Luminescent oxygen channeling immunoassays

A microfluidics device, comprising:

• (a) an inlet channel through which a sample may be applied;
• (b) at least one compartment capable of being in fluidic communication with the inlet channel, the at least one compartment containing:
• (i) a composition comprising a singlet oxygen-activatable chemiluminescent compound that directly or indirectly binds a target analyte;
• (ii) a sensitizer that directly or indirectly binds the target analyte and that generates singlet oxygen in its excited state, and wherein a sandwich complex is formed by binding of the sensitizer and the singlet oxygen-activatable chemiluminescent compound to target analyte;
• and
• (iii) a composition comprising a singlet oxygen quencher that interacts with unbound sensitizer but does not interact with sensitizer bound in the sandwich complex formed of chemiluminescent compound, sensitizer, and target analyte, and wherein:
• the composition comprising the singlet oxygen quencher is further defined as a high molecular weight composition comprising a singlet oxygen quencher, wherein the singlet oxygen quencher or a conjugate containing the singlet oxygen quencher has a molecular weight of at least 100 Da, and wherein steric hindrance substantially prevents the singlet oxygen quencher of the high molecular weight composition from quenching specific signal resulting from the sandwich complex.

A method for detecting the presence and/or concentration of a target analyte in a sample, comprising the steps of:

• (a) combining, either simultaneously or sequentially;
• (1) a sample suspected of containing the target analyte;
• (2) a composition comprising a singlet oxygen-activatable chumiluminescent compound that directly or indirectly binds the target analyte;
• and
• (3) a sensitizer that directly or indirectly binds the target analyte and that generates singlet oxygen in its excited state;
• (b) allowing the binding of (2) and (3) to target analyte present in the sample, whereby a sandwich complex is formed and the sensitizer is brought into close proximity to the chemiluminescent compound;
• (c) adding a composition comprising a singlet oxygen quencher that interacts with unbound sensitizer that is not part of the sandwich complex but does not interact with sensitizer bound in the sandwich complex, and wherein:
• the composition comprising the singlet oxygen quencher is further defined as a high molecular weight composition comprising a singlet oxygen quencher, wherein the singlet oxygen quencher or a conjugate containing the singlet oxygen quencher has a molecular weight of at least 100 Da, and wherein steric hindrance substantially prevents the singlet oxygen quencher of the high molecular weight composition from quenching specific signal resulting from the sandwich complex;
• (d) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the sandwich complex causes the activation of the chemiluminescent compound present in the sandwich complex;
• (e) determining the amount of chemiluminescence generated by the activated chemiluminescent compound;
• (f) optionally repeating steps (b)-(e);
• and
• (g) detecting the presence and/or concentration of the target analyte by analyzing the amount of chemiluminescence so produced, wherein the amount of chemiluminescence is directly proportional to the amount of target analyte in the sample.