Affinity dialysis

Another common sample pre-concentration method is dialysis which serves to remove small molecules. For instance, affinity dialysis and pre-concentration of aflatoxins were achieved in a copolyester chip (see Figure 5.10). After affinity binding to the aflatoxin Bi antibody, various aflatoxins (Bj, B2, Gi> G2, G2J in a sample were retained, while the other small molecules passed through a PVDF dialysis membrane. Thereafter, the sample solution was exposed to a countercurrent flow of dry air, leading to water evaporation and analyte concentration. The concentrated and desalted sample was used in subsequent MS analysis [821], More details for MS analysis are described in Chapter 7, section 7.3. 

FIGURE 5.10 Side-view schematic of miniaturized affinity dialysis and pre-concentration system. I, II, and III indicate top, middle, and bottom imprinted copolyester pieces, respectively. Piece II is imprinted on both sides. Two PVDF membranes separate the copolyester channels [821]. Reprinted with permission from the American Chemical Society... 

A very recent series of publications by Locascio and others [54-56] demonstrated successful applications of various plastic materials, such as polydi-methylsiloxane (PDMS), polymethylmethacrylate (PMMA), copolyester, and their combinations, for assembling integrated fluidic structures to perform online sample pretreatment by affinity dialysis and concentration for fast and sensi-... 

Plastic microdevices for high-throughput screening with MS detection were also prepared for detection of aflatoxins and barbiturates. These devices incorporated concentration techniques interfaced with electrospray ionization MS (ESI-MS) through capillaries [2], The microfluidic device for aflatoxin detection employed an affinity dialysis technique, in which a poly (vinylidene fluoride) (PVDF) membrane was incorporated in the microchip between two channels. Small molecules were dialyzed from the aflatoxin/antibody complexes, which were then analyzed by MS. A similar device was used for concentrating barbiturate/antibody complexes using an affinity ultrafiltration technique. A barbiturate solution was mixed with antibodies and then flowed into the device, where uncomplexed barbiturates were removed by filtration. The antibody complex was then dissociated and electrokinetically mobilized for MS analysis. In each case, the affinity preconcentration improved the sensitivity by at least one to two orders of magnitude over previously reported detection limits. 

Hu Shangxu. Selective Removal of Metals from Wastewater Using Affinity Dialysis. In Emerging Technologies in Hazardous Waste... 

Selective separation and concentration of both cations and anions using water-soluble polymer solutions LM as carriers and hoUow-fiber units (artificial kidneys) as membrane barrier were tested. The authors termed the process as affinity dialysis [74]. Hollow fiber units of Spectrum Medical Industries, Inc. with fibers of 5000 molecular weight cutoff and 150 cm surface area from Spectrapor were used in the experiments. 

Figure 6.13 Schematic of a complete affinity dialysis miniplant with polymer solution stripping, regeneration, and recycle. From Ref. [74] with permission.

The affinity and cross-reactivity of the whole serum and Fab fragments were determined using equilibrium dialysis for the affinity determination and RIA for the cross-reactivity studies. The average intrinsic affinity constant (Ko) of the antibody (Nisonoff and Pressman 1958) changed very little throughout the... 

Competition for binding to heparin (in essentially salt-free solutions) has been studied for most physiological cations, by ion-exchange,372 equilibrium dialysis,373 c.d.,365 and 23Na-n.m.r. spectroscopy.370 The following order of affinity was generally observed Na+ < K+ < Mg2+ < Ca2+. 

The micropipette tip containing solid phases is a relatively new sample preparation technique that permits handling of microliter to submicroliter amounts of liquid samples, using the techniques of SPE, dialysis, and enzyme digestion. Various phases (reversed-phase, affinity, size-exclusion, etc.) are packed, embedded, or coated on the walls of pipette, permitting liquid samples to be transferred without undue pressure drop or plugging (Fig. 2.5). 

Equilibrium dialysis of homogenates of kidneys of rats given mercuric chloride, revealed that over 99% of the mercury was not diffusible [40]. Diffusible compounds of mercury have the opportunity to cross the capillary membrane and enter the tissue spaces however, due to chemical affinities for cellular binding sites and the diffusible complex, and the ability to penetrate the cell membrane, not all diffusible complexes of mercury present in plasma lead to tissue accumulation. 

An optical immunosensor for continuous T4 measurement has been described, in which the fluorescent indicator protein is separated from the sample flow chamber by a dialysis membrane.024) The indicator is T4-binding globulin (TBG), the intrinsic fluorescence (ex. 290 nm) of which is quenched by T4binding. Due to the high affinity of the TBG for thyroxine, the immunosensor is not reversible, but multiple measurements can be made until the TBG is saturated. Sensitivity is inadequate for clinically useful concentrations of T4, but suggestions for improvement of the method are made. 

In the absence of crystallographic data, there exist many other experimental criteria that allow the determination of the binding mode [30,38]. They can be classified into two categories the measurement of physical effects on DNA and spectroscopic studies (Table 1, first column, C and A respectively). Irrespective of the binding mode, the binding parameters (the affinity constant and the number n of occupied base pairs per molecule) can be determined from equilibrium dialysis and Scatchard plots [43 46]... 

The equilibrium dialysis experiment revealed that histidine-substituted salicylamide was selected as an RNA ligand. Subsequent binding analysis by UV titrations and Job plot revealed the histidine-substituted salicylamide Cu + complex bound the target RNA hairpin with an apparent dissociation constant of 150 nM. This binding constant likely reflects more complex binding processes than a simple 1 1 interaction, as the observed binding curve saturates well below the concentration of the histidine-substituted salicylamide, and thus the actual affinity of the complex for targeted RNA is probably lower. Importantly, however, titrations with the... 

Equilibrium Dialysis and Affinity Dilution. Equilibrium dialysis (34) and affinity (35) dilution experiments followed standard procedures. 

The antisera which were collected at ca. 100 days were first characterized for STXOL affinity (33). Equilibrium dialysis (34) gives a good measure of both antibody-to-hapten affinity ( gj useful range for RIA development 10 L/mole) and antibody... 

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