Hung Lam

Optical instrumentation for bioprocess monitoring.

In this chapter the optical sensors for oxygen, pH, carbondioxide and optical density (OD) which are essential for bioprocess monitoring are introduced, their measurement principles are explained and their realization and applications are shown. In addition sensors for ethanol and GFP are presented. With the exception of the optical density sensor all others employ certain fluorophores that are sensitive to the designated parameter. These fluorophores along with their optical properties, the sensing mechanisms and their mathematical formulations are described. An important part of this chapter covers the development of the optoelectronic hardware for low cost systems that are able to measure the fluorescence lifetime and fluorescence intensity ratio. The employment of these probes in the bioprocess monitoring is demonstrated in different fermentation examples.

Dual optical sensor for oxygen and temperature based on the combination of time domain and frequency domain techniques

In measuring specific conditions in the real world, there are many situations where both the oxygen concentration and the temperature have to be determined simultaneously. Here we describe a dual optical sensor for oxygen and temperature that can be adapted for different applications. The measurement principle of this sensor is based on the luminescence decay times of the oxygen-sensitive ruthenium complex tris-4,7-diphenyl-1,10-phenanthroline ruthenium(III) [Rudpp] and the temperature-sensitive europium complex tris(dibenzoylmethane) mono(5-amino-1,10-phenanthroline)europium(III) [Eudatp]. The excitation and emission spectra of the two luminophores overlap significantly and cannot be discriminated in the conventional way using band pass filters or other optical components. However, by applying both the frequency and time domain techniques, we can separate the signals from the individual decay time of the complexes. The europium complex is entrapped in a poly(methyl methacrylate) (PMMA) layer and the ruthenium complex is physically adsorbed on silica gel and incorporated in a silicone layer. The two layers are attached to each other by a double sided silicone based tape. The europium sensing film was found to be temperature-sensitive between 10 and 70°C and the ruthenium oxygen-sensitive layer can reliably measure between 0 and 21% oxygen.

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.

A luminescence lifetime assisted ratiometric fluorimeter for biological applications

In general, the most difficult task in developing devices for fluorescence ratiometric sensing is the isolation of signals from overlapping emission wavelengths. Wavelength discrimination can be achieved by using monochromators or bandpass filters, which often lead to decreased signal intensities. The result is a device that is both complex and expensive. Here we present an alternative system--a low-cost standalone optical fluorimeter based on luminescence lifetime assisted ratiometric sensing (LARS). This paper describes the principle of this technique and the overall design of the sensor device. The most significant innovation of LARS is the ability to discriminate between two overlapping luminescence signals based on differences in their luminescence decay rates. Thus, minimal filtering is required and the two signals can be isolated despite significant overlap of luminescence spectra. The result is a device that is both simple and inexpensive. The electronic circuit employs the lock-in amplification technique for the signal processing and the system is controlled by an onboard microcontroller. In addition, the system is designed to communicate with external devices via Bluetooth.

Low-cost optical lifetime assisted ratiometric glutamine sensor based on glutamine binding protein.

Here we report a reagentless fluorescence sensing technique for glutamine in the submicromolar range based on the glutamine binding protein (QBP). The S179C mutant is labeled with the short-lived acrylodan (lifetime<5ns) and the long-lived tris(dibenzoylmethane) mono(5-amino-1,10-phenanthroline)europium(III) (lifetime > 300 micros) at the -SH and the N-terminal positions, respectively. In the presence of glutamine the fluorescence of acrylodan is quenched, while the fluorescence of europium complex remains constant. In this report we describe an innovative technique, the so called lifetime assisted ratiometric sensing to discriminate the two fluorescence signals using minimal optics and power requirements. This method exploits the large difference between the fluorescence lifetimes of the two fluorophores to isolate the individual fluorescence from each other by alternating the modulation frequency of the excitation light between 300 Hz and 10 kHz. The result is a ratiometric optical method that does not require expensive and highly attenuating band pass filters for each of the dyes, but only one long pass filter for both. Thus, the signal to noise ratio is enhanced, and at the same time, the optical setup is simplified. The end product is a simple sensing device suitable for low-cost applications such as point-of-care diagnostics or in-the-field analysis.

High Resolution Non-contact Fluorescence Based Temperature Sensor for Neonatal Care

To date, thermistors are used to continuously monitor the body temperature of newborn babies in the neonatal intensive care unit. The thermistor probe is attached to the body with a strong adhesive tape to ensure that the probe stays in place. However, these strong adhesives are shown to increase microbial growth and cause serious skin injuries via epidermal stripping. The latter compromises the skins ability to serve as a protective barrier leading to increase in water loss and further microbial infections. In this article a new approach is introduced that eliminates the need for an adhesive. Instead, two kinds of fluorophores are entrapped in a skin friendly chitosan gel that can be easily wiped on and off of the skin, and has antimicrobial properties as well. A CCD camera is used to detect the temperature dependent fluorescence of the fluorophore, tris(1,10-phenthroline)ruthenium(II) while 8-aminopyrene-1,3,6-trisulfonic acid serves as the reference. This temperature sensor was found to have a resolution of at least 0.13°C.

Low-cost fluorescence-based temperature sensing system for neonatal care

A remotely-measured fluorescence-based temperature measurement system designed for neonatal care is built and presented using a low-cost, off-the-shelf camera-phone image sensor. The fluorophore-based sensor salve removes the need of attaching thermistor probes using adhesives to the neonates skin; and therefore reduces the risk of epidermal stripping, microbial infections, etc. The system performs low-cost yet precise remote ratiometric measurements of our thermometric fluorescent salve using only a 10-bit, 120-FPS CMOS imager. An FPGA handles control and capture of image data in the region of interest with frames synchronized with a pulsing illuminator. A minimalistic algorithm using basic pixel-summations and division to compute a ratio is presented, suitable for implementation on a minimal embedded system. The resolution of the system is reported to be at least 0.18 degrees Celcius.

High resolution fluorescence-based temperature sensor for stand-off detection in physiological range

Fluorescence ratiometric temperature sensing system for stand-off temperature measurements in the physiological range is presented. It is based on the use of fluorescence dyes and an integrating photodetector system. The system is intended for use in neonatal intensive care units (NICU). It utilizes a measurement and a reference fluorescence dyes encapsulated in a film. Fluorescence is excited using LEDs and the emission is detected using photodiodes equipped with optical band pass filters. The current generated by the photodiodes is integrated and the charge is digitized. The fluorescence intensity is calculated as the difference in the measured intensities when the LED is on or off. The ratio of the two emission intensities depends linearly on the temperature. The system operates under room-light illumination with resolution <;0.1°C at a rate 1 sample per second. It is capable to better suppress the influence of the ambient light intensity as compared with previous camera based systems.