Viral identification by generation and detection of protein signatures

• US 20050014134A1
• Filed: 03/08/2004
• Published: 01/20/2005
• Est. Priority Date: 03/06/2003
• Status: Abandoned Application

• Current Assignee: Sandia National Laboratories (Government of the United States of America)
• Original Assignee: Sandia National Laboratories (Government of the United States of America)
• Inventors: West, Jason Andrew Appleton, Vandernoot, Victoria A., Lane, Todd William, Fruetel, Julia Ann, Shokair, Isaac Ramzy, Renzi, Ronald F., Wiedenman, Boyd J., Stamps, James Frederick
• Application Number:
  US10/795,549
• Publication Number:
  US 20050014134A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: G01N 2550/00 Electrophoretic profiling, e.g. for proteome analysis
• CPC Class Codes: G01N 27/44713 Particularly adapted electric power supply
• CPC Class Codes: G01N 27/44721 by optical means
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G01N 33/56983 Viruses
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins

Abstract

The present invention provides systems and processes for the collection and identification of macromolecules, such as biologically-derived macromolecules (e.g., proteins and nucleic acids), by measuring and comparing the molecular weight signatures of macromolecular samples. Reproducible molecular weight signatures provides reliable sample identification. In the case of viruses, proteomic molecular weight signatures can be used for identifying viral agents.

Claims

1. A process for identifying a virus comprising proteins, said process comprising:

solubilizing at least a portion of the proteins of the virus to provide solubilized proteins;

providing a microfluidic chip;

optionally preconcentrating said solubilized proteins on said microfluidic chip;

labeling at least a portion of said solubilized proteins to provide labeled proteins on said microfluidic chip;

electrokinetically injecting at least a portion of said labeled proteins into at least one microchannel electrophoretic separator on said microfluidic chip;

electrophoretically separating at least a portion of said labeled proteins in a constant-current mode to provide separated proteins on said microfluidic chip;

detecting at least a portion of said separated proteins on said microfluidic chip using a detector, wherein said detecting generates signals correlate to the concentration and separation time of said separated proteins; and

analyzing said signals to identify the virus.

2. The process of claim 1, wherein said detector is a laser-induced fluorescence detector.

3. The process of claim 1, wherein said proteins include viral coat proteins.

4. The process of claim 1, wherein the solubilized proteins are labeled with a fluorescent dye.

5. The process of claim 1, wherein said analyzing includes correlating said concentration and said separation time to a viral protein signature.

6. The process of claim 1, wherein said electrophoretically separating includes using at least two parallel electrophoretic separations.

7. The process of claim 6, wherein said at least two parallel electrophoretic analyses individually comprise capillary gel electrophoresis and capillary zone electrophoresis.

8. The process of claim 1, wherein said preconcentrating comprises:

providing a solution comprising solubilized proteins and ions in a first channel residing in said microfluidic chip; and

conducting ions from said first channel through a porous surface to a second channel residing in said microfluidic chip.

9. The process of claim 8, wherein said porous surface comprises a cover material bonded to a rough surface.

14. The process of claim 7, wherein said biological agent comprises a biotoxin, a bacterium, a virus, a nucleic acid, a portion of biotoxin, a portion of a bacterium, a portion of a virus, a nucleic acid, or any combination thereof.

10. A process, comprising:

solubilizing components of a sample, said sample comprising a chemical or a biological agent to provide solubilized components;

optionally preconcentrating said solubilized components;

labeling at least a portion of said solubilized components with a fluorescent dye to provide labeled components;

injecting said labeled components electrokinetically into at least one microchannel electrophoretic separator;

separating the labeled components electrophoretically using a controlled electric field, said controlled electric field operating in a constant-current mode;

detecting the separated components with a detector, said detector generating signals, the generated signals being correlated to the concentration and separation time of the labeled components;

generating an agent component signature comprising said concentration and said separation time; and

correlating said agent component signature to the identity of the chemical or biological agent.

11. The process of claim 10, wherein said detector is a laser-induced fluorescence detector.

12. The process of claim 10, wherein said preconcentrating comprises:

providing a solution comprising solubilized proteins and ions in a first channel residing in said microfluidic chip; and

conducting ions from said first channel through a porous surface to a second channel residing in said microfluidic chip.

13. The process of claim 12, wherein said porous surface comprises a cover material bonded to a rough surface.

15. A process, comprising:

solubilizing components of at least two samples comprising a chemical agent, a biological agent, or both, to provide solubilized components;

optionally preconcentrating said solubilized components;

individually labeling the solubilized components with a fluorescent dye;

individually injecting the solubilized components electrokinetically into at least one microchannel electrophoretic separator;

individually electrophoretically separating the labeled components using a controlled electric field operating in a constant-current mode to provide separated components;

individually detecting the separated components with a detector capable of generating signals correlatable to the concentration and separation time of the labeled components;

individually generating an agent component signature comprising said concentration and said separation time; and

identifying a chemical agent or biological agent isoform among the individual agent component signatures.

16. The process of claim 15, wherein said detector is a laser-induced fluorescence detector.

17. The process of claim 15, wherein said biological agent comprises a biotoxin, a bacterium, a virus, a nucleic acid, a portion of biotoxin, a portion of a bacterium, a portion of a virus or any combination thereof.

18. The process of claim 15, wherein said preconcentrating comprises:

providing a solution comprising solubilized proteins and ions in a first channel residing in said microfluidic chip; and

conducting ions from said first channel through a porous surface to a second channel residing in said microfluidic chip.

19. The process of claim 18, wherein said porous surface comprises a cover material bonded to a rough surface.

20. A system, comprising:

a microfluidic chip, comprising;

an injection port for receiving samples comprising protein;

an optional preconcentrator;

an electrokinetic pump for transporting proteins to an electrophoretic microchannel separator, said electrophoretic microchannel separator capable of separating proteins using a controlled electric field, said controlled electric field operating in a constant-current mode;

a detector giving rise to signals correlatable to the concentration and separation time of the separated proteins; and

a data processor for correlating said signals to the protein signatures of known biological samples.

21. The system according to claim 20, wherein said preconcentrator comprises a porous surface in fluid communication between a first channel provided in said microfluidic chip and a second channel provided in said microfluidic chip.

22. The process of claim 21, wherein said porous surface comprises a cover material bonded to a rough surface.

23. The system according to claim 20, further comprising at least one power supply, said power supply capable of generating at least one full-scale stepped voltage in at least 20 milliseconds and capable of measuring at least one current in at least 20 milliseconds.

24. The system according to claim 23, wherein said power supply further comprises an embedded microprocessor capable of measuring an electric current at least once every 100 milliseconds and capable of updating at least one voltage at least once every 100 milliseconds.

25. The system according to claim 24, wherein said embedded microprocessor is capable of measuring an electric current at least once every 50 milliseconds and is capable of updating at least one voltage at least once every 50 milliseconds.

26. The system according to claim 24, wherein said embedded microprocessor is capable of measuring at least ten electric currents at least once every 100 milliseconds and is capable of individually updating at least ten voltages at least once every 100 milliseconds.

27. The system according to claim 24, wherein the embedded microprocessor comprises a current control feedback algorithm and a timer interrupt, said feedback algorithm operating on the updated voltages and the current measurements by operation of a digital-to-analog converter coupled to said timer interrupt.

28. The system according to claim 20, wherein said proteins comprise viral proteins.

29. A process, comprising:

providing a sample comprising macromolecules derived from a biological entity;

solubilizing at least a portion of said macromolecules to provide solubilized macromolecules;

optionally preconcentrating said solubilized macromolecules;

labeling at least a portion of said solubilized macromolecules with a fluorescent dye to provide labeled macromolecules;

electrokinetically injecting at least a portion of said labeled macromolecules into a microchannel electrophoretic separator;

electrophoretically separating said labeled macromolecules using a controlled electric field operating in a constant-current mode to provide separated macromolecules;

detecting said separated macromolecules using a detector capable of generating signals, said signals capable of being correlated to the concentration and separation time of said separated macromolecules;

generating a macromolecular signature, said signature comprising said concentration and macromolecular separation time; and

analyzing said macromolecular signature to identify the biological entity.

30. The process of claim 29, wherein said detector is a laser-induced fluorescence detector.

31. The process of claim 29, wherein said macromolecules include amino acids, nucleic acids, or both.

32. The process of claim 29, wherein said preconcentrating comprises:

providing a solution comprising solubilized proteins and ions in a first channel residing in said microfluidic chip; and

conducting ions from said first channel through a porous surface to a second channel residing in said microfluidic chip.

33. The process of claim 32, wherein said porous surface comprises a cover material bonded to a rough surface.

34. A system, comprising:

an injection port for receiving biological samples comprising biological macromolecules;

a microfluidic chip in fluid communication with said injection port, said microfluidic chip comprising;

an optional preconcentrator in fluid communication with said injection port;

an electrokinetic pump in fluid communication with said injection port capable of transporting said biological macromolecules to an electrophoretic microchannel separator comprising a controlled electric field, said controlled electric field operating in a constant-current mode;

said electrophoretic microchannel separator capable of separating said biological macromolecules;

a detector capable of detecting the presence of said separated biological macromolecules, said detector giving rise to signals being correlatable to the concentration and separation time of said separated biological macromolecules; and

a data processor for correlating said signals to a biological macromolecular signature of a biological entity.

35. The system of claim 34, wherein said preconcentrator comprises a porous surface in fluid communication between a first channel provided in said microfluidic chip and a second channel provided in said microfluidic chip.

36. The process of claim 35, wherein said porous surface comprises a cover material bonded to a rough surface.

37. A system, comprising:

a microfluidic sample injection port capable of receiving a liquid comprising proteomic substances;

a microfluidic chip, comprising;

a preconcentrator in fluidic communication with said injection port, said preconcentrator comprising;

a porous surface in fluid communication between a first channel provided in said microfluidic chip and a second channel provided in said microfluidic chip, wherein the first and second channels comprise deep etched portions in said microfluidic chip and a shallow etched portions in said deep etch portions, said porous surface comprising a cover material bonded to a rough surface, said rough surface being contiguous to said shallow etched portions;

a microchannel capillary zone electrophoresis separator or a microchannel capillary gel electrophoresis separator in fluid communication with said preconcentrator; and

a detector capable of detecting the presence of proteomic substances, said detector capable of generating signals correlatable to the concentration and separation time of said proteomic substances.

38. The system of claim 37, further comprising a data processor for receiving said signals and generating a proteomic signature of a biological entity.

39. The system of claim 38, wherein the data processor is contained within the housing of the system.

40. The system of claim 39, further comprising an information display coupled to said data processor.

41. A system, comprising:

at least one separation module, comprising;

a microfluidic sample injection port capable of receiving a liquid under pressure, said liquid comprising proteomic substances;

a fluidic system capable of electrokinetically transporting said liquid and capable of separating said proteomic substances by molecular size; and

a microfluidic fluorescence detector capable of detecting the concentration and separation time of said proteomic substances; and

a power supply capable of monitoring and controlling electric currents and voltages of said fluidic system, said power supply capable of generating at least one full-scale stepped voltage in at least 20 milliseconds and capable of measuring at least one current in at least 20 milliseconds.

42. The system according to claim 41, wherein said power supply further comprises a microprocessor capable of measuring an electric current at least once every 100 milliseconds and capable of updating at least one voltage at least once every 100 milliseconds.

43. The system according to claim 41, wherein said embedded microprocessor is capable of measuring an electric current at least once every 50 milliseconds and is capable of updating at least one voltage at least once every 50 milliseconds.

44. The system according to claim 41, wherein said embedded microprocessor is capable of measuring at least ten electric currents at least once every 100 milliseconds and is capable of individually updating at least ten voltages at least once every 100 milliseconds.

45. The system according to claim 42, wherein said microprocessor comprises a current control feedback algorithm and a timer interrupt, said feedback algorithm operating on the updated voltages and the current measurements by operation of a digital-to-analog converter coupled to said timer interrupt.

46. The system of claim 41, further comprising a power source.

47. The system of claim 46, further comprising a housing enclosing said separation module and said power supply, said power source being located within said housing or external to said housing.

48. The system of claim 47, wherein said power source includes a battery, a fuel cell, or both, said power source being located within said housing.

49. The system of claim 41, wherein said fluidic system is capable of separating the proteomic substances using capillary gel electrophoresis or capillary zone electrophoresis.

50. The system of claim 41, wherein said fluidic system comprises a microfluidic chip for electrokinetically transporting said liquid, said microfluidic chip comprising a separation channel for separating said proteomic substances and a preconcentrator.