Peter Harms

Low‐cost microbioreactor for high‐throughput bioprocessing

The design of a microbioreactor is described. An optical sensing system was used for continuous measurements of pH, dissolved oxygen, and optical density in a 2 mL working volume. The K(L)a of the microbioreactor was evaluated under different conditions. An Escherichia coli fermentation in both the microbioreactor and a standard 1 L bioreactor showed similar pH, dissolved oxygen, and optical density profiles.%The low cost of the microbioreactor, detection system, and the small volume of the fermentation broth provide a basis for development of a multiple-bioreactor system for high-throughput bioprocess optimization.

Dual Excitation Ratiometric Fluorescent pH Sensor for Noninvasive Bioprocess Monitoring: Development and Application

The development and application of a fluorescent excitation‐ratiometric, noninvasive pH sensor for continuous on‐line fermentation monitoring is presented. The ratiometric approach is robust and insensitive to factors such as source intensity, photobleaching, or orientation of the patch, and since measurements can be made with external instrumentation and without direct contact with the patch, detection is completely noninvasive. The fluorescent dye 8‐hydroxy‐1,3,6‐pyrene trisulfonic acid was immobilized onto Dowex strongly basic anion‐exchange resin, which was subsequently entrapped into a proton‐permeable hydrogel layer. The sensor layer was polymerized directly onto a white microfiltration membrane backing that provided an optical barrier to the fluorescence and scatter of the fermentation medium. The ratio of emission intensity at 515 nm excited at 468 nm to that excited at 408 nm correlated well with the pH of clear buffers, over the pH range of 6–9. The sensor responded rapidly (<9 min) and reversibly to changes in the solution pH with high precision. The sterilizable HPTS sensor was used for on‐line pH monitoring of an E. coli fermentation. The output from the indwelling sensor patch was always in good agreement with the pH recorded off‐line with an ISFET probe, with a maximum discrepancy of 0.05 pH units. The sensor is easily adaptable to closed‐loop feedback control systems.

Low cost phase-modulation measurements of nanosecond fluorescence lifetimes using a lock-in amplifier

The use of a 200 MHz lock-in amplifier was demonstrated as a low cost instrument for frequency domain measurements of nanosecond fluorescence lifetimes. The lock-in directly provided both the dc bias and the ac signal used to modulate the intensity of a blue light emitting diode excitation source. The emission was measured by a photomultiplier tube and the resulting signal was sent back through a dc block to the lock-in with no external signal processing or heterodyning required. The system was highly accurate at measuring phase and modulation up to 80 MHz and moderately so up to 100 MHz. The fluorescence lifetimes of several standard fluorophores (Fluorescein, Rhodamine B, and [Ru(bpy)3]2+) were measured by the lock-in, and the results agreed closely with those made on a research grade fluorometer. The entire lock-in based system costs less than US

Ratiometric oxygen sensing: detection of dual-emission ratio through a single emission filter

10,000 to build and can be controlled by any standard computer through a GPIB or serial connection. The system is also portable, consumes little power, and c...

Unique Oxygen Analyzer Combining a Dual Emission Probe and a Low-Cost Solid-State Ratiometric Fluorometer:

A new method and device for the ratiometric measurement of oxygen concentration are presented. They are based on the use of a dual-emission oxygen-sensitive dye. The method allows the exclusion of the influence of emission overlap. The detection of the dual-emission ratio is performed using a single long-pass emission filter. The device described is simpler than the widely used lifetime instruments and could easily be a stand-alone low-cost instrument.

Low-cost phase-modulation fluorometer for measuring nanosecond lifetimes using a lock-in amplifier

A new oxygen analyzer combining a polymer-encapsulated dual-emission probe and a diode-based ratiometric fluorometer is described. The ratiometric fluorometer was configured to measure the relative fluorescence and phosphorescence intensity of the short-lived singlet and long-lived oxygen-quenchable triplet of [(dppe)Pt{S2C2(CH2CH2–N–2-pyridinium)}](BPh4), where dppe is 1,2-bis(diphenylphosphino)ethane. This luminescent dye was immobilized at 0.3% by weight in cellulose acetate/75% triethylcitrate and cast into a 0.5 mm-thick film. The dye molecule was excited with a blue light-emitting diode (LED) and the singlet and triplet emissions monitored by individual photodiodes at 570 and 680 nm, respectively. Oxygen can be accurately measured without interference from nonanalyte-induced intensity changes by monitoring the relative output of the two photodiodes. The output of the device was linear with oxygen concentration. The PO2(1/2) from the ratio-adapted Stern–Volmer plot was 9.6% oxygen (≈73 torr), offering a dynamic range of 0–90% O2. The device is stable and the oxygen measurements reproducible. Intentional photobleaching of ≈20% of the sensor dye had little effect upon the reproducibility of the oxygen measurements.

Noninvasive measurement of dissolved oxygen in shake flasks.

A Stanford Research Systems SR844 lock-in amplifier was used to build a sub

Design and Performance of a 24-Station High Throughput Microbioreactor

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.