Filtration probes

J. Sefconicova, J. Kahlik, V. Stefuca, et al, A filtration probe-free on-line monitoring of glycerol during fermentation by a biosensor device. Enzyme Microb. Technol, 42, 434 39 (2009). 

Figure 20.2 A schematic diagram of the set-up using a filtration probe for continuous withdrawal of sample from a fermentor. The filtered sample is led to the FIA system with the heat-sensitive split-flow biosensor. A personal computer was used for controlling the system and for calculation of concentrations. (Reproduced from [25] with permission.)... 

When used in biotechnology, the first part of a FIA system (see Figure 22-4) is a filtration probe [28-32]. A cell-free sample is drawn from the bioprocess unit (eg, a fermenter) via this probe. Thus, these probes are sterile barriers between the bioprocess and the analysis system, protecting the bioprocess from infections and the analysis system from clogging by cells. Only a few FIA applications utilizing cell-containing samples are known [33]. In those cases, cells and medium are separated inside the FIA system. 

Different filtration probes can be used. In Figure 22-5, a filtration probe for sampling in a fermenter bypass is shown [28, 34]. Since flat membranes are used in this probe, many types of micro- and ultrafiltration membranes can be used. On the other hand, the increased risk of infection can make bypass sampling problematic. The membranes used should be selected for cut-off size and material (eg, adsorption characteristics) as required by the process conditions. They should provide a sample stream that has the same analyte concentrations as the liquid in the fermenter. An in situ probe employing tubular membranes is shown in Figure 22-6. Thbular membranes are not available in the same variety as flat membranes, but this probe offers the advantages of a very small dead volume and simple connection to a fermenter via an ordinary sensor port. 

In situ filtration probe utilizing tubular membranes (membrane not shown). The probe can be connected to the fermenter via standard ports. The tubular membrane fits directly on the inner support. Channels in this support are neccessary to collect the cell-free medium (developed at the Institut fiir Technische Chemie of the Universitiit Hannover, Hannover, Germany). 

FIA systems can be recalibrated frequently by injecting calibration solutions instead of a sample. The entire analytical system (i.e., enzyme and sensor) is calibrated under real analysis conditions. Deactivated enzyme preparations or sensors can be exchanged without any problems since the fermenter is protected by the sterility barrier of the filtration probe. 

Such a filtration probe has been tested with very good results in a number of analyses with a special enzyme thermistor version designed for use in industrial environments. Figure 2 is a schematic illustration of an enzyme thermistor arranged for process monitoring by repeated flow injection analysis. The interval between sample injections is chosen with respect to how fast the analyte concentration changes. A typical figure is four to five sample injections per hour. The injection valve and a sample selector valve for selection of different samples or calibration solutions and the pumps are controlled with a personal computer with a 386 processor, which also... 

SWNTs (HiPco, Carbon Nanotechnologies Incorporated) were shortened by ultrasonication with a probe-type sonicator in mixed acids (H2SO4 and HNO3) under ice-cooling. After diluting the mixture with water (MiliQ), the shortened SWNTs were purified by filtration through a PTFE membrane filter (pore size 1 pm or 0.2 pm) or by chromatography (Sepadex G-50). 

Dissolve the purified SPDP-modified dendrimer of step 5 in 50 mM sodium phosphate, 0.15M NaCl, pH 7.5, or in DMSO at a concentration of at least lOmg/ml. Add a 10-20 X molar excess of an amine-reactive fluorescent molecule (i.e., NHS-rhodamine or a hydrophilic NHS-Cy5 derivative see section on fluorescent probes). React with mixing for 1 hour at room temperature. Purify the fluorescently labeled SPDP-modified dendrimer using gel filtration or ultrafiltration. Follow the method of either step 7 or 8 to conjugate the dendrimer to another protein or molecule. 

Remove excess fluorescent probe by gel filtration using a desalting resin. 

Purify the hydrazide-labeled oligo by gel filtration on desalting resin using 10 mM sodium phosphate, 0.15M NaCl, lOmM EDTA, pH 7.2. The probe now may be used to conjugate with an aldehyde-containing molecule. 

Remove excess reagents from the modified oligo by gel filtration on a desalting resin. The modified probe now may be used to conjugate with a sulfhydryl-containing molecule, or it may be reduced to create a thiol for conjugation with sulfhydryl-reactive molecules. 

Purify the thiolated oligonucleotide from excess DTT by dialysis or gel filtration using 50mM sodium phosphate, ImM EDTA, pH 7.2. The modified probe should be used immediately in a conjugation reaction to prevent sulfhydryl oxidation and formation of disulfide crosslinks. 

Isolate the biotinylated probe by ethanol/salt precipitation as described in Section 1 (this chapter) for nick-translation modification of DNA probes. Alternatively, dialysis, gel filtration, or w-butanol extraction may be used to remove excess reagents. 

Purify the biotinylated DNA probe by ethanol precipitation, gel filtration, w-butanol extraction, or dialysis as discussed in previous sections. 

Filtration of the dissolution sample aliquot is usually needed prior to quantitation. Filtration of the dissolution samples is usually necessary to prevent undissolved drug particles from entering the analytical sample and dissolving further. Also, filtration removes insoluble excipients that may otherwise cause a high background or turbidity. Prewetting of the filter with the medium is usually necessary. Filters can be in-line, at the end of the sampling probe, or both. The... 

Determining the kinetics and mechanisms of intercalation reactions is not trivial. Quenching studies (in which an aliquot of the reaction suspension is removed and the sohd product recovered through filtration) have frequently proved to be unreliable. The material isolated is often atypical of the reaction matrix as a whole, having been affected by the quenching process. Therefore, it is desirable to use a non-invasive probe to observe the reaction in situ, in real time. This allows the extraction of both qualitative and quantitative information on the kinetics of a process and the exact steps lying on the reaction pathway. A variety of techniques have previously been applied to monitor re-... 

Liposomes (SUVs) were prepared by probe sonication according to standard procedures (31) in the presence of STPP. A mixture of lecithin, cholesterol, and STPP (PC/Ch/STPP = 65/15/20, molar ratio final total lipid 25 mg/ mL) was dissolved in chloroform followed by removal of the organic solvent using a rotary evaporator. After adding 5 mM HEPES (pH 7.4) to the dry lipid film, the sample was probe sonicated with a Sonic Dismembrator (Model 100, Fischer Scientific) at a power output of approximately 10 W for 30 minutes. To remove any titanium particles, which have been shed from the tip of the probe during sonication, the sample was centrifuged for 10 minutes at 3000 X g. The formed liposomes were separated from free, i.e., nonincorporated, STPP by gel filtration chromatography on a Sephadex G-15 column. 

What issues concerning sample system or probe fouling and process stream filtration or coalescing need... 

Much of fhe early work relied upon hand spotting or manual application of probes using vacuum filtration devices such as the DotBlot apparatus (BioRad Laboratories) that allowed the formation of more xmiform spotting of probes in fhe form of small dofs or rectangular slots. The use of membranes for prinfed DNA arrays (often referred to as "grid" arrays) was subsequently developed. 

Probe sonication extraction with (CH3)2C0 centrifugation filtration... 

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