US3826734A - Apparatus for use in liquid sample analysis - Google Patents

Abstract

An electrophoretic cell having an elongate electrolytic expanse is enveloped between an overlayer and a substrate, the overlayer including a closable opening through which a liquid sample may be applied to a preselected location on the elongate electrolytic expanse. The cell provides for separation of the constituents of the sample on application or preselected electric voltage to the cell electrodes.

Description

F. W. GODSEY, JR

APPARATUS FOR USE IN LIQUID SAMPLE ANALYSIS Filed June 26. 1972 FIG.

,10 ,12 I4a\ lflflhlll [I43 WI IHHM! i 4 will 14c 5W Noam Ib III 4 ll HI II! L mb "W 206-1 '1 United States Patent 3,826,734 APPARATUS FOR USE IN LIQUID SAMPLE ANALYSIS Frank W. Godsey, In, St. Petersburg, Fla., assignor to Bio-Medical Sciences, Inc., Fairfield, NJ. Filed June 26, 1972, Ser. No. 266,449 Int. Cl. 301k /00 U.S. Cl. 204-499 Claims ABSTRACT OF THE DISCLOSURE An electrophoretic cell having an elongate electrolytic expanse is enveloped between an overlayer and a substrate, the overlayer including a closable opening through which a liquid sample may be applied to a preselected location on the elongate electrolytic expanse. The cell provides for separation of the constituents of the sample on application or preselected electric voltage to the cell electrodes.

FIELD OF THE INVENTION 1 This invention relates to apparatus for use in the analysis of fluid samples and more particularly to test apparatus for use in determining protein fractions in human body fluids such as blood.

BACKGROUND OF THE INVENTION Various procedures are presently known for separating the protein fractions of blood serum for quantification thereof, e.g., salting-out of the fractions, precipitation of the fractions using predetermined concentrations of organic solvents, isoelectric, cationic and anionic precipitation, and the use of centrifuges and electrophoresis. As presently implemented, all of these methods are practiced with laboratory discipline and involve the collection of a sample of whole blood in the order of cubic centimeters, transportation of the collected sample to a laboratory facility, separation of blood serum from the whole blood sample and finally, the qualitative and quantitative analysis of the serum. In the most widely practiced of the foregoing procedures, namely, that involving electrophoresis, the whole blood sample together with a clotting agent is placed in a centrifuge and the resulting blood serum is applied to a medium many inches in length and pre-wetted with an electrolyte of pH level sufficient to enable the medium to support the migration of protein fractions. The medium is disposed across a trough-like electrolyte container with the opposed medium end portions immersed in container electrolyte. An electric voltage of one hundred or more volts is applied between a pair of electrodes supported by the container and electrically connected by container and medium electrolyte. An electrical field is thus applied to the serum and induces migration of the protein fractions along the medium, fractions involving smaller molecules of higher electrical charge moving at a faster rate than other fractions involving larger molecules of lesser electrical charge. With the passage of time, the participating protein fractions of different type migrate to distinct zones having definite boundaries. Upon completion of a preselected time, the applied voltage is discontinued and the medium is subjected to treatment which fixes the protein fractions in their respective migra tion zones. Quantification of the separated and fixed fractions is attained by chromatography, i.e., by staining the medium and comparing color therein with a standard, or by passing the medium through a densitometer.

As is evident from the foregoing, the presently known procedures for blood serum analysis require apparatus not ordinarily available for use outside a laboratory and demand the discipline of a skilled technician. In this re- 3,826,734 Patented July 30, 1974 SUMMARY OF THE INVENTION It is an object of the present invention to provide improved apparatus for use in the analysis of fluid samples.

It is a more particular object of the invention to provide improved electrophoretical apparatus for use in analyzing blood serum.

In the efficient attainment of these and other objects, the present invention provides elec'trophoretical apparatus usable in non-laboratory environment for the receipt of a fluid sample and for separating fractions thereof into distinct zones for quantification. In its particularly preferred form, such apparatus includes, on a common substrate, a blood serum protein fraction-discriminating cell disposed in a sealed container openable at the time of ap paratus use and a further cell adapted to supply all operating power required by the protein fraction discriminating cell.

The foregoing and other objects and features of the invention will be evident from the following detailed description of preferred embodiments thereof and from the drawings wherein like parts are identified by like reference numerals throughout.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a plan view of one embodiment of apparatus in accordance with the invention.

FIG. 2 is a front elevational view of the FIG. 1 apparatus taken in section along the plane II-II of FIG. 1.

FIG. 3 is a partial view taken in section of an alternate form of the FIG. 1 apparatus.

FIG. 4 is a plan view of a second embodiment of apparatus in accordance with the invention.

FIG. 5 is a plan view of a third embodiment of apparatus in accordance with the invention.

FIG. 6 is a side elevational view of the FIG. 5 apparatus taken in section along the plane VI-VI of FIG. 5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Test apparatus 10 of FIGS. 1 and 2 includes a substrate 12 to which is secured the marginal portions 14a-d of a film 14. The substrate and film are comprised of chemically inert, electrically on-conductive materials. Film 14 is substantially transparent to light and the substrate may also have this characteristic, Film 14 includes an opening 14:: extending therethrough for purposes discussed below. This opening is closed by removable sealing strip 16 preferably equipped with a tab 16a to facilitate removal thereof. First and second electrodes 18 and 20 are spacedly disposed on substrate 12 interiorly of film 14 and are separated by elongate means in the form of a strip member 22. Electrodes 18 and 20 are preferably comprised of like or dissimilar metals and strip member 22 is typically a strip of paper, cellulose acetate or polyethylene foam, opposed end surfaces thereof preferably abutting the facing surfaces of electrodes 18 and 20, respectively. In assembling the FIG. 1 apparatus, electrodes 18 and 20 are preferably formed on substrate 14, e.g., by vacuum deposition or spraying techniques, and strip member 22 is treated to provide electrolytic connection of the electrodes of pH level supporting migration of the constituents of a liquid sample under study, e.g., the protein fractions of blood serum, upon application of preselected electrical voltage to the electrodes. In the case of blood serum, the

electrolyte may comprise sodium diethylbarbiturate buffer, known commercially by the trademark Veronal and having a pH of 8.6. Such treatment of the strip member may comprise pre-wetting the strip throughout or applying electrolyte to surface 22a thereof to provide a film of electrolyte thereon, constituent migration desirably occurring in such electrolyte film and not within the strip member. Film 14 may be fabricated so as to provide nominal clearance between itself and surface 22a. Since the film is secured to the substrate along its marginal portions, the film comprises a containment layer for the electrophoretic cell comprised of electrodes 18 and 20 and the elongate means therebetween. By virtue of the seals between members 12 and 14 and between 14 and 16, loss of electrolyte is minimized and apparatus shelf life is enhanced.

Various alternative modes of assembling the apparatus of FIGS. 1 and 2 may be employed. For example, the assembly of elements may be accomplished with strip member 22 in dry state, electrolyte being added prior to securement of sealing strip 16 to film 14 or at the time of use of the apparatus.

Conductors 18a and 20a extend from electrodes 18 and 20, respectively, through film 14 to provide for the connection of a voltage source 24 to the electrodes.

In use of the apparatus of FIGS. 1 and 2, sealing strip 16 is peeled away from film 14 at the time of use and pre-measured quantity of blood serum, less than a drop as contrasted with above-discussed cubic centimeters laboratory quantity, is applied to opening 14c of the cell containment layer. After a short time period, the residue of the drop is wiped away from opening 14c. At this time, conductors 18a and 20a are connected to voltage source 24, which provides a voltage of magnitude sufficient to provide the field intensity (volts/cm.) throughout the electrolyte film on strip member 22 required for protein fraction separation.

The elongate means supporting protein fraction separation in the apparatus of FIGS. 1 and 2 may comprise a strip member of several centimeters in length, as contrasted with the separation supporting means in prior laboratory-type apparatus of many inches in length. Accordingly, to attain sufficient field intensity, e.g., 20 volts per centimeter, apparatus of FIGS. 1 and 2 requires a voltage source providing tens of volts rather than the hundreds of volts required in prior laboratory-type apparatus. Strip member width is likewise reduced from that of the prior laboratory apparatus. The apparatus of FIGS. 1 and 2 provides for the clear separation of protein fractions in shortened migration distances and in consequently shortened time periods. Such separation occurs on completion of a time period predeterminable in accordance with the distance between electrodes 18 and 22, the voltage provided by source 24, the electrolyte employed, and like factors influencing migration. Following separation, the elongate means is subjected to fixing, and may thereafter be stained and inspected by chromatography whereby each separated fraction is quantized.

Apart from such staining technique, the invention contemplates optical reading of the separated constituent magnitudes. In this connection, substrate 12, film 14 and strip member 22 may all be comprised of material substantially transparent to light whereby light diffraction through the apparatus, attributable to protein fraction magnitude, provides a basis for quantizing. 'As discussed above, blood serum quantity employed in samples analyzed by the apparatus of the invention is quite small. As-a result, a separation of the blood serum sample from whole blood .may be effected by the use of filter 3, microporous member 26 may be disposed in the aperture 14e of film 14. Where whole blood is applied to the member, clotting thereof occurs in the member and only the serum passes therethrough to strip member 22. Such microporous member may incorporate a clotting agent to enhance the clotting of whole blood therein.

means integral with the test apparatus. Referring to FIG.

Test apparatus 28 of FIG. 4 includes on its substrate 30 an electrophoretic cell 32 constructed as in the case of the electrophoretic cell of the apparatus of FIGS. 1 and 2. While in the apparatus of FIGS. 1 and 2, conductors 18a and 20a were connected to a voltage source 24, not integral with apparatus 10, corresponding conductors 18b and 20b of apparatus 28 are connectedto a galvanic cell 34 disposed on substrate 30. The FIG. 4 apparatus is accordingly self-contained as respects all elements needed in providing for the separation of the constituents of a liquid sample. In the particular application of the apparatus to blood serum analysis, galvanic cell 34 is required tov apply to conductors 18b and 20b a voltage in the order of tens of volts, as discussed above, and is thus preferably comprised of a plurality of discrete cells connected in series circuit between conductors 18b and 20b each discrete cell providing a complement of such total voltage. In the illustrated embodiment of apparatus 28, three such cells are illustrated each being commonly structured. By way of example, cell 36 may include first and second electrodes 38 and 40, respectively fractions of milligrams of carbon and zinc spacedly disposed on substrate 30 with suitable electrolytic connection therebetween. Such electrolytic connection may be provided by the use of an electrolyteabsorbent member 42 comprised of paper or the like. The electrolyte for activating cell 36 may be introduced into member 42 at the time of use as is illustrated schematically by removable sealing member 43.

Use of the Fig. 4 apparatus parallels that of the FIGS. 1 and 2 apparatus discussed above, the application of electrical voltage to electrophoretic cell 32 of FIG. 4 being accomplished by activating galvanic cell 34. In the galvanic cell form illustrated schematically, activation occurs on addition of electrolyte to all the individual units, e.g., 36. Where the galvanic cell is provided beforehand with electrolyte, means may be incorporated for providing selective connection of the galvanic cell to the electrophoretic cell to control application of voltage to the electrophoretic cell. 1

Referring to FIGS. 5 and 6, apparatus 44 comprises a substrate 46 having a surface depression 461: covered by cell containment layer 48. Such depression defines a trough-like receptacle for an electrophoretic cell including electrodes 52 and 54 and elongate means comprising a body of electrolyte 50. Layer 48 includes therein an opening 48a in communication with a channel 56 extending to depression 46a. Layer opening 48a is closed by removable sealing member 58 having tab 58a. On removal of member 58, the sample is applied to layer opening 48a and thence through channel 56 into the electrolyte in depression 46a. Upon application of voltage to electrode conductors 52a and 54a, separation of the sample constituents occurs in electrolyte 50 between the electrodes.

In the apparatus of FIGS. 4 and 5, the dimensions of depression 46a are selected with a view toward minimizing mixing of the sample with the electrolyte and toward avoiding any migration of the sample constituents'in the absence of applied voltage. The mobilities of sample constituents are increased from sample constituent mobilities in the previously-discussed embodiments of apparatus in accordance with the invention in view of the increased migration-supporting electrolyte area. Accordingly, lessened time for sample analysis is required. The greater ratio of surface area to volume in the apparatus of FIGS. 4 and 5 as compared to the previously-discussed appara tus serves effectively to reduce temperature rises from electrical sources. In addition, lesser sample volume is required. Where substrate 46 and cell containment layer 48 are comprised of materials substantially transparent to light, the aforementioned optical reading of sample constituents may be practiced upon completion of constituent migration.

It is to be appreciated that various changes may be made in the foregoing particularly disclosed preferred embodiments of apparatus according with the invention.

For example, any of the vast number of materials having the aforementioned characteristics may be used in respect of the apparatus substrates, cell containment members, sealing strips, electrodes, electrolytes and the like. Immobilizing additives, such as gelling agents, for the electrolytes are Within the contemplation of the invention. Accordingly, the particularly disclosed embodiments are intended in an illustrative and not in a limiting sense. The true spirit and scope of the invention is defined in the following claims.

What is claimed is:

1. A self-contained electrophoretic cell comprising a chemically inert, electrically non-conducting substrate member, a pair of electrodes fixed to said substrate member, a thin strip member disposed on said substrate member between and in electrical contact with said electrodes, said strip being operable to support an electrophoretic medium, and a chemically inert electrically non-conducting transparent film overlying said strip and said electrodes and having its marginal portions sealed to said substrate member, said film having an opening operable to permit introduction of a sample to said strip and closure means operable to seal said opening before and after sample introduction.

2. A cell as defined in claim 1 wherein said strip member is wetted throughout by an electrolyte prior to sealing said film.

3. A cell as defined in claim 1 including a pair of conductors in electrical connection with said electrodes and extending to the exterior of said cell.

4. A cell as defined in claim 3 wherein said conductors are adapted for electrical connection with an outside voltage source.

5. A cell as defined in claim 3 including a self-contained voltage source in electrical contact with said electrodes through said conductors.

6. A cell as defined in claim 1 wherein said strip member comprises paper, cellulose acetate or polyethylene.

7. A cell as defined in claim 1 wherein said substrate- UNITED STATES PATENTS 794,864 7/1905 Kamperdylc 136-952 795,325 7/1905 Winters 136-1252 968,154 8/1910 Hite 136-127 X 3,432,414 3/1969 Rand 204- G 3,582,490 6/1971 Zemel 204-180 G 3,594,263 7/1971 Dwyer et a1. 204-180 S X 3,635,808 1/1972 Elevitch 204-180 G 3,674,678 7/1972 Post, Jr. et al. 204-299 3,691,054 9/1972 CaWlcy 204-299 JOHN H. MACK, Primary Examiner A. C. PRESCOTT, Assistant Examiner US. Cl. X.R. 204-180 G, 180 S

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