A specific chemiluminescent reaction initiated electrochemically on the electrode surface is used to label Ab with the electrochemiluminescent agent ruthenium tripyridyl [Ru(bpy)3]2+, and the Ag or Ab to be measured is quantified/qualified according to the intensity of light emitted by ruthenium tripyridyl on the electrode through the Ag-Ab reaction and magnetic bead separation technique.

Appliance

Hormones
Tumor Markers
Endocrine Function
Infectious Diseases

Operation method

electrochemiluminescence immunoassay (ECLI)

Materials and Instruments

【Materials】TSH antibody coupled with activated terpyridine ruthenium derivative, i.e., [Ru(bpy)3]2+N-hydroxysuccinimidyl ester (NHS); biotin-conjugated TSH antibody; Magnetic beads; Serum; TPA-containing buffer.
[Instruments, Consumables] Peristaltic pump

Move

Step 1: TSH antibody coupled with activated terpyridine ruthenium derivative i.e. [Ru(bpy)3]2+N-hydroxysuccinimidyl ester (NHS) and biotin-conjugated TSH antibody are added to a reaction cup and incubated simultaneously with serum to be tested.Step 2: Magnetic beads coated with streptavidin are added to the reaction cup and incubated again so that the binding of biotin to the affinity element connects the magnetic beads, TSH antibody as one to form a double antibody sandwich.In step 3, the peristaltic pump sucked the formed [Ru(bpy)3]2+ antibody-antigen-antibody-magnetic bead complex into the flow measurement chamber, at which time the magnetic beads were adsorbed on the electrode surface by the magnet under the working electrode. At the same time, free TSH antibodies (biotin-conjugated and [Ru(bpy)3] 2+-conjugated antibodies) are drawn out of the measurement chamber.Immediately afterward, the peristaltic pump adds a buffer containing tripropylamine (TPA) while the electrode is energized to initiate the ECL reaction process.The luminophore [Ru(bpy)3]2+ and the electron donor TPA on the anode surface, can each lose one electron at the same time and undergo an oxidation reaction, so that the 2-valent [Ru(bpy)3] 2+ is oxidized to a 3-valent, which is a strong oxidizing agent; on the other hand, TPA is oxidized to the cationic radical TPA ..., which is very unstable, and can spontaneously lose 1 proton (H+) to form the radical TPA-, which is a strong reducing agent that can give 1 electron to the 3-valent [Ru(bpy)3)3] 2+ to form the excited state [Ru(bpy)32+, while TPA itself is oxidized to dipropylamine and propionaldehyde.The excited state of [Ru(bpy)3] 2+ emits a photon at a wavelength of 620 nm through a fluorescence decay mechanism, regenerating the ground state of [Ru(bpy)3] 2+. This process takes place week after week on the electrode surface, generating many photons, and the photomultiplier tube detects the light intensity, which is linearly related to the concentration of [Ru(bpy)3] 2+, so the amount of antigen to be measured can be measured.

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