System and Method for Single Protein Detection by Digital Plasmonic Nanobubbles

A method for digital detection of a multivalent target analyte in a sample without enzymatic amplification or physical compartmentalization, comprising:

• (a) mixing the sample with a plurality of pairs of plasmonic nanoparticles, wherein each member of a pair is conjugated to a different binding agent that recognizes a different epitope on the multivalent target analyte, and wherein binding of the multivalent target analyte to both binding agents of a pair induces aggregation of the plasmonic nanoparticles;
• (b) exposing the mixture to pulsed laser light sufficient to generate plasmonic nanobubbles preferentially around aggregated plasmonic nanoparticles compared to non-aggregated plasmonic nanoparticles;
• (c) detecting transient scattering signals from the plasmonic nanobubbles using a continuous-wave probe laser and a photodetector;
• and (d) digitally quantifying the target analyte by counting scattering events that satisfy a threshold criterion indicative of aggregation, wherein the method distinguishes aggregated from non-aggregated plasmonic nanoparticles based on at least one of:
• scattering signal amplitude, scattering signal duration, nanobubble generation threshold fluence, or nanobubble lifetime.

A system for digital detection of a target analyte without enzymatic amplification or physical compartmentalization, comprising:

• (a) solid supports configured to capture the target analyte;
• (b) plasmonic nanoparticles configured to label the captured target analyte;
• (c) a pulsed laser configured to generate plasmonic nanobubbles around the plasmonic nanoparticles;
• (d) a detector configured to detect scattering signals from the plasmonic nanobubbles;
• and (e) a processor configured to digitally quantify the target analyte based on the scattering signals.

A system for digital detection of a target analyte without enzymatic amplification or physical compartmentalization, comprising:

• (a) antibody-conjugated plasmonic nanoparticles configured to aggregate upon binding to the target analyte;
• (b) a pulsed laser configured to generate plasmonic nanobubbles around the aggregated plasmonic nanoparticles;
• (c) a detector configured to detect scattering signals from the plasmonic nanobubbles;
• and (d) a processor configured to digitally quantify the target analyte based on the scattering signals distinguishing aggregated from non-aggregated plasmonic nanoparticles.

A method for digital detection of a viral protein in a lysed virus sample without enzymatic amplification or physical compartmentalization, comprising:

• (a) lysing a virus sample to release a viral protein;
• (b) capturing the viral protein on a solid support using a binding agent forming a captured viral protein;
• (c) labeling the captured viral protein with a plasmonic nanoparticle forming a labeled solid support;
• (d) exposing the labeled solid support to a pulsed laser light generating plasmonic nanobubbles around the plasmonic nanoparticles;
• (e) detecting scattering signals from the plasmonic nanobubbles;
• and (f) digitally quantifying the viral protein based on the detected scattering signals, wherein the method achieves detection at a concentration of about 6 PFU/mL or lower.