Microdialysis cutoff

The dialysis chambers are assembled. We use microdialysis chambers in which the volume of the two compartments is 0.35-0.4 ml. The chambers are separated by 50,000 molecular weight cutoff SpectraPor dialysis tubing. 

A straightforward way to collect solutes from the interstitial fluid (ISF) space would be to have a semipermeable, hollow fiber, membrane-based device as originally described by Bito et al.1 Two semipermeable membrane-based devices that have been used to collect different types of analytes from various mammalian tissues include microdialysis sampling probes (catheters) and ultrafiltration probes. The heart of each of these devices is the semipermeable polymeric membrane shown in Figure 6.1. The membranes allow for collection of analytes from the ISF that are below the membrane molecular weight cutoff (MWCO). Each of these devices provides a sample that has a significantly reduced amount of protein when compared to either blood or tissue... 

Microdialysis was achieved in a fused silica chip with in situ photopattemed porous membrane, as shown in Figure 5.13. Phase-separation polymerization of the membrane (7-50 pm thick) was formed between posts. The posts maximize the mechanical strength of the membrane so that it can withstand a pressure drop of 1 bar. Low MW cutoff (MWCO) membrane, which can be formed by using less organic solvent, 2-methoxyethanol, appears to be more transparent (see Figure 5.13). This low MWCO membrane can be used to dialyze away low MW molecules, such as rhodamine 560, but not fluorescently labeled proteins (insulin, BSA, anti-biotin, and lactalbumin). Fligh MWCO membrane, which was formed by more organic solvent, allows diffusion of lactalbumin [347]. 

In brain research, microdialysis sampling employing a miniaturized dialysis unit (probe) containing a dialysis membrane of a few millimeters length has become popular. The probe is implanted into the tissue or organ of the test animal and is infused with an isotonic solution (typically at 0.5-25 L/min). A steady-state osmotic flux across the membrane removes molecules with a mass below the cutoff of the membrane from the extracellular matrix. Microdialysis yields relatively clean samples of volumes in the range 20-100 jU-L. However, the recovery of neuropeptides can be as low as 0.5-15%, leading to a low neuropeptide concentration in the samples [5,6]. 

Dialysis membranes (12,000-mol-wt cutoff) for microdialysis apparatus (Gibco-BRL). 

Transfer each culture lysate to a well of a microdialysis apparatus (Gibco-BRL, 28-place apparatus set up with a 12,000 mol wt cutoff membrane). Dialyze samples against 6 L of 50 mM Tris, pH 7.2, 5.0 mM Na2EDTA, over 60-75 min at room temperature (buffer elevation approx 30 cm above the apparatus), samples will become slightly cloudy and volume will increase to approx 600 pL. 

In microdialysis, when the perfusion solution is pumped through the fiber capillary, there is ideally no change in volume of the perfusate and no fluid removed from the tissue. This requires a negligible pressure gradient across the membrane. The perfusate must be isosmotic with the tissue. If either the membrane capillary or the outlet capillary is very long, the back pressure at the membrane can become sufficient for it to leak. This can be minimized by using low flow rates and membranes that have relatively low molecular weight cutoffs. 

Two additional techniques that are used in sample preparation protocols are ultrafiltration and microdialysis. In ultrafiltration, pressure is applied to a membrane and those molecules smaller than the molecular-weight cutoff can pass through while molecules larger are retained. This technique can be used as a way of sample concentration or as a way to eliminate higher-molecular-weight compounds from an analytical scheme. Membranes are available with cutoffs ranging from 300 to 300,000 daltons. Microdialysis differs from the other techniques because it is in vivo sampling and has been applied to the... 

This is of particular interest for determination of drugs. Most drugs bind to proteins to a certain extent (up to 99%) and only the unbound fraction is biologically active. In such cases, microdialysis gives an estimate of realistic free concentration. Factors such as flow rate of the perfusate, diameter and length of the membrane, molecular mass cutoff, and membrane composition have influence on microdialysis. 

Although membranes with a cutoff between 5 and 35 kDa are mostly used, the higher molecular mass cutoff membranes are also commercially available. The values given suggest that analytes with a molecular mass up to the cutoff value can be isolated. However, this is not the case, since these values reflect the cutoff mass at equflibrium. The cutoff value during microdialysis will be considerably lower and it is therefore recommended not to choose a cutoff too close to the molecular mass of the analyte. For a membrane with a cutoff of 5 kDa, the recovery of compounds larger than 1 kDa is lowered due to lower diffusion. 

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