Homogeneous immunoassay based on two-photon excitation fluorescence resonance energy transfer.
A two-photon excitable small organic molecule (abbreviated as TP-NH 2) with large two-photon absorption cross section and competitive fluorescence quantum yield was prepared, which emitted fluorescence in the visible region upon excitation at 800 nm. Using the TP-NH 2 molecule as an energy donor, a two-photon excitation fluorescence resonance energy-transfer (TPE-FRET) based homogeneous immunoassay method was proposed. The donor and the acceptor (DABS-Cl, a dark quencher) were labeled to bovine serum albumin (BSA) separately, and anti-BSA protein was determined by employing an antibody bridging assay scheme. Rabbit anti-BSA serum containing other biomolecules was intentionally used as the sample to introduce interference. A parallel assay was performed using the traditional one-photon excitation FRET model, which failed to carry out quantitative determination due to the serious background luminescence arising from those biomolecules in the sample. The TPE-FRET model showed its strong ability to overcome the problem of autofluorescence and provided satisfying analytical performance. Quite good sensitivity and wide linear range (0.05-2.5 nM) for anti-BSA protein was obtained. The results of this work suggest that TPE-FRET could be a promising technique for homogeneous assays excluding separation steps, especially in complicated biological sample matrixes.
Liu
Lingzhi
L
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR China.
Shao
Mei
M
Dong
Xiaohu
X
Yu
Xuefeng
X
Liu
Zhihong
Z
He
Zhike
Z
Wang
Ququan
Q
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology; D002417:Cattle; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007118:Immunoassay / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry; D017785:Photons; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000276:immunology; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis
D000906:Antibodies; D009930:Organic Chemicals; D012710:Serum Albumin, Bovine