In biotechnology, the ability to instantly detect contaminants is key to running a reliable bioprocess. Bioprocesses are prone to be contaminated by cells that are abundant in our environment; detection and quantification of these cells would aid in the preservation of the bioprocess product. This paper discusses the design and development of a portable kinetics fluorometer which acts as a single-excitation, single-emission photometer that continuously measures fluorescence intensity of an indicator dye, and plots it. Resazurin is used as an indicator dye since the viable contaminant cells reduce Resazurin toResorufin, the latter being strongly fluorescent. A photodiode detects fluorescence change by generating current proportional to the intensity of the light that reached it, and a trans-impedance differential op-amp ensures amplification of the photodiodes’ signal. A microfluidic chip was designed specifically for the device. It acts as a fully enclosed cuvette, which enhances the Resazurin reduction rate. E. coli in LB media, along with Resazurin were injected into the microfluidic chip. The optical sensor detected the presence of E. coli in the media based on the fluorescence change that occurred in the indicator dye in concentrations as low as 10 CFU/ml. A method was devised to detect and determine an approximate amount of contamination with this device. This paper discusses application of this method to detect and estimate sample contamination. This device provides fast, accurate, and inexpensive means to optically detect the presence of viable cells.
Fluorescent proteins are often used as reporters of protein concentration in biology and biomedicine applications. They can be detected using a fluorimeter equipped with fiber optics for ease of access. However, small changes in the path length due to change in the position, or immersion depth of the optical fiber results in large changes in readings. To alleviate the situation, the fiber is equipped with a fixed-length-extension that provides constant path length. The operation of the fiber equipped fluorimeter is theoretically modelled and practically verified in this paper.
The main bioprocess variables that are continuously measured are pH, dissolved oxygen (DO), and dissolved car- bon dioxide (DCO2). Less common variables are redox, concentrations of substrate and product concentrations, product activity, etc. Recently, pH and DO have been measured using optical chemical sensors due to their small form factor and convenience in use. These sensors are typically interrogated using a lab grade spectrometer, or with the help of a low-cost, tailor-made optoelectronic transducer that is designed around the optical sensor. Recently, we proposed a new class of optoelectronic transducers that are capable of monitoring several different optical sensors without the need to switch the optics or hardware when changing the type of sensor. This allows flexibility closer to the lab-grade devices at a price point of a dedicated sensor.
In this work, we have demonstrated a universal optical platform capable of monitoring pH or DO sensors. It uses the principle of ratio-metric fluorescence measurements for pH and fluorescence lifetime measurements for DO. The platform is capable of seamlessly switching between these two modes. It is also capable of auto recognition of the sensor type. The platform can operate both with patch-type or fiber optic type of sensors. The platform has measurement accuracy of about 0.08 pH units and approximately 5 % air saturation with oxygen. Additionally, an approach to obtain identical calibrations between several devices is presented.
The described platform has been tested in actual bioprocesses and has been found adequate for continuous bioprocess monitoring.
A new method for surface-based fluoroimmunoassays that eliminates separation steps while still allowing high
sensitivity detection of biomolecular interactions is presented. The capture antibody is electrostatically immobilized on a
glass slide coated with a high density silver island film. The metal-enhanced fluorescence generated by the presence of
the islands allows the sensitive detection of bound reporter antibodies versus those free in solution. In order to perform
the measurement, phase-modulation fluorometry is employed which allows observation of the distinct fluorescence
signal of the bound antibodies with a shorter lifetime than unbound antibodies. Here, we show the use of metal-enhanced
fluorescence with phase-modulation fluorometry to quantify monoclonal antibody from a cell culture. The
results show the new technique produces very similar data upon analysis as measured with ELISA analysis. With further
optimization of the procedures, it is forecast that real time monitoring during bioprocessing will be feasible with the
described technique.
KEYWORDS: Glucose, Proteins, Luminescence, Sensors, Fluorescence resonance energy transfer, Green fluorescent protein, Fluorescent proteins, Yeast, In vivo imaging, Molybdenum
The glucose-mediated conformational changes in the glucose binding protein (GBP) have been exploited in the development of fluorescence based glucose sensors. The fluorescence response is generated by a polarity sensitive dye attached to a specific site. Such fluorescent sensors respond to submicromolar glucose at diffusion-controlled rates mimicking the wild type. However, such sensors have been limited to in vitro glucose sensing because of the preliminary dye-labeling step. In the study described here, the dye-labeling step is omitted by genetically encoding the GBP with two green fluorescent mutants namely, the green fluorescent protein (GFP) and the yellow fluorescent protein (YFP) in the N- and C-terminal ends, respectively. These two GFP mutants comprise a fluorescence resonance energy transfer (FRET) donor and acceptor pair. Thus, when glucose binds with GBP, the conformational changes affect the FRET efficiency yielding a dose-dependent response. A potential application for this FRET-based glucose biosensor is online glucose sensing in bioprocessing and cell culture. This was demonstrated by the measurement of glucose consumption in yeast fermentation. Further development of this system should yield in vivo measurement of glucose in bioprocesses.
A baculovirus expression system was used to produce DsRed fusion protein in insect larvae. As the baculovirus/insect larvae system requires precise harvest timing to achieve high yield of protein, a low-cost miniature all-solid state optical probe was used for detection of the protein concentrations in the frozen larvae. Three batches of infected larvae were monitored at different post-infection times. The calibration curve of the probe was obtained by simultaneous measurements both in laboratory fluorimeter and using gel electrophoresis analysis. The results show good correlation between the optical measurements and the standard laboratory technique.
Glucose is the major source of carbon, and glutamine is the major source of nitrogen in cell culture media. Thus, glucose and glutamine monitoring are important in maintaining optimal conditions in industrial bioprocesses. Here we report reagentless glucose and glutamine sensors using the E. coli glucose binding protein (GBP) and the glutamine binding protein (GlnBP). Both of these proteins are derived from the permease system of the gram-negative bacteria. The Q26C variant of GBP was labeled at the 26-position with anilino-naphthalene sulfonate (ANS), while the S179C variant of GlnBP was labeled at the 179-position with acrylodan. The ANS and acrylodan emissions are quenched in the presence of glucose and glutamine, respectively. The acrylodan-labeled GlnBP was labeled at the N-terminal with ruthenium bis-(2,2’-bipyridyl)-1,10-phenanthroline-9-isothiocyanate. The ruthenium acts as a non-responsive long-lived reference. The apparent binding constant, Kd’, of 8.0 μM glucose was obtained from the decrease in intensity of ANS in GBP. The reliability of the method in monitoring glucose during yeast fermentation was determined by comparison with the YSI Biochemistry Analyzer. The apparent binding constant, Kd’, of 0.72 μM glutamine was calculated from the ratio of emission intensities of acrylodan and ruthenium (I515/I610) in GlnBP. The presence of the long-lived ruthenium allowed for modulation sensing at lower frequencies (1-10 MHz) approaching an accuracy of ± 0.02 μM. The conversion of the GBP into a similar ratiometric sensor was described.
We describe a new method for fluorescence sensing based on measurements of the steady state polarization of an analyte- sensitive fluorophore in the presence of a reference fluorophore with known polarization. The basic concept is that the polarization of a mixture reflects a weighted average of the polarization of the emitting species. By use of reference fluorophores the starting values can be near zero, or near 0.9 for oriented films which contain the reference fluorophore. Changing intensities of the sensing fluorophore due to the analyte result in changes in the polarization of the combined emission. A wide dynamic range is available because of the freedom to select high or low starting polarization values. Polarization-based sensing was demonstrated for pH using 6- carboxy fluorescein. We also show that polarization sensing can be used for measurements of oxygen and glucose. Polarization sensing can have numerous applications in clinical and analytical chemistry.
A Stanford Research Systems SR844 lock-in amplifier was used to build a sub $10,000 phase-modulation fluorometer capable of measuring nanosecond fluorescence lifetimes. The lock-in directly provided both the DC bias and the AC signal used to modulate the intensity of a blue LED excitation source. A photomultiplier tube measured the emission, and the resulting signal was sent back through a DC block to the lock-in with no external signal processing or heterodyning required. A simple computer program was developed to automate the measuring process and correct for the most common sources of error, namely coherent pickup and stray ambient light. Several standard fluorophores were measured, and the results compare favorably with those from a research grade cross-correlation phase fluorometer up to frequencies of 100 MHz. This system can operate in several configurations, each with benefits and limitations. The system is particularly well suited for fluorescence lifetime based sensing applications, demonstrated by measuring dissolved carbon dioxide online in a bacterial fermentation.
We devised an optical assay for glucose based on the genetically-engineered glucose/galactose binding protein (GGBP) from E. coli and phase-modulation fluorometry. A single cysteine mutation was introduced at position 26 of GGBP. When labeled with the sulfhydryl-reactive probe I-ANS, GGBP showed a more than 50% decrease in florescence intensity with increasing glucose concentration (Kd approximately 1 (mu) M). This is consistent with the glucose-bound structure of GGBP where residue 26 becomes more exposed to the aqueous media. Since minimal lifetime changes were observed with glucose binding, a modulation sensor was devised wherein a long lifetime ruthenium metal-ligand complex (Ru) was painted on the surface of the cuvette containing ANS26-GGBP. Glucose binding resulted in changes in the relative intensities of ANS26-GGBP and Ru which were observed as dramatic changes in the modulation at a low frequency of 2.1 MHz. The modulation measured at 2.1 MHz accurately determines the glucose concentration to plus or minus 0.2 (mu) M.
Murine hybridomas were cultivated in tissue culture flasks. Dissolved oxygen tensions in the gas and liquid phases during cell growth were measured non-invasively by an optical oxygen sensor. Readings were made with caps both cracked open and completely closed. During cell growth, gas phase oxygen concentrations remained near atmospheric levels, while the oxygen tension at the bottom of the flasks eventually reached zero. These results suggest that the widespread practice of cracking open tissue culture flask caps during cell growth with a view to supplying adequate oxygen to cells is ineffective and unnecessary. The mass transfer characteristics of the tissue culture flask indicate the dominant resistance to oxygen mass transfer to the cells was the liquid media. The mass transfer rates through the liquid layer under standard laboratory conditions were found to be greater than those predicted by diffusion alone, suggesting microscale mixing. Volumetric and specific oxygen consumption rates were calculated from the sensor data, and were comparable to published values. A recently developed single fiber optic oxygen sensor is described. This new sensor will provide oxygen concentrations at various levels in the tissue culture flasks, allowing more accurate modeling of oxygen diffusion.
A directly modulated infrared diode laser was frequency doubled in an intracavity KTP quasi-phase matched waveguide, producing modulated laser light at 425 nm, which was subsequently employed for phase fluorometric detection of CO2 and O2 gases. The frequency-doubled source was coupled to a single optical fiber to deliver the excitation light to the sensor mounted at the fiber tip. Fluorescence from the sensor returned through the same fiber to a photodetector. This compact, low-power source provided continuously tunable modulation frequencies up to 200 MHz. In the detection of CO2 , the doubled infrared diode laser produced 425-nm light that was modulated to a depth of 27% at a modulation frequency of 95 MHz.
We describe the fabrication and testing of an optical pH sensor based on fluorescence lifetime measurements and sol-gel technology. The sensor is based on the phenomenon of fluorescence resonance energy transfer (FRET), from a pH-insensitive donor to a pH-sensitive acceptor. The pH-dependent increase in the bromothymol blue acceptor absorbance results in increased energy transfer, reducing the lifetime of the Texas red hydrazide donor. The lifetimes were measured by the phase and modulation of the emission, relative to the modulated incident light, and were found to be insensitive to the total signal level and fluctuations in light intensity. However, the present sensors are sensitive to salt concentration and/or ionic strength. Importantly, this sol-gel sensor is not fragile, providing stable readings for days and can be repeatedly autoclaved without loss of sensitivity to pH. The use of FRET as the pH transduction mechanism can be reliably extended to longer wavelengths, and allows the future use of laser diode excitation sources.
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