Free solution measurement of molecular interactions by backscattering interferometry

• US 8,445,217 B2
• Filed: 09/20/2008
• Issued: 05/21/2013
• Est. Priority Date: 09/20/2007
• Status: Active Grant

• Current Assignee: University of Pittsburgh of The Commonwealth System of Higher Education, Vanderbilt University
• Original Assignee: Vanderbilt University
• Inventors: Bornhop, Darryl J.
• Primary Examiner(s): Yang, N. C.
• Assistant Examiner(s): MOERSCHELL, RICHARD P
• Application Number:
  US12/674,610
• Publication Number:
  US 20120015376A1
• Time in Patent Office: 1,704 Days
• Field of Search: None
• US Class Current: 435/7.2
• CPC Class Codes: G01N 2021/0346 Capillary cells; Microcells
• CPC Class Codes: G01N 2021/058 Flat flow cell
• CPC Class Codes: G01N 2021/4709 Backscatter
• CPC Class Codes: G01N 21/05 Flow-through cuvettes G01N21/09 takes precedence; handling fluid samples G01N1/10
• CPC Class Codes: G01N 21/45 using interferometric methods; using Schlieren methods
• CPC Class Codes: G01N 2201/06113 Coherent sources; lasers
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/536 with immune complex formed in liquid phase

Abstract

Disclosed are methods, systems, and apparatuses for the free solution measurement of molecular interactions by backscattering interferometry (BSI). Molecular interaction can be detected between analytes in free-solution wherein at least one of the analytes is label-free and detection is performed by back-scattering interferometry. Further, molecular interaction can be detected between analytes in free-solution, wherein at least one of the analytes is label-free, wherein one of the analytes is present in a concentration of less than about 5.0×10⁻⁷M. Also disclosed are label-free, free-solution, and/or real-time measurements of characteristic properties and/or chemical events using the disclosed techniques. The disclosed methods can have very low detection limits and/or very low sample volume requirements. Also disclosed are various biosensor applications of the disclosed techniques. This abstract is intended as a scanning tool for purposes of searching in the particular art and is not intended to be limiting of the present invention.

Claims

1. A method for free-solution determination of molecular interactions comprising the steps of:

(a) providing a substrate having a channel formed therein for reception of a fluid sample to be analyzed;

(b) introducing a first sample comprising a first non-immobilized analyte to be analyzed into the channel;

(c) introducing a second sample comprising a second non-immobilized analyte to be analyzed into the channel;

(d) allowing the first analyte to interact with the analyte to form one or more interaction products;

(e) directing a coherent light beam onto the substrate such that the light beam is incident on the channel to generate backscattered light through reflective and refractive interaction of the light beam with a substrate/channel interface and the sample, the backscattered light comprising interference fringe patterns including a plurality of spaced light bands whose positions shift in response to changes in the refractive index of the fluid sample;

(f) detecting positional shifts in the light bands; and

(g) determining an equilibrium constant of the interaction from the detected shifts.

2. The method of claim 1, further comprising the step of determining the formation of the one or more interaction products of the first analyte with the second analyte from the positional shifts of the light bands in the interference patterns.

3. The method of claim 1, wherein detecting positional shifts in the light bands comprises determining a change in a physical or chemical property of the fluid sample corresponding to the formation of the one more interaction products of the first analyte with the second analyte.

4. The method of claim 1, wherein the first analyte and/or the second analyte is/are unlabeled.

5. The method of claim 1, wherein the substrate further comprises a reference channel.

6. The method of claim 1, wherein the interaction is the formation of one or more covalent bonds, electrostatic bonds, hydrogen bonds, or hydrophobic interactions.

7. The method of claim 1, wherein the interaction is a binding event between small molecule-protein.

9. The method of claim 1, further comprising the step of performing a chromatographic or electrophoretic separation on the sample prior to the determining the characteristic property step.

10. The method of claim 7, wherein interaction is a binding event involving a protein associated with plasma or organelle membranes.

11. The method of claim 7, wherein the interaction is a binding event involving calmodulin.

8. The method of clam 1, wherein the substrate and channel together comprise a capillary tube.

12. A method for free-solution determination of molecular interactions comprising the steps of:

(a) providing a substrate having a channel formed therein for reception of a fluid sample to be analyzed;

(b) introducing a first sample comprising a first non-immobilized analyte to be analyzed into the channel;

(c) introducing a second sample comprising a second non-immobilized analyte to be analyzed into the channel;

(d) allowing the first analyte to interact with the second analyte to form one or more interaction products;

(e) directing a coherent light beam onto the substrate such that the light beam is incident on the channel to generate backscattered light through reflective and refractive interaction of the light beam with a substrate/channel interface and the sample, the backscattered light comprising interference fringe patterns including a plurality of spaced light bands whose positions shift in response to changes in the refractive index of the fluid sample;

(f) detecting positional shifts in the light bands; and

(g) determining an end-point value of the interaction from the detected shifts.

13. The method of claim 12, further comprising the step of determining the formation of the one or more interaction products of the first analyte the second analyte from the positional shifts of the light bands in the rote interference patterns.

14. The method of claim 12, wherein detecting positional shifts in the light bands comprises determining a change in a physical or chemical property of the fluid sample corresponding to the formation of the one or more interaction products of the first analyte with the second analyte.

15. The method of claim 12, wherein the first analyte and/or the second analyte is/are unlabeled.

16. The method of claim 12, wherein the substrate further comprises a reference channel.

17. The method of claim 12, wherein the interaction is the formation of one or more covalent bonds, electrostatic bonds, hydrogen bonds, or hydrophobic interactions.

18. The method of claim 12, wherein the interaction is a binding event between small molecule-protein.

19. The method of claim 12, wherein the substrate and channel together comprise a capillary tube.

20. The method of claim 12, further comprising the step of performing a chromatographic or electrophoretic separation on the sample prior to the determining the characteristic property step.

21. The method of claim 18, wherein the interaction is a binding event involving a protein associated with plasma or organelle membranes.

22. The method of claim 18, wherein the interaction is a binding event involving calmodulin.