A microdialysis system for the dialysis of small sample volumes of protein, nucleic acid, peptide, and other biomolecules has been developed. The device may contain a built-in magnet to permit remote rotation of the device during dialysis. Double sided dialysis chambers can be used for electrodialysis, electroelution, or electroconcentration. Two or more chambers can be joined either by a union or directly for equilibrium dialysis, or other applications.

BACKGROUND OF THE INVENTION 
This invention relates to a device and method for the dialysis and 
concentration of macromolecules (including proteins and nucleic acids) in 
small sample volumes. A number of methods are currently available or 
published: 
1. Floating membrane dialysis: A membrane is floated on water or buffer and 
a small droplet is placed on the membrane for dialysis. In this method, 
the volume of the sample can be changed by evaporation and it is difficult 
to handle the sample after dialysis or even an overflow of the sample can 
mix it with the buffer. 
2. A device to handle multiple samples is developed by Pierce, Inc., which 
as a capability of micro-dialysis but the exchange buffer volume is 
limited and it is an open system, which allows a cross-flow of the sample 
from one well to another. 
3. Hong described in U.S. Pat. No. 5,183,564, the dialysis of samples by 
shaking the sample compartment. The dialysis chambers are fused with the 
membrane, however, giving the user no choice of different membranes. The 
system is an open system and an increase in the volume is possible. The 
volume of the dialysate chamber is fixed therefore, in some cases a 
frequent exchange of buffers in the dialysis chamber is required. 
The principle objective of the invention is to provide a simple, efficient 
method for the dialysis of biological samples specially in small volumes 
in microliter range. This invention gives flexibility in selecting the 
volume of the samples as well as the pore size and type of the membrane. 
Further, the same unit can be used for the concentration of the sample 
without transferring the sample from one system to another. 
Another advantage is that of using inert material, for example, TEFLON.TM., 
which does not bind most of the biomolecules as compared to other plastic 
materials. TEFLON.TM. dialysis chambers are autoclaveable and easy to 
clean. 
Another advantage is each chamber be separately dialyzed as compared to 
multi-chamber of shaking dialysis system as described above, the small 
molecule can contaminate the other sample by back dialysis from buffer to 
the dialysis chamber. 
Another advantage is that dialysis and concentration can be done in the 
same chamber, so there is no loss of sample during the transfer from one 
container to the other. The sample is concentrated on a TEFLON.TM. surface 
so there is high recovery, as compared to the centrifugal method, where 
the sample concentrates on the membrane and it is known that membranes may 
bind many proteins and biomolecules. Further, centrifugal force may cause 
the biomolecules to pass through the membrane, reducing recovery. 
Another advantage is that these dialysis chambers can be used for 
electroelution, electrodialysis, electroconcentration, on-line dialysis, 
on-line electrodialysis, on-line electroconcentration. 
Another advantage is that these chambers can be joined together either by a 
union or directly with male-female connector. By using two or more 
chambers, a very selective filtration can be achieved by using membranes 
of different molecular weight cut-off between two chambers. 
Another advantage of this dialysis chamber is that it can be immersed in 
any buffer and it is leakproof. Therefore, without any extra unit this can 
be immersed in a bath of any suitable temperature for temperature 
controlled dialysis. This is not possible in other commercially available 
units. 
The advantage of a serpentine dialyser is that it can run at a very small 
flow rate of 1-1000 ml/min. The flow rate of the sample and type of 
membrane can determine the percentage of desalting. The total volume of 
the serpentine dialyser can be controlled by using the different length 
and depth of the serpentine. In combination with liquid chromatography, 
this serpentine dialyser is a very useful tool. However, without 
serpentine dialyser, sample should be collected in fractions and dialyzed 
in dialysis chambers individually.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENT 
The electroelution chambers described in the co-pending application can 
also be used for the dialysis and/or concentration of samples. The 
dialysis of small sample volumes (10-5000 .mu.l or larger) can be achieved 
by this method. The advantage of this method over the prior art is that it 
is very simple, easy to use and requires no gaskets. 
In this invention a dialysis chamber has been described for the dialysis of 
small sample volumes (in .mu.l to ml range). Furthermore, same device can 
be used for the concentration of the sample. The dialysis chamber is made 
of any suitable plastic material, including but not limited to plastic or 
glass. Suitable plastics include TEFLON.TM., acrylics and the like. The 
outer surface of dialysis chamber can be conical, round or any other shape 
is made of TEFLON.TM., glass or other plastic materials which do not react 
with the solutions used for the dialysis or buffer. For example, the 
chamber can be cylindrical for the use with a round gasket 4 to prevent 
leakage. Further, the chamber can have a cubical or rectangular shape to 
prevent it from rotating or rolling away from a specific position within a 
tank or other containers. 
The dialysis chamber 1 has a through-hole 7, which can be of different 
shape, sizes and diameters. This hole determines the volume of the 
dialysis sample. On both sides of the hole 7, there are bigger holes 
(wells) 3 and 3a, which are tapered and may be sealed by seals (5 and 6). 
In the case of dialysis, the wells are sealed by membrane 5 and 6, which 
can be of the same or of different types, such as different materials or 
different molecular size exclusions, described below. These membranes can 
be of different materials, for example, cellulose acetate, nitrocellulose, 
PVDF, or TEFLON.TM.. The membranes are semipermeable so as to trap 
macromolecules while allowing small molecules to pass through depending on 
the molecular weight cutoff of these membranes. Membranes 5, 6 can be 
placed at platform 2 and 2a, respectively, via a tapered screw 9, 9a. The 
screws 9 and 9a and the chamber can be joined, for example, by a threaded 
fitting (as in FIG. 1) or by a tapered quickfit or snap-type fitting. In 
addition, the chamber 1 can be provided with threads 12 on its exterior to 
cooperate with threads 13 on the screws 9, 9a as shown in FIG. 2. This 
configuration is advantageous as it positions the membranes 5, 6 in close 
contact with the liquid in the tank to allow faster interaction. 
The device may also be used as a macromolecule crystal growth system by 
sealing one well with a suitable transparent window (6) from which crystal 
growth may be observed. Buffer solution is slowly flowed through inlet 
(20) and circulated through outlet (21), thereby causing macromolecules to 
precipitate due to changes in pH. 
The screws 9, 9a are tapered to fit the holes 3 and 3a so that they can be 
screwed into the holes 3 and 3a, respectively. 
Screws 9, 9a are made of the same or compatible material as the chamber 1. 
Screws 9, 9a have a smooth surface 10, 10a which fit on the membranes 5 or 
5a and tighten on the surface 2, 2a in such a way that no fluid can leak 
from the hole 7. Leak-proof tightening can also be achieved by using 
o-ring. The membranes 5, 6 have the same diameter as the surface 10, 10a. 
Screw 9 or 9a has a through-hole 8 or 8a,respectively. The hole 8 and 8a 
can be conical or cylindrical, and can be of a different size and shape. 
As shown in FIG. 1, if in the dialysis chamber, one of the screws (8 or 9a) 
is replaced by a screw without through hole (11), the dialysis chamber can 
be used for the concentration of the samples. Further, this configuration 
can be kept under vacuum for further concentration of the sample in the 
sample compartment (7). The advantage of this type of concentration over 
centrifugal concentration of the sample is that the sample is concentrated 
on the TEFLON.TM. surface as compared to the centrifugal method where the 
sample is concentrated on the membrane. This method yields a much higher 
recovery of the sample as the sample is not in contact with the membrane 
after replacement with the screw (11) and therefore cannot stick to the 
membrane. 
As shown in FIG. 3, a magnet 14 can be placed inside the chamber wall in 
such a way that it has no contact with the liquid. This allows use of a 
magnetic stirrer 15 for dialysis or exchange of buffer. The magnet 
arrangement depicted in FIG. 5 is advantageous because the centrifugal 
force created by the stirrer 15 and magnet 14 interaction accelerates the 
exchange of molecules at the membranes 5, 6. 
With reference to FIG. 4, a single-sided chamber with built-in magnet can 
be used for simple dialysis as well as concentration of samples. 
The chamber in FIG. 1 can be used for electrodialysis, by placing the 
dialysis chamber in any electrophoresis tank in such a way that the 
chamber separates the anode and cathode buffer and the current flows 
through hole 7. 
Two or more dialysis chambers can be joined either by a union 16 as in FIG. 
5, or by the configuration as shown in FIG. 6a and 6b. By using the 
attachment 17, two chambers joined without the union have no dead volume. 
This two or more chamber configuration can be used for the 
electroconcentration of the samples. Further, using a throughflow chamber 
as in FIGS. 7a & 7b, the electrodialysis or electroconcentration of larger 
volumes can be achieved. 
FIG. 8 shows two chambers 19 and 19a, which contain serpentine channels (18 
& 18a) as flow path for the samples. These can be superimposed and the 
volume of the serpentine 18 or 18a can be changed by changing the depth of 
the serpentine. However, the length of the serpentine is constant. These 
two serpentines are held together (separated by a membrane 20 between 
them) using a clamp. The sample flows in from one end and flows out from 
the other end in one of the serpentines. In the other serpentine separated 
by a membrane, a constant flow of the buffer is maintained using a pump. 
This serpentine dialyser can be used in combination with a liquid 
chromatography column for the desalting or concentration of small 
molecules, which can pass through the membrane. 
EXAMPLES OF APPLICATIONS 
1. DIALYSlS OF SALTS FROM PROTEINS 
Take a 100 .mu.1 dialysis chamber (1). A membrane with a molecular weight 
cut off less than protein's molecular weight should be taken. First place 
membrane 6 and tighten with screw 9a and then fill the well 7 with the 
protein solution. Put another membrane of the same molecular weight 
cut-off on the other side and tighten with screw (9). Drop the dialysis 
chamber in a beaker with a desired buffer. If the dialysis chamber does 
not have a built-in magnet, a stirring bar and magnetic stirrer can be 
used to accelerate the dialysis process. However, if the dialysis chamber 
has a built-in magnet, it will further accelerate the dialysis. After the 
dialysis is complete, the sample can be concentrated by removal of the 
membrane of one side and the replacement of through hole screw (9 or 9a) 
with the solid screw (11). Place the chamber under vacuum. The water or 
solvent will diffuse through the membrane and the sample will be 
concentrated on the chamber bottom. 
2. EQUILIBRIUM DIALYSIS 
Two equilibrium dialysis chambers as shown in FIG. 6a are used for the 
study of equilibrium dialysis. The chambers can be opened at any time and 
a small sample aliquot can be taken for the determination of free 
biomolecules or drugs. Furthermore, three chambers can also be joined for 
the study of protein binding assays (FIG. 6b). The middle chamber is 
filled with the drug; the chamber on one side is filled with the protein 
and the chamber on the other side with the control buffer. A small aliquot 
is taken time to time to analyze the binding of biomolecule or drug to the 
protein. If the drug concentration is higher in the protein chamber than 
in the control chamber, it shows that the protein is binding the drug. 
This is a very simple and time saving method for the protein binding assay 
and it could be very useful in testing of new drugs. 
3. ELECTRODIALYSIS 
Charged molecules can be concentrated by electrodialysis. Chambers of 
different volume are used for the concentration of samples. A sample can 
be placed in a large volume chamber, the whole unit assembled as in FIG. 5 
and placed in an electrophoretic tank. The buffers are chosen according to 
the application, using criteria known to those skilled in the art. A 
current is applied in such a direction that the biomolecule moves from the 
larger chamber to the smaller chamber according to the charge of the 
molecule. Within 5-10 min., most of the sample concentrates in the small 
chamber. By using a through flow chamber (FIG. 7a and 7b) a continuous 
concentration can be achieved. Before injecting the sample in High 
Performance Liquid Chromatography (HPLC), the sample can be concentrated, 
desalted, or partially filtered using appropriate membrane or using 
through flow cell chambers in combination with an HPLC injection valve. 
4. ON-LINE DIALYSIS 
A serpentine chamber can be used on-line with the HPLC for the removal of 
salts on-line. Serpentine chambers of different volume are available and 
the flow rate at each side can also be controlled. This facilitates the 
concentration of the dialysate for protein binding assays. The serpentine 
system is also useful for the removal of unbound radioactive label 
compound in affinity chromatography, and sample purification during the 
separation. 
While a specific embodiment of the invention has been shown and described 
in detail to illustrate the application of the principles of the 
invention, it will be understood that the invention may be embodied 
otherwise without departing from such principle and that various 
modifications, alternate constructions, and equivalents will occur to 
those skilled in the art given the benefit of this disclosure. Thus, the 
invention is not limited to the specific embodiment described herein, but 
is defined by the appended claims.