Scheme 1. Target antigen-binding antibody conjugated with single stranded DNA (20 – 100 bp) and a luminescent dye. 1: Pacific Blue, 2: 6-Carboxyfluorescein (6-FAM), 3. Fluorescein.
Scheme 2. Phenylglyoxal derivative chemiluminescence in All-in-One immunoassays. 1. Cytosine, 2: Adenine, 3: Guanine, 4: Thymine, 5. Pacific Blue under the ground state, 6: High-energy intermediate formed from the reaction of guanine and TMPG in the presence of TPA and DMF, 7: Pacific Blue under the excited state.
A chemical reaction between a phenylglyoxal derivative (e.g., 3-methoxylphenylglyoxal (3-MPG) and 3, 4, 5-trimethoxylphenylglyoxal (TMPG)) and guanine in single-stranded DNA (or RNA) produces a high-energy intermediate. This intermediate emits weak chemiluminescence. Also, the high-energy intermediate transfers energy to a luminescent dye based on the principle of chemiluminescence resonance energy transfer (CRET). The luminescent dye excited due to the CRET emits bright chemiluminescence. The brightness and color of chemiluminescence emitted in this reaction depend on the properties of luminescent dye excited due to the CRET. Based on the principle, the all-in-one immunoassay with phenylglyoxal derivative chemiluminescence detection uses antibodies conjugated with single-stranded DNA (or RNA) linked to a luminescent dye, as shown in Scheme 1. The single-stranded DNA (or RNA) must have multiple guanines (5 – 15) with other components (e.g., adenine, cytosine, and thymine for DNA, adenine, cytosine, uracil for RNA) to produce a strong high-energy intermediate from the reaction of guanine and a phenylglyoxal derivative. Also, guanine in single-stranded DNA (or RNA) must be adjacent to a luminescent dye to transfer the intense energy from the high-energy intermediate formed by the reaction of guanine with a phenylglyoxal derivative to the luminescent dye. For example, trace levels of D-dimers in human serum are quantified from an all-in-one immunoassay using TMPG chemiluminescence detection based on the reaction mechanism shown in Scheme 2. In addition, the all-in-one immunoassay with a phenylglyoxal derivative chemiluminescence detection can be used as a multiplex immunoassay tool. For example, in an all-in-one immunoassay with a luminometer with three different filtered photon multiplier tubes capable of measuring the specific color of chemiluminescence, three different target antigens present in a sample can be quantified simultaneously as shown in Scheme 3.
Scheme 3. Multiplex all-in-one immunoassay with phenylglyoxal derivative chemiluminescence detection capable of simultaneously quantifying three different target antigens in a sample. 1: Pacific Blue, 2: 6-FAM, 3: Rhodamine 101 (or ROX).