Label-free detection of small and large molecule interactions, and activities in biological systems

• US 9,909,993 B2
• Filed: 12/14/2015
• Issued: 03/06/2018
• Est. Priority Date: 12/15/2014
• Status: Active Grant

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Tao, Nongjian, Guan, Yan
• Primary Examiner(s): Lefkowitz, Sumati
• Assistant Examiner(s): Wang, Carol
• Application Number:
  US14/968,331
• Publication Number:
  US 20160169873A1
• Time in Patent Office: 813 Days
• CPC Class Codes: G01N 21/77 by observing the effect on a chemical indicator
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G02B 21/365 Control or image processing arrangements for digital or video microscopes G02B21/361, G02B21/362 take precedence

Abstract

A system for quantitative detection and analysis of the interactions of molecules with molecular receptors on the surfaces of biological cells based on detecting a mechanical deformation in the membrane of a cell associated with the molecular interactions, which works for both large and small molecules. The mechanical deformation can be detected with high precision in real time from an optical image of the cell with a differential detection method. The system can be also used to detect the electrical activities, such as ion channel opening and closing, as well as action potential propagation in neuronal cells.

Claims

1. A system for quantitative detection and analysis of the binding of molecules with molecular receptors on the surfaces of a biological object, the system comprising:

a microscope positioned to image the biological object and transmit a time sequence of images of the biological object;

a camera located to receive the transmitted time sequence of images of the biological object from the microscope and transmit data representing the time sequence of images;

a plurality of molecules introduced to bind with the molecular receptors;

a processor, coupled to receive the data representing the time sequence of images from the camera;

the processor including an image processing algorithm that quantitatively determines mechanical deformation of the biological object from the time sequence of images, wherein the mechanical deformation is due to an interaction of the biological object with the plurality of molecules, wherein the image processing algorithm determines mechanical deformation by using an edge detector to determine an edge of the biological object and a time sequence tracker for tracking a plurality of movements of the edge by measuring positions of the edge at different times, and

wherein the image processing algorithm also determines molecular binding kinetic values of the biological object as proportional to the mechanical deformation of the biological object.

2. The system of claim 1 wherein the time sequence tracker for tracking the plurality of movements of the edge of the biological object comprises a differential image intensity analysis algorithm.

3. The system of claim 1 wherein the biological object comprises a biological cell, tissue or virus.

4. The system of claim 1 wherein the plurality of molecules are selected from the group consisting of proteins, nucleic acids, amino acids, peptides, hormones, drugs, metabolites, minerals, and ions.

5. The system of claim 1 wherein the microscope comprises an optical microscope.

6. The system of claim 5 wherein the optical microscope comprises a phase contrast microscope.

7. The system of claim 1 wherein the biological object comprises a neuron and the plurality of molecules comprise neuron stimulator molecules that stimulate an electrical activity of the neuron.

8. The system of claim 7 wherein the neuron stimulator molecules comprise agonists or antagonists.

9. The system of claim 1 wherein the microscope comprises a z-modulation temporal Fast Fourier Transform (FFT) microscope.

10. The system of claim 1 wherein the biological object is a cell and the image processor operates to measure the plurality of movements of the edge of the biological object by:

detecting a cell edge from an optical image of the cell;

selecting a region of interest (ROI) at a location of the cell edge;

dividing the ROI into two equal halves, wherein one half is inside of the cell, and wherein the second half falls outside of the cell; and

determining the edge movement by computing (A−

B)/(A+B), where A and B are the intensities of the two halves of the image.

11. A method for quantitative detection and analysis of the binding of molecules with molecular receptors on the surfaces of a biological object, the method comprising:

operating a microscope and a sensor to capture a time sequence of images of a biological object;

introducing a plurality of molecules to interact with the biological object;

operating a processor to quantitatively determine the mechanical deformation of the biological object from the time sequence of images due to interaction of the biological object with the plurality of molecules, wherein determining the mechanical deformation comprises operating an edge detector for determining an edge of the biological object and tracking a plurality of movements of the edge by measuring positions of the edge at different times; and

operating the processor to determine molecular binding kinetic values of the biological object as proportional to the mechanical deformation of the biological object and to determine binding kinetic constants, including ka, kd and KD from the time sequence of images.

12. The system of claim 11 wherein tracking the plurality of movements of the edge of the biological object further comprises running a differential image intensity analysis algorithm on the processor.

13. The method of claim 11 wherein the biological object comprises a biological cell, tissue or virus.

14. The method of claim 11 wherein the plurality of molecules are selected from the group consisting of proteins, nucleic acids, amino acids, peptides, hormones, drugs, metabolites, minerals, and ions.

15. The method of claim 11 wherein the microscope comprises an optical microscope and the sensor comprises a MOS imager or CCD imager.

16. The method of claim 15 wherein the optical microscope comprises a phase contrast microscope.

17. The method of claim 11 wherein the biological object comprises a neuron and the plurality of molecules comprise neuron stimulator or inhibitor molecules that stimulate or inhibit electrical activity of the neuron.

18. The method of claim 17 wherein the neuron stimulator molecules comprise agonists or antagonists.

19. The method of claim 11 wherein the microscope comprises a z-modulation temporal Fast Fourier Transform (FFT) microscope to produce images.

20. A system for quantitative detection and analysis of binding of neuron stimulator molecules with molecular receptors on the surfaces of a neuron, the system comprising:

a microscope positioned to image the neuron and transmit a time sequence of images of the biological object;

an image sensor located to receive transmitted images of the neuron from the microscope and transmit data representing the time sequence of images;

neuron stimulator molecules introduced to bind with the molecular receptors;

a processor coupled to receive the data representing the time sequence of images from the image sensor, the processor including an image processing algorithm that quantitatively determines mechanical deformation of the neuron from the time sequence of images, wherein the mechanical deformation is due to interaction of the neuron with the plurality of neuron stimulator molecules, wherein the image processing algorithm determines mechanical deformation by using an edge detector to determine an edge of the biological object and a time sequence tracker for tracking a plurality of movements of the edge by measuring positions of the edge at different times, and

wherein the image processing algorithm also determines molecular binding kinetic values of the neuron as proportional to the mechanical deformation of the neuron.

21. The system of claim 20 wherein the neuron stimulator molecules comprise nicotinic acetylcholine receptors.

22. A method for quantitative detection and analysis of the binding of neuron stimulator molecules with molecular receptors on the surfaces of a neuron, the method comprising:

operating a microscope and a sensor to capture a time sequence of images of a biological object;

introducing a plurality of neuron stimulator molecules to interact with the neuron;

operating a processor to quantitatively determine mechanical deformation of the neuron from the time sequence of images due to interaction of the neuron with the plurality of neuron stimulator molecules, wherein determining the mechanical deformation comprises operating an edge detector for determining an edge of the biological object and tracking a plurality of movements of the edge by measuring positions of the edge at different times; and

operating the processor to determine molecular binding kinetic values of the neuron as proportional to the mechanical deformation of the neuron.

23. The method of claim 22 wherein the neuron stimulator molecules comprise nicotinic acetylcholine receptors.