Free solution electrophoresis-membrane filters trapping assay apparatus and method

• US 5,427,664 A
• Filed: 07/22/1993
• Issued: 06/27/1995
• Est. Priority Date: 07/22/1993
• Status: Expired due to Fees

• Current Assignee: Cvetan Strshenovsky, Stoyan V. Stoev
• Original Assignee: Cvetan Strshenovsky, Stoyan V. Stoev
• Inventors: Stoev, Stoyan V., Strshenovsky, Cvetan
• Primary Examiner(s): Niebling, John
• Assistant Examiner(s): STARSIAK, JOHN S
• Application Number:
  US08/095,960
• Time in Patent Office: 705 Days
• Field of Search: 204/299 R, 204/182.8, 204/180.1, 204/301, 204/182.3, 204/182.6
• US Class Current: 204/516
• CPC Class Codes: G01N 15/0272 with screening; with classification by filtering B01D takes precedence
• CPC Class Codes: G01N 27/447 using electrophoresis

Abstract

A free solution electrophones-membrane filter trapping assay apparatus includes a container and at least one porous membrane. The porous membrane and container define a chamber capable of holding run solution. A specimen containing particles to be classified is juxtaposed with the run solution. A voltage is placed across the specimen, run solution and membrane to cause particles to be released from the specimen. Particles enter the run solution and readily pass therethrough. Particles that are smaller in size than the pores of the membrane pass through the membrane while particles having a larger particle size do not. A method of classifying particles includes the steps of providing a sample of particles and a porous membrane, positioning a run solution between the sample and the membrane and applying an initial voltage across the sample, run solution and membrane.

Claims

1. A method of classifying, by size, electrically charged particles contained in a specimen, the method comprising the steps of:

providing a first porous membrane having a first pore size that permits a portion of the particles to pass therethrough;

positioning a run solution adjacent to the membrane;

applying an initial voltage across the specimen, run solution and membrane; and

reducing the initial voltage.

2. The method of claim 1 further comprising the step of recirculating the run solution.

3. A method of classifying molecules contained in a sample by size, the method comprising the steps of:

(1) preparing a sample containing molecules wherein the molecules are obtained from cells having a nucleus, the preparation comprising the steps of;

loading cells on a cushion;

centrifuging the cushion and cells under conditions effective to permit the nuclei to pass through the cushion; and

(2) providing a porous membrane;

(3) positioning a run solution adjacent to the membrane; and

(4) applying an initial voltage across the sample, run solution and membrane.

4. The method of claim 3 further comprising the step of recirculating the run solution.

5. A vertical, free solution electrophoresis, membrane filter trapping apparatus suitable for classifying of electrically charged particles by size, shape and weight, the particles, prior to classifying, being contained in a specimen, the apparatus comprising:

a container having an elongate shape having upper and lower ends;

a holder suitable to hold a specimen positioned on the upper end of said container, whereby the releasing of particles from a specimen is steady and causes preliminary separation of the particles by size, starting with releasing of smallest and finishing with largest particles;

a cascade of membrane filters, including first and last membrane filters, received in the container and having pore sizes, the membrane filters being arranged in sequence by pore size, with the first membrane filter having the largest pore size being adjacent the specimen holder on the upper end of the container to form a first chamber for free solution movement of particles between the specimen holder, the first membrane filter and the inner wall of the container, and last membrane filter having the smallest pore size and being adjacent to the lower end of the container and forming a last chamber with side walls of the lower end of the container;

electrodes, connectable to a suitable power supply of direct current, for creating an electrical field across said container; and

means for assembling and disassembling of said specimen holder and said cascade membrane filters into and from said container, whereby membrane filters in said cascade can be very easily varied in numbers, depending on the number of different kinds of particles in the specimen to be classified and can be very easily disassembled for final recovery, investigation or other needs, the apparatus, the apparatus being capable of containing a free run liquid solution as a run buffer with predetermined strength, pH, temperature and constituents suitable to run electrophoretical movement of concrete particles, whereby particles can move freely in said solution with relatively high speed at very low viscosity.

6. The apparatus of claim 5 further comprising means for recirculating a free run liquid solution between said first chamber with said last chamber.

7. The apparatus of claim 6 wherein the recirculating means comprises a peristaltic pump.

8. The apparatus of claim 6 further comprising means for maintaining a free run liquid solution at a constant temperature.

9. The apparatus of claim 5 wherein the electrodes are vertically positioned, wound about tubes extended from the bottom and from the cover along an axis of the container, whereby a more homogeneous electrical field is created.

10. The apparatus of claim 5 further comprising means for assembling and disassembling the apparatus capable of being inserted into the container, the assembling and disassembling means comprise:

an elongate carder having a retainer in a lower part of the carder for retaining the last membrane filter;

a plurality of spacers with suitable dimensions, whereby determining the volume of the chambers and the length particles travel between membranes; and

a pair of tubes having holes to receive said carrier positioned one extended from the bottom of the container and the other from the cover.

11. The apparatus of claim 5 wherein said membrane filters are built from synthetic polycarbonate material.

12. The apparatus of claim 5 further comprising means for recirculating the free run liquid solution.

13. A free solution electrophoresis membrane trapping assay method of classifying, by size and shape, electrically charged particles contained in a specimen, the method comprising the steps of:

providing a container having therein at least one porous membrane having a predetermined, well defined pore size;

providing a specimen holder;

providing a specimen containing electrically charged particles;

placing the specimen on the specimen holder;

positioning a free run solution in the container; and

applying an initial high voltage across the specimen, run solution and at least one porous membrane, the voltage being in the range of about 0.5 to about 10 volts per centimeter, the initial high voltage being decreased a predetermined amount at predetermined time intervals to facilitate steady and consistent release of macromolecules by size.

14. A free solution electrophoresis membrane trapping assay method of classifying, by size and shape, electrically charged particles contained in a specimen, the method comprising the steps of:

providing a container having therein at least one porous membrane having a predetermined, well defined pore size;

providing a specimen holder;

providing a specimen containing electrically charged particles;

placing the specimen on the specimen holder;

positioning a free run solution in the container;

applying an initial appropriate voltage across the specimen, run solution and at least one porous membrane; and

calibrating the classification of the particles using commercially available DNA having a known length.

15. A free solution electrophoresis membrane trapping assay method of classifying, by size and shape, electrically charged particles contained in a specimen, the method comprising the steps of:

providing a container having therein at least one porous membrane having a predetermined, well defined pore size;

providing a specimen holder;

providing a specimen containing DNA macromolecules obtained from whole cells or purified cellular nuclei;

embedding the cells or nuclei in age plug containing about 0.35 to about 0.75 wt % agarose gel;

placing the embedded specimen on the specimen holder;

positioning a free run solution in the container; and

applying an initial voltage across the specimen, run solution and at least one porous membrane.