Biomolecules & Biotechnology
microSI-LOV and FIAlab 3200 instruments
are compatible with all applications listed in this section.
They are available in Plug and Play configuration
Fermentation monitoring first used FI, and later SI , for continuous assay of nutrients and products in both industrial and laboratory setting. Presently FI and SI techniques are also widely applied in food analysis and for clinical and biochemical assays. The common thread of these applications is the use of enzymatic assays, since they are automated and miniaturized in the microSI-LOV format in a superior way, using UV-VIS spectrophotometry or fluorescence detection.
The Universal Bioanalyzer
Assays of Biomolecules, including Enzymatic Assays, Bioligand Interaction Assays and Chromatography of Biomolecules are traditionally carried out on different instruments, which necessitates mastering various types of hardware and software. This fragmented approach supports a view of an apparent incompatibility of these techniques, thus obscuring similarities of their underlying biochemical reactions and their complex kinetic behavior.
It is the versatility of programmable flow, combined with the ability of the LOV platform to accommodate UV/VIS , fluorescence and chemiluminescence detection, that allows the gap between various analytical techniques to be bridged by a single MicroSIA instrument as illustrated by the following examples.
Enzymatic Assays
Enzymatic assays are based on reaction rate measurements, carried out in a stopped-flow mode, where the reaction mixture is held for monitoring within the flow through cell. Most frequently performed assays include glucose, lactate, urea, glycerol and ethanol.
Referencing out the initial absorbance reading at the start of each assay removes any optical matrix effects such as natural color or salinity (index of refraction). A typical assay cycle takes less than 1 min.
microAffinity Chromatography
This miniaturized variant of conventional affinity chromatography uses the Lab-On-ValveŽ platform to carry out separation of biomolecules on automatically renewable microcolumns that are packed within the LOV using Sepharose beads. Availability of many functional groups on Sepahrose make this revolutionary technique suited not only for immunoassays (Protein A or G) but also for a wide variety of target biomolecules including enzymes and nucleotides. Native proteins are monitored by UV spectrophotometry @280nm, colored boimolecules ( e.g. B12) by spectrophotometry. In addition fluorescence and chemiluminescence can be carried out in the same instrument.
Separation of mouse IgG
from bovine serum albumin
Bioligand Interaction Assay
This method often seen as the exclusive domain of Surface Plasmon Resonance (SPR), can be successfully carried out by the microSI-LOV technique. The protocol starts by injecting microliter volumes of Sephadex or Sepharose beads into the flow cell, where the beads are captured and monitored while the analyte solution is passed through. Native biomolecules typically absorb at 260 and 280 nm, while labeled molecules absorb visible light (and some also emit fluorescence). Thus both nonlabeled and labeled biomolecules can be measured simultaneously. Since Sephadex and Sepharose beads are readily available with a wide variety of functional groups, this method is faster, more versatile and far less expensive than SPR based techniques.
Binding and elution of monoclonal
anti-insulin Ab from
Sephadex-Protein A beads
Fermentation Monitoring
Continuous monitoring of small scale fermentors in a laboratory setting by using by the microSI-LOV system required only 20 micro L of sample per data point and produced less than 30 mL of waste during 6 hours of monitoring. The small footprint of the instrument allowed its placement in the immediate vicinity of the 20 L Bioflo fermentor, furnished with a 19 mm Flownamic sampling probe. Ammonia (3-1200 ppm) was determined by the indophenol blue method, glycerol (20-120 ppm) by GPO-Trinder reagent, glucose (35-1000 ppm) by Infinity (Sigma) reagent and free iron (80-400 ppm) by the ferrozine method (Sigma 565-A kit).
