We present a novel dual-mode fluorescent and colorimetric immunosensor based on conventional immunoassay platforms by utilizing a gold nanoflower (AuNF)-loaded fluorescein molecule (AuNF@Fluorescein) as signal output. The AuNFs were modified with thiolated carboxyl ligand, which consisted of a hydrophobic alkane chain as hydrophobic wallet for fluorescein encapsulation, a tetra (ethylene glycol) unit for biocompatibility and solubility, and a functional carboxyl group for the conjugation of biorecognition molecules for biosensing. The resultant AuNFs showed a high loading capacity of 3.74 × 106 fluorescein molecules per AuNF because of its flower-like shape with many complex branches. By adjusting the solution pH to 8.0, the fluorescein molecules can almost entirely be released from the hydrophobic wallet of AuNF@Fluorescein, which led to strong fluorescent-signal amplification. Under the optimal detection conditions, the proposed immunoassay based on fluorescent signal exhibited ultrahigh sensitivity for alpha-fetoprotein (AFP) detection, with a limit of detection (LOD) of 29 fg/mL. This value is approximately 9.3 × 103-fold lower than that of corresponding horseradish peroxidase (HRP)-based immunoassay (LOD = 270 pg/mL). The fluorescein molecule also had intrinsic peroxidase-like activity to catalyze 3,3',5,5'-tetramethylbenzidine oxidation with hydrogen peroxide for colorimetric signal. The proposed method with colorimetric mode further exhibited a sensitivity with a LOD of 17.7 pg/mL, which is about 15-fold lower than that of conventional HRP-based immunoassay. The recoveries of the proposed dual-mode immunoassay for AFP spiked serum samples ranged within 89.85%-100.0%, with the coefficient of variations ranging from 0.5% to 2.4%, indicating acceptable accuracy and precision for AFP quantitative detection. The reliability of the developed dual-mode immunoassay was further compared with a commercial chemiluminescence immunoassay kit by analyzing 20 clinical serum samples, showing that the two methods well agreed with each other, with high correlation coefficients of 0.997 and 0.986 based on recorded fluorescence and colorimetric signals, respectively. In summary, the proposed method was highly suitable for the ultrasensitive analysis of biomarkers or infectious diseases by fluorescence mode and can be used for routine clinical diagnosis by colorimetric mode.

中文翻译：

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用于超灵敏检测血清样品中甲胎蛋白的双模式荧光和比色免疫分析

我们利用负载金纳米花 (AuNF) 的荧光素分子 (AuNF@Fluorescein) 作为信号输出，提出了一种基于传统免疫测定平台的新型双模式荧光和比色免疫传感器。AuNFs用硫醇化羧基配体修饰，其由疏水烷链作为荧光素封装的疏水钱包、生物相容性和溶解性的四（乙二醇）单元和用于生物传感的生物识别分子缀合的功能性羧基组成。所得的 AuNF 显示出每个 AuNF 具有 3.74 × 106 荧光素分子的高负载能力，因为它的花状形状具有许多复杂的分支。通过将溶液 pH 值调整为 8.0，荧光素分子几乎可以完全从 AuNF@Fluorescein 的疏水钱包中释放出来，这导致了强烈的荧光信号放大。在最佳检测条件下，所提出的基于荧光信号的免疫测定对甲胎蛋白 (AFP) 检测具有超高灵敏度，检测限 (LOD) 为 29 fg/mL。该值比相应的基于辣根过氧化物酶 (HRP) 的免疫测定 (LOD = 270 pg/mL) 低约 9.3 × 103 倍。荧光素分子还具有内在的过氧化物酶样活性，可催化 3,3',5,5'-四甲基联苯胺用过氧化氢氧化以获得比色信号。所提出的比色模式的方法进一步表现出灵敏度，LOD 为 17.7 pg/mL，比传统的基于 HRP 的免疫测定法低约 15 倍。建议的双模式免疫测定法对 AFP 加标血清样品的回收率范围在 89.85%-100.0% 范围内，变异系数范围从 0.5% 到 2.4%，表明 AFP 定量检测的准确性和精密度可接受。通过分析 20 个临床血清样本，进一步将开发的双模式免疫测定法的可靠性与商业化学发光免疫测定法试剂盒进行了比较，表明两种方法相互吻合，基于记录的荧光和比色法的相关系数分别为 0.997 和 0.986。信号，分别。总之，该方法非常适用于荧光模式下生物标志物或传染病的超灵敏分析，可用于比色模式下的常规临床诊断。变异系数在 0.5% 到 2.4% 之间，表明 AFP 定量检测的准确度和精密度可以接受。通过分析 20 个临床血清样本，进一步将开发的双模式免疫测定法的可靠性与商业化学发光免疫测定法试剂盒进行了比较，表明两种方法相互吻合，基于记录的荧光和比色法的相关系数分别为 0.997 和 0.986。信号，分别。总之，该方法非常适用于荧光模式下生物标志物或传染病的超灵敏分析，可用于比色模式下的常规临床诊断。变异系数在 0.5% 到 2.4% 之间，表明 AFP 定量检测的准确度和精密度可以接受。通过分析 20 个临床血清样本，进一步将开发的双模式免疫测定法的可靠性与商业化学发光免疫测定法试剂盒进行了比较，表明两种方法相互吻合，基于记录的荧光和比色法的相关系数分别为 0.997 和 0.986。信号，分别。总之，该方法非常适用于荧光模式下生物标志物或传染病的超灵敏分析，可用于比色模式下的常规临床诊断。通过分析 20 个临床血清样本，进一步将开发的双模式免疫测定法的可靠性与商业化学发光免疫测定法试剂盒进行了比较，表明两种方法相互吻合，基于记录的荧光和比色法的相关系数分别为 0.997 和 0.986。信号，分别。总之，该方法非常适用于荧光模式下生物标志物或传染病的超灵敏分析，可用于比色模式下的常规临床诊断。通过分析 20 份临床血清样本，进一步将开发的双模式免疫测定的可靠性与商业化学发光免疫测定试剂盒进行了比较，表明两种方法相互吻合，基于记录的荧光和比色法的相关系数分别为 0.997 和 0.986。信号，分别。总之，该方法非常适用于荧光模式下生物标志物或传染病的超灵敏分析，可用于比色模式下的常规临床诊断。