Method for identifying biochemical and chemical reactions and micromechanical processes using nanomechanical and electronic signal identification

• US 5,620,854 A
• Filed: 03/13/1995
• Issued: 04/15/1997
• Est. Priority Date: 08/25/1993
• Status: Expired due to Fees

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Holzrichter, John F., Siekhaus, Wigbert J.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/402,800
• Time in Patent Office: 764 Days
• Field of Search: 435/6, 435/5, 435/91.2, 435/7.1-7.9, 436/182, 436/183, 250/561
• US Class Current: 435/6.19
• CPC Class Codes: B82Y 35/00 Methods or apparatus for measurement or analysis of nanostructures
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]
• CPC Class Codes: G01Q 60/04 STM [Scanning Tunnelling Microscopy] combined with AFM [Atomic Force Microscopy]
• CPC Class Codes: G01Q 60/42 Functionalisation
• CPC Class Codes: Y10S 977/853 Biological sample

Abstract

A scanning probe microscope, such as an atomic force microscope (AFM) or a scanning tunneling microscope (STM), is operated in a stationary mode on a site where an activity of interest occurs to measure and identify characteristic time-varying micromotions caused by biological, chemical, mechanical, electrical, optical, or physical processes. The tip and cantilever assembly of an AFM is used as a micromechanical detector of characteristic micromotions transmitted either directly by a site of interest or indirectly through the surrounding medium. Alternatively, the exponential dependence of the tunneling current on the size of the gap in the STM is used to detect micromechanical movement. The stationary mode of operation can be used to observe dynamic biological processes in real time and in a natural environment, such as polymerase processing of DNA for determining the sequence of a DNA molecule.

Claims

1. A method for detecting time-varying motions created on a microscopic scale using a scanning probe microscope having a physical tip for mechanical signal identification, comprising:

(a) locating a single site comprising a molecule or molecules of interest having microscopic dimensions by using the scanning probe microscope in a locator mode, wherein the molecule or molecules have time-varying motions due to a biological, chemical, mechanical, optical, or electrical process;

(b) positioning the physical tip of the scanning probe microscope in a stationary mode at the single site; and

(c) monitoring physical movements of the tip as a function of time as the process occurs by using the scanning probe microscope in a stationary, non-scanning mode, thereby detecting micromotions created at the site.

2. The method as recited in claim 1, further comprising positioning the tip in physical contact with at least one molecule at the site, and wherein the physical movements of the tip are caused by micromotions of the molecule.

3. The method as recited in claim 1, further comprising surrounding the site with a transmitting medium, and positioning the tip in physical contact with the medium, wherein the micromotions created at the site are transmitted through the medium as acoustic or pressure waves, and the physical movements of the tip are caused by the waves.

4. The method as recited in claim 1, further comprising attaching at least one molecule at the site to a supporting substrate.

5. The method as recited in claim 1, wherein the scanning probe microscope is selected from the group consisting of atomic force microscopes and scanning tunneling microscopes.

6. The method as recited in claim 1, further comprising attaching an intermediary body to at least one molecule at a site of interest, whereby the micromotions of the site of interest cause micromotions at the intermediary body, and wherein the physical movements of the tip are caused by micromotions created at the intermediary body.

7. The method as recited in claim 6, further comprising positioning the tip in physical contact with the intermediary body, and wherein the physical movements of the tip are caused by micromotions of the intermediary body.

8. The method as recited in claim 6, further comprising surrounding the intermediary body with a transmitting medium, and positioning the tip in physical contact with the medium, wherein the micromotions created at the intermediary body are transmitted through the medium as acoustic or pressure waves, and the physical movements of the tip are caused by the waves.

9. The method as recited in claim 6, wherein the intermediary body is selected from the group consisting of a membrane and a chemically inert metallic sphere.

10. The method as recited in claim 1, wherein the tip comprises a tip and cantilever assembly.

11. The method as recited in claim 1, wherein the site is an enzyme having polymerase activity.

12. The method as recited in claim 1, wherein the site is a living cell.

13. A method for detecting time-varying motions created on a microscopic scale by an enzyme using a scanning probe microscope having a physical tip for mechanical signal identification, comprising:

(a) attaching an enzyme to a supporting matrix, wherein the enzyme is in solution and has time-varying micromotions due to interaction with a substrate;

(b) locating the enzyme and positioning the physical tip of the scanning probe microscope in a stationary mode at the enzyme;

(c) introducing the substrate into the solution, wherein the solution is allowed to come to rest and the substrate and the enzyme interact; and

(d) monitoring the physical movements of the tip as a function of time as the interaction occurs, by using the scanning probe microscope in a stationary, non-scanning mode, thereby detecting micromotions caused by the enzyme'"'"'s interaction with the substrate.

14. The method as recited in claim 13, further comprising positioning the tip in physical contact with the enzyme, and wherein the physical movements of the tip are caused by micromotions of the enzyme.

15. The method as recited in claim 13, further comprising surrounding the enzyme with a transmitting medium, and positioning the tip in physical contact with the medium, wherein the micromotions created at the enzyme are transmitted through the medium as acoustic or pressure waves, and the physical movements of the tip are caused by the waves.

16. The method as recited in claim 13, further comprising attaching an intermediary body to the enzyme, whereby the micromotions of the enzyme cause micromotions at the intermediary body, and wherein the physical movements of the tip are caused by micromotions created at the intermediary body.

17. The method as recited in claim 16, further comprising positioning the tip in physical contact with the intermediary body, and the physical movements of the tip are caused by micromotions of the intermediary body.

18. The method as recited in claim 16, further comprising surrounding the intermediary body with a transmitting medium, and positioning the tip in physical contact with the medium, wherein the micromotions created at the intermediary body are transmitted through the medium as acoustic or pressure waves, and the physical movements of the tip are caused by the waves.

19. A method for detecting time-varying motions created on a microscopic scale using a scanning probe microscope having a physical tip for mechanical signal identification, comprising:

(a) locating a single site comprising a molecule or molecules of interest having microscopic dimensions by using the scanning probe microscope in a locator mode, wherein the molecule or molecules have time-varying motions due to a biological, chemical, mechanical, optical, or electrical process;

(b) attaching an intermediary body to the site of interest, whereby micromotions of the site of interest cause micromotions at the intermediary body; and

(c) monitoring the physical movements of the intermediary as a function of time as the process occurs using laser interferometry, thereby detecting micromotions created at the site.