Title of Invention

A SIMPLE AND EFFICIENT MICRODIALYSIS ASSEMBLY FOR DIALYSIS OF SAMPLES IN MICROLITER VOLUMES

Abstract

A simple and efficient microdialysis assembly for dialysis of samples in microlitre volumes is described. The assembly is prepared from microcentrifuge tube. The invented assembly is simple in design and easy to use. The chances of formation of air-pockets between sample and dialysand are very low and even if air pockets are formed, it is convenient to remove them without risk of rupturing membrane. Dialysis is highly efficient and removes 97% of small molecules in 2 hours. Recovery of large size molecules such as protein is in the range of 85 to 95%. Sample loading and sample recovery are convenient.

Full Text

This invention relates to construction of microdialysis assembly for dialysis of samples in microlitre volumes wherein free flow of dialysand is maintained in the vicinity of dialysis membrane by creating holes in assembly at defined positions. The free flow of dialysand does not allow trapping of air in the vicinity of membrane and thus enhances efficiency of dialysis. Prior Art: Dialysis method removes selectively small molecular weight components from solution. Dialysis sacs of different diameters are commercially available to suit wide volume range of samples. Use of these sacs for volumes less than 500 u,l is not preferred because of poor sample recovery. Alternative devices have been developed for dialysis of samples in microliter volumes with quantitative recoveries. These devices/ methods include • Cellophane is placed over glass-wool-dialysis-wick through which dialysand flows between two beakers. Sample in the form of drop is added over the cellophane. Sample during dialysis spreads and to avoid spreading of sample, cellophane membrane is boiled for 5 days. Further, colourless samples are difficult to visualize and therefore difficult to recover. Evaporation of sample may also occur during dialysis. • Microchambers are prepared from lid and rim of microcentrifuge tube. A hole is made in lid with heated wire and dialysis membrane is placed between lid and rim. After sample application, hole is sealed with adhesive tape. These chambers are placed in a beaker containing dialysand. After dialysis, adhesive tape is removed and sample is recovered through hole. These chambers have following disadvanges. (i) While recovering sample, there is accidental loss due to rupturing of membrane, (ii) During stirring, microchambers tumble and if chamber is not completely filled with sample, air-pockets will hinder dialysis, (iii) Because of small size, handling of microchamber during sample recovery is inconvenient. • Sample is dispensed in microcentriftige tube and is dialysed across dialysis-membrane secured over the tube opening by a perforated microcentrifuge cap. The retentate is recovered by centrifugation. However, air-pocket between dialysis membrane and dialysand solution is usually formed and removal of air-pocket is essential for efficient dialysis. Care is to taken not to rupture membrane during air-pocket removal. Detailed Description of Invention- A microdialysis assembly, which we have invented, is constructed from microcentrifuge tube. The chances of formation of air-pockets between dialysis membrane and dialysand are least and even if these pockets are formed, it is very convenient to remove them. The construction of dialysis assembly is described below. Microdialysis assembly is constructed from microcentrifuge tube (Fig.la). These tubes have a recess inside the lid that forms a dialysis chamber. The lid (Fig.lb) and tube (Fig.lc) are obtained from microcentrifuge tube (Fig.la) by making a cut at indicated points (Fig.la). The obtained lid (Fig.Ib) is used for dialysis chamber. Holes are made in tube (Fig.lc) below the flange and other places to obtain holed-tube (Fig. 1 d) for free flow of dialysand during dialysis. Lid is placed over horizontal surface, with recess portion facing upward (Fig.le). Sample (to be dialysed) is added to recess portion of the lid and over this dialysis membrane is placed (Fig.le'). This is referred as dialysis chamber. Holed-tube in inverted position is then placed over dialysis chamber and the assembly is referred as dialysis assembly (Fig.If). The dialysis assembly is placed in a float which is made to stuck to walls of beaker containing dialysand. After dialysis, dialysis assembly is placed in inverted position (Fig.lh). Then holed-tube is detached (Fig.li) and membrane is removed (Fig.lj). Dialysed sample is then collected from recess portion of lid. The recovery of protein (large size molecule) after dialysis is in the range of 85 to 95 %. More than 97% small molecules are removed in 2 hour. These data indicates good recovery of large size molecules and excellent efficiency in removal of small molecules. The assembly is simple in construction and convenient to use. Air-pockets between membrane and dialysand are rarely formed and even if these are formed, these can be removed by just tilting the assembly while it was immersed in dialysand. This feature of assembly avoids chances of air pocket formation and membrane is not damaged during air-pocket removal. We claim 1. An assembly for microdialysis having interconnectivity of recessed face of lid, dialysis membrane and holed-tube wherein holes are made at positions just lower to flange for preventing air-pocket formation between dialysis membrane and dialysand and at further lower positions for enhanced dialysis. 2. An assembly for microdialysis as claimed in claim 1 wherein the lid is obtained from microcentrifuge tube. 3. An assembly for microdialysis as claimed in claim 1 wherein holed- tube is prepared from microcentrifuge tube. 4. Accordingly, fci assembly for microdialysis as claimed in claim 1 has been described with reference to accompanying drawings.

Documents:

245-del-2002-abstract.pdf

245-del-2002-claims.pdf

245-del-2002-correspondence-others.pdf

245-del-2002-correspondence-po.pdf

245-del-2002-description (complete).pdf

245-del-2002-drawings.pdf

245-del-2002-form-1.pdf

245-del-2002-form-19.pdf

245-del-2002-form-2.pdf

245-del-2002-form-3.pdf