Real time microarrays

• US 9,133,504 B2
• Filed: 06/05/2007
• Issued: 09/15/2015
• Est. Priority Date: 06/05/2006
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Hassibi, Arjang, Hassibi, Babak, Vikalo, Haris, Riechmann, Jose Luis
• Primary Examiner(s): Gross, Christopher M
• Application Number:
  US11/758,621
• Publication Number:
  US 20080039339A1
• Time in Patent Office: 3,024 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

This invention provides methods and systems for measuring the binding of analytes in solution to probes bound to surfaces in real-time. The method involves contacting a fluid volume having a plurality of different analytes with a solid substrate having a plurality of different probes. The probes are capable of specifically binding to the analytes. The method also involves measuring signals at multiple time points while the fluid volume is in contact with the substrate. The signals measured at multiple time points can be correlated with the amount of binding of the analytes with the probes. The method eliminates the need to wash the probes before measuring the binding characteristics.

Claims

1. A method of measuring at least one parameter of a binding reaction of an analyte with a probe on a microarray, comprising the steps of:

contacting a fluid volume comprising an initial concentration C of at least one analyte that is labeled with a quencher with a solid substrate comprising a number Po of unbound molecules of at least one probe that has been labeled with a donor and affixed thereto at a probe spot, said at least one probe and said at least one analyte configured to bind together, wherein the at least one analyte is not labeled with a metal cluster;

measuring kinetically in real-time a plural number of values for the number P(t) of unbound molecules of said probe at time t, during a time interval when said reaction of said at least one probe and said at least one analyte is occurring, wherein said reaction is not competitive;

determining by using said measured plural number of values P(t) fitted to an exponential curve at least one parameter descriptive of a binding reaction of said at least one probe and said at least one analyte; and

performing a selected one of storing, manipulating and displaying to a user data comprising said at least one parameter,

wherein said contacting and measuring are performed in the absence of a washing step.

2. The method of claim 1, wherein said at least one parameter descriptive of a binding reaction of said at least one probe and said at least one analyte comprises a selected one of the following parameters:

a forward binding reaction rate;

a backward binding reaction rate;

C which is an original analyte quantity in said fluid volume;

C−

(P₀−

P(t)) which is an available analyte density at time t;

t which is a time constant;

P_∞

which is a steady-state value of P(t);

n(t) which is a number of analytes in said fluid volume that are specific to said probe molecule;

P_near which is a probability that said analyte is in close proximity to said probe molecule;

P_h which is a probability that, in a unit interval of time, said analyte binds to said probe once said analyte is near;

P_r which is a probability that, in a unit interval of time, said analyte bound to said probe molecule is released;

N which is a total number of analyte molecules;

q(t) which is a number of unbound molecules of said at least one analyte as a function of time;

a reaction rate between said analyte and said probe; and

a cross-hybridization parameter.

3. The method of claim 1, wherein said measuring step occurs before said reaction reaches an equilibrium.

4. The method of claim 1, wherein said measuring step uses an optical signal.

5. The method of claim 4, wherein said optical signal is a selected one of a fluorescence signal, a luminescence signal, and an absorption signal.

6. The method of claim 4, wherein said optical signal involves fluorescence resonance energy transfer.

7. The method of claim 1, further comprising the step of determining how many analyte molecules are bound to said probe spot.

8. The method of claim 1, wherein said fluid volume further contains a plurality of different analytes and said solid substrate further comprises a plurality of different probes.

9. The method of claim 1, wherein said measuring a plural number of values for the number P(t) occurs in a time interval of one millisecond or less.

10. The method of claim 1, wherein said donor is a fluorophore.