Method of analysis for determining a specific protein in blood samples using fluorescence spectrometry

A method of analysis for determining the presence, absence or amount of a specific protein in a blood sample, comprising the steps of:

• (a) providing a processed sample, wherein the processed sample has been prepared by
• (i) extracting serum or plasma from the blood sample;
• (ii) contacting the serum or plasma with a selective capture agent specific for the protein, wherein the capture agent is immobilized or becomes immobilized on a surface;
• (iii) adding a fluorescently-labeled specific binding partner for the protein, wherein the binding partner and the protein associate to form a protein-binding partner complex;
• (iv) removing labeled binding partner that has not associated with the protein;
• (v) releasing the protein-binding partner complex into solution or dissociating the labeled binding partner from the protein-binding partner complex so that the dissociated labeled binding partner moves into solution;
• (b) sampling a portion of the processed sample, wherein the sampling is performed by a sampling system that comprises a pressure source to move the portion of the processed sample into a detection channel of a single molecule analyzer,
• wherein the detection channel is substantially transparent to at least some wavelengths of light and provides a channel through which the portion of the processed sample may flow, and the analyzer comprises a laser for providing excitation light, wherein the light is within the wavelengths to which the detection channel is substantially transparent;
• and
• (c) passing the portion of the processed sample through the detection channel of the analyzer using positive or negative pressure;
• (d) focusing the excitation light on a portion of the detection channel, so that the light excites the labeled specific binding partner if present in the portion of the detection channel so that the labeled specific binding partner produces emitted light;
• (e) passing the emitted light through an aperture so that the focusing of excitation light and passing of emitted light through the aperture define a molecule detection volume within the portion of the detection channel of between about 0.1 pL and about 25 pL;
• (f) detecting, in discreet time bins, the emitted light that has passed through the aperture with a detector, wherein the detector transforms the light into an electronic signal;
• and
• (g) determining the presence, absence or amount of the specific protein in a blood sample by determining a threshold photon bin value based upon a distribution of background noise signal and analyzing the electronic signal from a plurality of the bins with a data analysis system operably connected to the detector that compares the signal for each bin to the threshold value to determine whether or not the labeled specific binding partner is present in the molecule detection volume.