Determination of glucose by affinity adsorption solid substrate-room temperature phosphorimetry based on concanavalin agglutinin labeled with fluorescein using 1.5-generation dendrimers as sensitizer

摘要

In the presence of the heavy atom perturber Pb(Ac)₂, fluorescein (HFin) can emit strong and stable room temperature phosphorescence (RTP) on the surface of a nitrocellulose membrane (NCM) at λ_ex max/λ_em max = 480/648 nm. It can be spiked with the 1.5-generation polyamidoamine dendrimers (abbreviated: D-1.5) to emit stronger RTP. It was found that a quantitative specific affinity adsorption (AA) reaction between concanavalin agglutinin (abbreviated: Con A) labeled with D-1.5-HFin and N-acetylglucosamine (G) could be carried out on the surface of NCM. The product of the reaction (D-1.5-HFin- Con A-G) could emit strong and stable RTP, and the ΔI_p was proportional to the content of G. According to the above facts, a new method for determination of G by affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) was established, based on Con A labeled with fluorescein using D-1.5 dendrimers molecules as sensitizer. The linear range of the sandwich method was 0.040–60 pg G spot−1 (corresponding concentration range: 0.10–150 ng mL−1; sample volume: 0.40 µL spot−1). The regression equation of the working curve was ΔI_p = 6.880 + 5.610 m_G pg spot−1, r = 0.9987. The working solutions containing 0.10 and 150 ng mL−1 G were determined repeatedly 11 times, respectively. The corresponding RSDs were 2.9 and 3.8%. The detection limit of this method calculated by 3Sb/k was 0.021 pg spot−1 (5.2 × 10−11 g mL−1). Compared with the direct method (detection limit: 0.078 pg spot−1, linear range: 0.40–40 pg G spot−1), the sensitivity was obviously improved and the linear range was wider. This method has been successfully applied to the determination of G in human plasma, as well as to the supervision and forecast of human diseases, for it is of good sensitivity, accuracy and precision.