Electrophoresis extraction device

• US 20090301885A1
• Filed: 05/05/2009
• Published: 12/10/2009
• Est. Priority Date: 11/07/2003
• Status: Active Grant

• Current Assignee: Princeton Biochemicals, Inc.
• Original Assignee: Princeton Biochemicals, Inc.
• Inventors: Guzman, Norberto A.
• Granted Patent: US 8,268,247 B2
• Time in Patent Office: Days
• US Class Current: 204/601
• CPC Class Codes: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 27/44708 Cooling
• CPC Class Codes: G01N 27/44743 Introducing samples
• CPC Class Codes: G01N 27/44756 Apparatus specially adapted therefor
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/561 Immunoelectrophoresis

Abstract

An electrophoresis apparatus is generally disclosed for sequentially analyzing a single sample or multiple samples having one or more analytes in high or low concentrations. The apparatus comprises a relatively large-bore transport capillary which intersects with a plurality of small-bore separation capillaries and includes a valve system. Analyte concentrators, having antibody-specific (or related affinity) chemistries, are stationed at the respective intersections of the transport capillary and separation capillaries to bind one or more analytes of interest. The apparatus allows the performance of two or more dimensions for the optimal separation of analytes. The apparatus may also include a plurality of valves surrounding each of the analyte concentrators to localize each of the concentrators to improve the binding of one or more analytes of interest.

Claims

1-321. -321. (canceled)

322. An electrophoresis extraction device, comprising:

a base having a transport passage first port, a transport passage second port, a separation passage first port and a separation passage second port;

the base having an elongated passage portion communicating the separation passage first port with the separation passage second port;

the transport passage first and second ports intersecting as a staggered configuration intersection with the elongated passage portion; and

the elongated passage portion defining an area for holding a means for concentrating an analyte of interest in a sample passing from the transport passage first port through the elongated passage portion to the transport passage second port and for allowing the concentrated analyte of interest to be released from the elongated passage portion and delivered out the separation passage second port by pressure, electrophoresis migration or a combination of electrophoresis migration and pressure and to an analyte detection system.

323. The extraction device of claim 322 wherein the analyte of interest is a simple or complex analyte, the concentrating means includes a first affinity ligand component and a different second affinity ligand component, and the first and second affinity ligand components are attracted to different portions of the simple or complex analyte which thereby increases the capturing of the analyte of interest by the concentrating means.

324. The extraction device of claim 322 wherein the concentrating means includes a combination of an antibody, a lectin and/or another affinity ligand to capture the analyte of interest (a) through a selective peptide and/or epitope or through a selective sugar present on the analyte of interest or (b) through both a selective peptide and/or epitope and a selective sugar present on the analyte of interest.

325. The extraction device of claim 322 wherein the concentrating means permits capture of the analyte of interest present in simple or complex mixtures of the sample for purification, desalting and enrichment.

326. The extraction device of claim 325 wherein the concentrating means isolates and separates the analyte of interest from the simple or complex mixture.

327. The extraction device of claim 322 wherein the sample is introduced by pressure from a sample reservoir into and through the transport passage first and second ports and to a waste reservoir.

328. The extraction device of claim 322 wherein the concentrating means includes beads interconnected to each other by at least one chemical bond within a matrix and to inner walls of the elongated passage portion.

329. The extraction device of claim 322 wherein the analyte of interest is a first analyte of interest, and the concentrating means also concentrates a second analyte of interest in the sample in the elongated passage portion and allows the concentrated second analyte of interest to be released from the elongated passage portion and delivered out the separation passage second port by pressure, electrophoresis migration or a combination of electrophoresis migration and pressure and to the analyte detection system.

330. The extraction device of claim 322 wherein the concentrating means includes beads having antibody fragments covalently attached to surfaces of the beads or intact antibodies covalently attached to surfaces of the beads.

331. The extraction device of claim 330 wherein the concentrating means includes an altered condition in the elongated passage portion to improve binding of the analyte of interest to the antibodies or the antibody fragments.

332. The extraction device of claim 331 wherein the altered condition is temperature.

333. The extraction device of claim 331 wherein the altered condition is a gentle shaking, a microwave pulse or an acoustic micromixing.

334. The extraction device of claim 322 wherein the concentrating means is adapted to perform on-line enzymatic cleavage of proteins to generate peptides.

335. The extraction device of claim 331 wherein the altered condition is a gentle shaking, a microwave pulse or an acoustic micromixing which is adapted to accelerate the work of proteases immobilized to a matrix of the concentrating means and reduce the time required to cleave a protein into peptide components thereof.

336. The extraction device of claim 322 wherein the concentrating means includes microstructures selected from a group consisting of beads, platelets, chips, fibers, polymers, globules and filaments.

337. The extraction device of claim 322 further comprising localizing means for localizing the concentrating means in the elongated passage portion to improve concentration of the analyte of interest by the concentrating means.

338. The extraction device of claim 337 wherein the localizing means includes first and second valves operatively associated with the transport passage first and second ports, respectively, and third and fourth valves operatively associated with the separation passage first and second ports, respectively.

339. The extraction device of claim 338 wherein the valves are motor operated and controlled remotely by a processor based on a predetermined set of instructions.

340. The extraction device of claim 322 wherein the staggered configuration intersection includes bulging members configured to retain the concentrating means in the elongated passage portion.

341. The extraction device of claim 322 wherein the staggered configuration intersection includes frit structures configured to retain the concentrating means in the elongated passage portion.

342. The extraction device of claim 322 wherein the staggered configuration includes means other than bulging members or frit structures for retaining the concentrating means in the elongated passage portion.

343. The extraction device of claim 322 wherein the analyte detection system is a component of an electrophoresis apparatus:

and further comprising (a) first coupling means for coupling the transport passage first and second ports to a transport passage of the electrophoresis apparatus, (b) second coupling means for coupling the separation passage first and second ports to a separation passage of the electrophoresis apparatus and (c) the analyte detection system is operatively associated with the separation passage.

344. The extraction device of claim 343 wherein the first coupling means includes threaded connectors and the second coupling means includes threaded connectors.

345. The extraction device of claim 322 wherein the base includes threaded openings, each associated with a respective one of the ports.

346. The extraction device of claim 322 wherein the elongated passage portion is longitudinally aligned with the separation passage first and second ports.

347. The extraction device of claim 322 wherein the base includes an opening for inserting the concentrating means into and replacing the concentrating means in the elongated passage portion.

348. The extraction device of claim 322 wherein the base includes four side-edge threaded openings for receiving therein a respective transport/separation passage connector.

349. The extraction device of claim 322 wherein the elongated passage portion is embedded and enclosed in the base.

350. An electrophoresis extraction device, comprising:

a base having a transport passage first port, a transport passage second port, a separation passage first port and a separation passage second port;

the base having an elongated passage portion communicating the separation passage first port with the separation passage second port;

the transport passage first and second ports intersecting as a staggered configuration intersection with the elongated passage portion;

means in the elongated passage portion for concentrating an analyte of interest in a sample passing from the transport passage first port through the elongated passage portion to the transport passage second port and for allowing the concentrated analyte of interest to be released from the elongated passage portion and delivered out to the separation passage second port by pressure, electrophoresis migration or a combination of electrophoresis migration and pressure and to an analyte detection system of an electrophoresis apparatus;

the extraction device being a component of the electrophoresis apparatus;

a connector which operatively connects the separation passage second port to a separation passage of the electrophoresis apparatus; and

the separation passage being operatively associated with the analyte detection system.

351. The extraction device of claim 350 wherein the connector defines a first connector, and further comprising a second connector which operatively connects the transport passage second port to a transport passage of the electrophoresis apparatus.

352. The extraction device of claim 351 wherein the extraction device defines a first extraction device and the transport passage connects to a connector of a second extraction device of the electrophoresis apparatus.

353. The extraction device of claim 351 wherein the transport passage is operatively communicable with a waste container.

354. The extraction device of claim 351 wherein the transport passage has a larger diameter than the separation passage.

355. The extraction device of claim 351 wherein the transport and separation passages are both capillaries.

356. The extraction device of claim 351 wherein the separation passage is a channel and the transport passage is a channel along at least a substantial portion of a length of the transport passage.

357. The extraction device of claim 356 wherein a portion of an inlet side of the transport passage is a capillary tube.

358. The extraction device of claim 351 further comprising a third connector which operatively connects the separation passage first port to a portion of the separation passage and a fourth connector which operatively connects the transport passage first port to a portion of the transport passage.

359. The extraction device of claim 358 wherein the portion of the separation passage is operatively communicable with a source of eluting and/or separation buffer.

360. The extraction device of claim 358 wherein with the analyte of interest concentrated in the elongated passage portion, the portion of the separation passage is operatively communicable with a conditioning buffer solution, then a separation buffer solution and then an elution buffer solution.

361. The extraction device of claim 358 wherein the portion of the separation passage is operatively communicable with a source of a cleaning solution with the analyte of interest concentrated in the elongated passage portion, the cleaning solution being adapted to wash away at least some of the salts and unwanted materials in the elongated passage portion.

362. The extraction device of claim 358 wherein the portion of the transport passage is operatively communicable with a source of the sample.

363. The extraction device of claim 351 wherein the sample is introduced by pressure from a sample reservoir into and through the transport passage and to a waste reservoir.

364. The extraction device of claim 351 wherein the elongated passage portion is embedded and enclosed in the base.

365. The extraction device of claim 351 wherein the concentrating means captures the analyte of interest present in simple or complex mixtures of the sample for purification, desalting and enrichment.

366. The extraction device of claim 351 wherein the concentrating means isolates the analyte of interest from simple or complex mixtures of the sample.

367. The extraction device of claim 351 wherein the concentrating means includes beads having antibody fragments covalently attached to surfaces of the beads or intact antibodies covalently attached to surfaces of the beads.

368. The extraction device of claim 367 wherein the concentrating means includes an altered condition in the elongated passage portion to improve binding of the analyte of interest to the antibodies or the antibody fragments.

369. The extraction device of claim 368 wherein the altered condition is temperature.

370. The extraction device of claim 368 wherein the altered condition is a gentle shaking, a microwave pulse or an acoustic micromixing.

371. The extraction device of claim 368 wherein the altered condition is a gentle shaking, a microwave pulse or an acoustic micromixing that is adapted to accelerate the work of proteases immobilized to a matrix of the concentrating means and reduce the time required to cleave a protein into peptide components thereof.

372. The extraction device of claim 351 wherein the concentrating means performs on-line enzymatic cleavage of proteins to generate peptides.

373. The extraction device of claim 351 wherein the base includes an opening for inserting the concentrating means into and replacing the concentrating means in the elongated passage portion.

374. The extraction device of claim 351 wherein the staggered configuration intersection includes bulging members positioned and sized to retain the concentrating means in the elongated passage portion.

375. The extraction device of claim 351 wherein the staggered configuration intersection includes frit structures configured to retain the concentrating means in the elongated passage portion.

376. The extraction device of claim 351 wherein the staggered configuration includes means other than bulging members or frit structures for retaining the concentrating means in the elongated passage portion.

377. The extraction device of claim 351 wherein the concentrating means includes microstructures selected from a group consisting of beads, platelets, chips, fibers, polymers, globules and filaments.

378. The extraction device of claim 351 wherein the concentrating means includes affinity ligands that are immobilized onto surfaces of magnetic beads.

379. The extraction device of claim 351 wherein the concentrating means includes a matrix assembly having a fixed architecture defined by interconnected polymeric microstructures that are formed from a monolithic lattice or a sol-gel lattice.

380. The extraction device of claim 351 wherein the concentrating means includes affinity ligands that are selected from a group consisting of whole antibodies, antibody fragments, lectins, aptamers, chemical dyes, protein A, protein G, substrates, enzymes, proteins, peptides, DNA, RNA, oligonucleotides, carbohydrates, cation exchange resins, anion exchange resins, immobilized metal affinity capture resins, mixed-mode resins, ions, aminoacids, monossacharides, fatty acids, vitamins, metabolites, viruses, bacteria, cells and subcellular organelles.

381. The extraction device of claim 351 wherein the concentrating means includes controlled porous glass beads having antibody fragments covalently attached to surfaces thereof.

382. The extraction device of claim 351 wherein the extraction device forms an analyte concentrator microreactor.

383. The extraction device of claim 351 wherein the concentrating means includes at least one of beads, platelets, chips, fibers, polymers, monolithic, sol-gels, filaments, cells, subcellular organelles, viruses, phages, micro-particles, nano-particles and cellular receptors.

384. The extraction device of claim 351 wherein the concentrating means includes entrapping cells, subcellular organelles or cellular receptors to perform metabolic studies and degradation products of small molecular weight substances and biomolecules.

385. The extraction device of claim 351 further comprising localizing means for localizing the concentrating means to thereby improve concentration of the analyte of interest by the concentrating means.

386. The extraction device of claim 385 wherein the localizing means includes first and second valves operatively associated with the transport passage first and second ports, respectively, and third and fourth valves operatively associated with the separation passage first and second ports, respectively.

387. The extraction device of claim 386 wherein the valves are motor operated and remotely controlled by a processor based on a predetermined set of instructions.

388. The extraction device of claim 351 further comprising means for cleaning sample solution from a sample solution supply before passing the sample to the transport passage first port.

389. The extraction device of claim 388 wherein the cleaning means to remove unwanted analytes of the sample solution that can be bound non-specifically to the concentrating means and to the transport passage by a cleaning solution passed through the transport passage.

390. The extraction device of claim 351 further comprising an auxiliary buffer passage communicating with the separation passage downstream of the concentrating means.

391. The extraction device of claim 351 wherein the base defines a first base, the elongated passage portion defines a first elongated passage portion, the analyte of interest defines a first analyte of interest, the concentrating means defines a first concentrating means, and the separation passage defines a first separation passage;

and wherein the electrophoresis apparatus includes a second base, a second elongated passage portion in the second base, a second separation passage, and second concentrating means for concentrating a second analyte of interest in the sample passing through the second elongated passage portion and for allowing the concentrated second analyte of interest to be released from the second elongated passage portion to the second separation passage and delivered by pressure, electrophoresis migration or a combination of electrophoresis migration and pressure to the analyte detection system.

392. The extraction device of claim 391 wherein the electrophoresis apparatus includes an exit passage into which the first and second separation passages merge and relative to which the analyte detection system is operatively aligned.