A novel approach for monitoring extracellular acidification rates: based on bead injection spectrophotometry and the lab-on-valve systemElectronic supplementary information (ESI) available: Derivation of eqn. (4). See http://www.rsc.org/suppdata/an/b3/b315007k/

Abstract

Monitoring extracellular acidification rates (ECARs) is important for the study of cellular activities, since it allows for the evaluation of factors that alter metabolic function, such as stimulants, inhibitors, toxins as well as receptor and non-receptor mediated events. While the light addressable potentiometric sensor (Cytosensor Microphysiometer) has been the principal tool for ECARs measurement in the past, this work introduces a novel method that exploits an immobilized pH indicator on the surface of microcarrier beads (Sephadex) and is probed with a fiber optic coupled spectrophotometer. Likewise, live cells under investigation were also immobilized on microcarrier beads (Cytopore). These beads are metered, transported and monitored within a microfluidic system, termed as the Lab-on-Valve (LOV). Use of carrier beads in conjunction with Bead Injection Spectrophotometry and a Lab-on-Valve module (BIS-LOV), makes ECAR measurements reliable and automated. The feasibility of the BIS-LOV approach is demonstrated measuring ECARs of the mouse hepatocyte cell line, TABX.2S, grown on Cytopore beads packed within the central channel of the LOV system. These immobilized cells were perfused in a phosphate buffer carrier solution (capacity: 1 mmol L(-1), pH 7.4). Protons extruded from 10(5) to 10(6) cells were accumulated during a stopped flow period of 220 s followed by a pH measurement, detected by changes in absorbance of the pH indicator bonded to the microcarrier beads. Addition of metabolic inhibitors (sodium azide, oxamic acid) to the carrier buffer solution can induced an increase or decrease of the basal proton extrusion rate in a very reproducible manner. Comparison of the BIS-LOV technique to the Cytosensor microphysiometer and literature confirms the validity of this novel approach, highlighting its advantages and suggesting future improvements that will make the BIS-LOV a practical tool for routine ECARs measurement.

Flow Injection Analysis in Industrial Biotechnology

“…The second approach has proven to be a very powerful tool in many contexts and implemented in the various generations of flow injection (14). Not the least for bioanalytical assays encompassing investigation of cellular activities (15,16) and enzymatic procedures for determination of both substrates and enzymatic activities (17,18), where the latter one traditionally has been very difficult to execute.…”

“…Nowadays, a wide array of bead particles (predominantly with a core of Sephadex type copolymers) with various surface characteristics are commercially available, and in the following section will be described procedures for real-time monitoring of cellular activities via immobilized live cells (15,72), isolation and on-column fluorimetric detection of DNA (15), optimization of immobilization protocols for proteins (74), as well as investigation of biomolecular association and dissociation processes as exploited in enzyme-linked immunosorbent assays (75) and affinity chromatographic methods (59,60,76). Yet before that, it might be of interest to compare the LOV with the socalled micro-total analysis systems (μTAS) (77), or as they lately have been termed Lab-on-a-Chips (LOC), which are exploited for bionanotechnological applications (78,79).…”

“…Combining LOV and bead injection (BI), Ruzicka and coworkers have in particular focused on cellular analyses and immunoassays (15,67,(74)(75)(76). The former are being used for screening of potential drugs by in-vitro evaluation of biological responses.…”

Recent developments in automatic solid-phase extraction with renewable surfaces exploiting flow-based approaches

“…For bioassays, antibodies or antigens [15,[21][22][23][24][25] have been immobilized on magnetic [21,22] or polysaccharide-based [24,25] beads. The latter have also been proven suitable as substrate for cell cultivation [26,27].…”

“…For example, consumption of glucose and extrusion of lactate could be detected sensitively and rapidly due to the high concentration of immobilized cells [47]. The exploration of cellular activities based on the release of proton from cells in LOV was also reported [27]. Though bead injections were done manually here, two micro-packed bead columns were uniquely created within one section of the LOV micro-channel, as shown in Figure 4A.…”