Flow-based enhancement of specificity for label-free biochemical assays

Patent metadata

Field	Value
Publication number	US-9599612-B2
Application number	US-71273110-A
Country	US
Kind code	B2
Filing date	Feb 25, 2010
Priority date	Feb 26, 2009
Publication date	Mar 21, 2017
Grant date	Mar 21, 2017

Abstract

A label-free biochemical assay, in which label-free interrogation of a target-receptor layer is performed while the target-receptor layer is subjected to a relatively strong flow of an analyte-containing fluid. The volumetric flow rate for the assay is selected based on calibration data corresponding to the target substance, which advantageously results in fewer and/or smaller false-positive signals corresponding to non-target substances compared to those produced with the fluid being stationary. In various embodiments, the label-free interrogation method can be electro-mechanical and/or optical.

First claim

What is claimed is:

1. A method of analyzing an analyte, the method comprising:
(A) exposing a target-receptor layer that comprises a plurality of target receptors adapted to bind a target substance selected from bacteria and spores in a fluid that comprises the analyte including the target substance; and
(B) performing label-free interrogation of the target-receptor layer for a presence of the target substance bound thereto while subjecting the target-receptor layer to a laminar flow of the fluid flowing at a flow speed in a range of from 2.7 mm/s to 4.05 mm/s that is controlled based on calibration data for the selectivity of binding of the target substance relative to binding of at least one non-target substance that can interfere with detection of the target substance by binding to the target receptors thereby providing a higher specificity of detection of the target substance in the analyte compared to a corresponding specificity of detection with the fluid being stationary, and
wherein the target substance is a spore of Bacillus anthraces.

2. The method of claim 1 , wherein:
the target-receptor layer is disposed on a surface of a planar structure; and
step (B) comprises:
placing the planar structure in a middle portion of a flow cell with the target-receptor layer being at least partially immersed in the fluid; and
pumping the fluid through the flow cell to produce the laminar flow of the fluid near the surface.

3. The method of claim 2 , wherein:
the planar structure comprises a first side and a second side opposite to the first side; and
each of the first and second sides has a respective portion of the target-receptor layer.

4. The method of claim 2 , wherein step (B) further comprises:
inducing a mechanical oscillation of the planar structure using a piezoelectric transducer attached to the planar structure; and
determining a resonance frequency of the oscillation which is dependent on the presence of the target substance.

5. The method of claim 2 , wherein:
the planar structure is a piezoelectric transducer; and
step (B) further comprises:
electrically inducing a mechanical oscillation of the piezoelectric transducer; and
determining a resonance frequency of the oscillation which is dependent on the presence of the target substance.

6. The method of claim 1 , wherein a target receptor from the plurality comprises an antibody corresponding to the target substance.

7. The method of claim 1 , wherein step (B) comprises selecting the flow speed for the flow based on calibration data corresponding to the target substance.

8. The method of claim 1 , wherein:
the target-receptor layer is disposed on a surface of a planar structure and comprises an antibody corresponding to the target substance; and
step (B) comprises:
(B1) placing the planar structure in a middle portion of a flow cell with the target-receptor layer being at least partially immersed in the fluid;
(B2) selecting the flow speed;
(B3) pumping the fluid through the flow cell at the selected flow speed;
(B4) inducing a mechanical oscillation of the planar structure using a piezoelectric transducer attached to the planar structure;
(B5) determining a resonance frequency of the oscillation; and
(B6) processing data collected in step (B5) to determine whether the target substance is present in the target-receptor layer.

9. The method of claim 8 , further comprising the step of: (C) determining a range of flow speeds for use in step (B) using a calibration procedure.

10. The method of claim 9 , wherein step (C) comprises:
(C1) exposing the target-receptor layer to a test fluid comprising a specific non-target substance;
(C2) performing label-free interrogation of the target-receptor layer for a false-positive signal corresponding to the specific target substance bound thereto while subjecting the target-receptor layer to a flow of the test fluid; and
(C3) repeating step (C2) for each flow speed selected from a predetermined plurality of flow speeds.

11. A bio-analytical system, comprising:
means for exposing a target-receptor layer that comprises a plurality of target receptors adapted to bind a target substance in a fluid that comprises the analyte including the target substance selected from bacteria and spores; and
means for performing label-free interrogation of the target-receptor layer for a presence of the target substance bound thereto while subjecting the target-receptor layer to a laminar flow of the analyte-containing fluid flowing at a flow speed in a range of from 2.7 mm/s to 4.05 mm/s that is controlled based on calibration data for selectivity of binding of the target substance relative to binding of at least one non-target substance that can interfere with detection of the target substance by binding to the target receptors thereby providing a higher specificity of detection of the target substance in the analyte compared to a corresponding specificity of detection with the fluid being stationary, and
wherein the target substance is a spore of Bacillus anthraces.

12. A method of analyzing an analyte, the method comprising:
(A) exposing a target-receptor layer that comprises a plurality of target receptors adapted to bind a target substance in a fluid that comprises the analyte including the target substance; and
(B) performing label-free interrogation of the target-receptor layer for a presence of the target substance bound thereto while subjecting the target-receptor layer to a laminar flow of the fluid flowing at a flow speed in a range of from 2.7 mm/s to 4.05 mm/s that is controlled based on calibration data for the selectivity of binding of the target substance relative to binding of at least one non-target substance that can interfere with detection of the target substance by binding to the target receptors thereby providing a higher specificity of detection of the target substance in the analyte compared to a corresponding specificity of detection with the fluid being stationary, and
wherein the target substance is a spore of Bacillus anthraces and the at least one non-target substance is selected from one or more of a spore of B. thuringiensis , a spore of B. cereus , and a spore of B. subtilis.

13. A bio-analytical system, comprising:
means for exposing a target-receptor layer that comprises a plurality of target receptors adapted to bind a target substance in a fluid that comprises the analyte including the target substance; and
means for performing label-free interrogation of the target-receptor layer for a presence of the target substance bound thereto while subjecting the target-receptor layer to a laminar flow of the analyte-containing fluid flowing at a flow speed in a range of from 2.7 mm/s to 4.05 mm/s that is controlled based on calibration data for selectivity of binding of the target substance relative to binding of at least one non-target substance that can interfere with detection of the target substance by binding to the target receptors thereby providing a higher specificity of detection of the target substance in the analyte compared to a corresponding specificity of detection with the fluid being stationary, and
wherein the target substance is a spore of Bacillus anthraces and the at least one non-target substance is selected from one or more of a spore of B. thuringiensis , a spore of B. cereus , and a spore of B. subtilis.

Assignees

• Shih Wan Y
• Shih Wei-Heng
• Mcgovern John-Paul
• Univ Drexel

Inventors

• Shih Wan Y
• Shih Wei-Heng
• Mcgovern John-Paul

Classifications

• G01N2333/32

  from Bacillus (G) · CPC title

• G01N33/56911

  Bacteria · CPC title

• G01N33/54373 Primary

  involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings · CPC title