基于革兰氏阳性菌以及金纳米粒子(gold nanoparticles, GNPs)对溶菌酶(lysozyme, LZM)的竞争结合实现了革兰氏阳性菌的广谱快速定量检测。在GNPs与LZM的孵育时间为15 min、孵育pH值为8.0、孵育温度为常温的条件下,通过紫外(ultraviolet-visible, UV-Vis)光谱以及激光光散射仪(dynamic light scattering, DLS)分别实现革兰氏阳性菌的定量检测。结果表明,相比UV-Vis光谱,DLS对粒子尺寸变化体现出了更高的灵敏度,LZM的添加量由UV-Vis法的40 μL(0.14 mg/mL)降低至DLS法的20 μL(0.14 mg/mL),定量限由1.26×107 CFU/mL降低至6.87×105 CFU/mL。与革兰氏阴性菌相比,2种定量方式均体现出对革兰氏阳性菌良好的选择性,均能实现真实样品中细菌的定量检测,其中回收率为83.0%~87.0%,相对标准偏差为2.4%~3.1%,且DLS定量法同样体现出更高的灵敏度。实验结果表明,在该竞争结合细菌检测体系中,DLS优于UV-Vis法,能够实现更灵敏的细菌检测。
A broad-spectrum quantitative detection method of Gram-positive bacteria was realized based on the competitive binding of Gram-positive bacteria and gold nanoparticles (GNPs) to lysozyme (LZM). LZM and GNPs were incubated under the optimized condition of 15 min, pH 8.0 and room temperature. The Gram-positive bacteria were quantitatively detected by ultraviolet-visible (UV-Vis) spectrum and laser light scattering (DLS). Compared with the UV-Vis spectrum, DLS showed higher sensitivity to particle size changes, and the addition of 0.14 mg/mL LZM solution could be reduced from 40 μL in UV-Vis method to 20 μL in DLS method when the signal intensity reached an acceptable range. Therefore, the limit of quantitation reduced from 1.26×107 CFU/mL to 6.87×105 CFU/mL. Compared with the Gram-negative bacteria, both quantitative methods showed good selectivity for Gram-positive bacteria, and both methods could realize the quantitative bacterial detection in real sample with the average recoveries of 83.0%-87.0% with the relative standard deviation of 2.4%-3.1%. The DLS quantification also showed higher sensitivity. Compared with the UV-Vis method, DLS showed the superiority in this competitive binding detection system, which could improve the sensitivity of detection.
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