometers, the variability in the brightness ratio of the blue-to-violet LEDs is the primary reason for the lack of precision. Other factors like LED light intensity distribution, optical properties of the filters and electronics also contribute to the coaster-to-coaster difference, but to a lesser extent. Two different methods for correcting the instrument variations were introduced. After correction, the collective reading errors for all the tested instruments were reduced to less than 0.2 pH units within the sensors useful sensing range. Based on this result, our lab is currently implementing further improvements in modifying the coasters to equalize the ratios of blue-to-violet LED brightness.

Portable system for the detection of micromolar concentrations of glucose.

Glucose in non-invasively collected biofluids is generally in the micromolar range and thus, requires sensing methodologies capable of measuring glucose at these levels. Here, we present a small fluorometer system that can quantify glucose in the range of 0-5 μM with resolution of ~0.07 μM. It relies on the glucose binding protein (GBP) fluorescently labeled with two fluorophores. Fluorescence signals from the dual-labeled GBP are utilized in a ratiometric mode, making the measurements insensitive to variations in protein concentration and other systematic errors. Fluorescence is quantified by a miniature, dedicated ratiometric fluorometer that is powered via USB. Concentration is calculated using an ultra-mobile personal computer (UMPC). The whole system is designed to be pocket sized suitable for point-of-care or bedside applications. Test results suggest that the system is a promising tool for accurate measurements of low glucose concentrations (0.1-10 μM) in biological samples.

Detection of trace glucose on the surface of a semipermeable membrane using a fluorescently labeled glucose-binding protein: a promising approach to noninvasive glucose monitoring.

Background: Our motivation for this study was to develop a noninvasive glucose sensor for low birth weight neonates. We hypothesized that the underdeveloped skin of neonates will allow for the diffusion of glucose to the surface where it can be sampled noninvasively. On further study, we found that measurable amounts of glucose can also be collected on the skin of adults. Method: Cellulose acetate dialysis membrane was used as surrogate for preterm neonatal skin. Glucose on the surface was collected by saline-moistened swabs and analyzed with glucose-binding protein (GBP). The saline-moistened swab was also tested in the neonatal intensive care unit. Saline was directly applied on adult skin and collected for analysis with two methods: GBP and high-performance anion-exchange chromatography (HPAEC). Results: The amount of glucose on the membrane surface was found (1) to accumulate with time but gradually level off, (2) to be proportional to the swab dwell time, and (3) the concentration of the glucose solution on the opposite side of the membrane. The swab, however, failed to absorb glucose on neonatal skin. On direct application of saline onto adult skin, we were able to measure by HPAEC and GBP the amount of glucose collected on the surface. Blood glucose appears to track transdermal glucose levels. Conclusions: We were able to measure trace amounts of glucose on the skin surface that appear to follow blood glucose levels. The present results show modest correlation with blood glucose. Nonetheless, this method may present a noninvasive alternative to tracking glucose trends.

Real‐time monitoring of shake flask fermentation and off gas using triple disposable noninvasive optical sensors

Bioprocess development is a data‐driven process requiring a large number of experiments to be conducted under varying conditions. Small‐scale upstream bioprocess development is often performed in shake flasks because they are inexpensive and can be operated in parallel. However, shake flasks are often not equipped to accurately monitor critical process parameters such as pH, dissolved oxygen, and CO2 concentrations. Therefore, there is no definitive information on oxygen supply of growing cells, CO2 formation, and pH changes. Here we describe several shake flask fermentations where all three parameters are monitored by disposable noninvasive optical sensors. The sensitive element of these sensors is a thin, luminescent patch affixed inside the flask. Small electronic devices for excitation and fluorescence detection are positioned outside the shake flask for noninvasive monitoring. By measuring the process parameters throughout the course of the E. coli fermentations, we obtain information that is not routinely available in shake flask fermentations. For example, for cultures with only a few millimeters liquid depth, oxygen limitation can occur at relatively low agitation speeds. Under certain conditions oscillations in dissolved oxygen can occur. An increase in shaker speed and a decrease in culture volume can increase the oxygen availability and reduce the duration of oxygen limitation.

Optimizing cell-free protein expression in CHO: Assessing small molecule mass transfer effects in various reactor configurations†

Cell‐free protein synthesis (CFPS) is an ideal platform for rapid and convenient protein production. However, bioreactor design remains a critical consideration in optimizing protein expression. Using turbo green fluorescent protein (tGFP) as a model, we tracked small molecule components in a Chinese Hamster Ovary (CHO) CFPS system to optimize protein production. Here, three bioreactors in continuous‐exchange cell‐free (CECF) format were characterized. A GFP optical sensor was built to monitor the product in real‐time. Mass transfer of important substrate and by‐product components such as nucleoside triphosphates (NTPs), creatine, and inorganic phosphate (Pi) across a 10‐kDa MWCO cellulose membrane was calculated. The highest efficiency measured by tGFP yields were found in a microdialysis device configuration; while a negative effect on yield was observed due to limited mass transfer of NTPs in a dialysis cup configuration. In 24‐well plate high‐throughput CECF format, addition of up to 40 mM creatine phosphate in the system increased yields by up to ∼60% relative to controls. Direct ATP addition, as opposed to creatine phosphate addition, negatively affected the expression. Pi addition of up to 30 mM to the expression significantly reduced yields by over ∼40% relative to controls. Overall, data presented in this report serves as a valuable reference to optimize the CHO CFPS system for next‐generation bioprocessing. Biotechnol. Bioeng. 2017;114: 1478–1486.

A unique noninvasive approach to monitoring dissolved O2 and CO2 in cell culture.

Although online monitoring of dissolved oxygen (DO) and carbon dioxide (DCO2) is highly desirable in bioprocesses, small‐scale bioreactors are usually not monitored due to the lack of suitable sensors. Traditional electrochemical sensors are usually not used because they are bulky and invasive. Disposable optical sensors are small and only partially invasive, but there are concerns regarding the toxicity of the patch and the phototoxicity of the illuminating light. Here we present a novel, noninvasive, rate‐based technique for monitoring DO and DCO2 in cell cultures. A silicone sampling loop which allowed the diffusion of O2 and CO2 through its wall was inserted inside a bioreactor, and then flushed with N2 until the CO2 and O2 inside the loop were completely removed. The gas inside the loop was then allowed to recirculate through gas impermeable tubing to the O2 and CO2 sensors. We have shown that by measuring the initial diffusion rate we were able to determine the partial pressures of the two gases in the culture. The technique could be readily automated and measurements could be made in minutes. It was tested in demonstration experiments by growing murine hybridoma cells in a T‐flask and a spinner‐flask at 37°C. The results were comparable to those measured with commercially available fluorescence‐based patch sensors. These results show that the rate‐based method is an effective way to monitor small‐scale cell cultures. This measurement mechanism can be easily built into disposable cell culture vessels for facile use. Biotechnol. Bioeng. 2015;112: 104–110.

Passive Diffusion of Transdermal Glucose: Noninvasive Glucose Sensing Using a Fluorescent Glucose Binding Protein

Background: The motivation for this study was to determine if a statistically significant correlation exists between blood glucose (BG) and transdermal glucose (TG) collected by passive diffusion. A positive outcome will indicate that noninvasive passive TG diffusion is a painless alternative to collecting blood through a break on the skin. Method: Sampling involves placing a small volume of buffer solution on the surface of membrane or skin for 5 minutes. The sample is then assayed with fluorescent GBP. In vitro testing was done on regenerated cellulose and a porcine skin model to determine diffusion of standard glucose solutions. In vivo testing was done on a healthy subject and a subject with type 2 diabetes. Results: Glucose diffused readily through the regenerated cellulose membrane with good correlation between surface and internal glucose concentrations (R2 = .997). But the porcine skin model required a surface prewash to achieve the same good correlation R2 = .943). Based on this, an optimum prewash step was determined for the in vivo studies. The resulting correlation coefficients between TG and BG after a 15-minute prewash in a healthy subject and type 2 subject were .87 and .93, respectively. Conclusions: Removal of the extraneous glucose in the skin by prewashing was an important step in achieving good correlation between TG and BG. The results suggest that passive collection of TG is a noninvasive alternative to current practice of breaking the skin. Further studies are under way to determine the lag time between TG and BG and for the sampling protocol to be more amenable to point-of-care application.