Glycoproteins are of significant value to liquid biopsy of human diseases. Herein, we present a universal electrochemical platform for the amplified detection of glycoproteins, taking advantage of the glycan-matchmade multivalent decoration of enzyme labels for the enzymatic signal amplification. Briefly, the glycan-matchmade multivalent decoration involves two steps, i.e., the site-directed decoration of the phenylboronic acid-coated gold nanoparticles (PBA-AuNPs) to the cis-diol-containing glycans of glycoproteins and the subsequent decoration of enzyme labels via the affinity cross-linking between the glycan moieties of enzyme labels and the remaining PBA groups on the PBA-AuNP cross-linkers. As the glycan matchmaking-based strategy enables the decoration of each glycoprotein with multiple enzyme labels, this electrochemical platform exhibits a high sensitivity toward glycoprotein detection. Using alkaline phosphatase (ALP) as the proof-of-concept enzyme label in combination with the solid-state voltammetric stripping assay of the enzymatically deposited metallic silver, the detection limits at the pg mL–1 level have been obtained for the electrochemical aptamer-based detection of thrombin and prostate-specific antigen. Overall, this work illustrates an efficient and versatile strategy for the multivalent decoration of enzyme labels for electrochemical detection of glycoproteins at ultralow concentration levels, holding the desirable advantages of simplicity and cost-effectiveness over sandwich enzyme-linked immunosorbent assays.
Zou Xueyan,Yang Fengbo,Sun Xin,Qin Mingming,Zhao Yanbao,Zhang Zhijun
Thiol-functionalized silica nanospheres (SiO 2 -SH NSs) with an average diameter of 460 nm were synthesized through a hydrothermal route. Subsequently, the prepared SiO 2 -SH NSs were modified by SnO 2 quantum dots to afford SnO 2 /SiO 2 composite NSs possessing obvious fluorescence, which could be used to trace the target protein. The SnO 2 /SiO 2 NSs were further modified by reduced glutathione (GSH) to obtain SnO 2 /SiO 2 -GSH NSs, which can specifically separate glutathione S -transferase-tagged (GST-tagged) protein. Moreover, the peroxidase activity of glutathione peroxidase 3 (GPX3) separated from SnO 2 /SiO 2 -GSH NSs in vitro was evaluated. Results show that the prepared SnO 2 /SiO 2 -GSH NSs exhibit negligible nonspecific adsorption, high concentration of protein binding (7.4 mg/g), and good reused properties. In the meantime, the GST-tagged GPX3 separated by these NSs can retain its redox state and peroxidase activity. Therefore, the prepared SnO 2 /SiO 2 -GSH NSs might find promising application in the rapid separation and purification of GST-tagged proteins.
Lin Yang,Shu Jun Zhen,Yuan Fang Li,Cheng Zhi Huang
Graphene oxide (GO) exhibits distinctive Raman scattering features for its high frequency D (disordered) and tangential modes (G-band), which are characteristically sharp at 1580 cm−1 and 1350 cm−1, respectively, but are too weak for sensitive quantitation purposes. By depositing silver nanoparticles on the surface of GO in this contribution, both D and G bands of GO become enhanced. The enzyme label of this method controls the dissolution of silver nanoparticles on the surface of GO through hydrogen peroxide which is produced by the oxidation of the enzyme substrate. With the dissolution of the silver nanoparticles a greatly decreased SERS signal of GO was obtained. This strategy involves dual signal amplification of the enzyme and nanocomposites to improve the detection sensitivity. As a proof of concept, prostate specific antigen (PSA), a biomarker for prostate cancer, is successfully detected as a target by forming a sandwich structure in immunoassay. The SERS immunoassay possesses excellent analytical performance in the range 0.5 pg mL−1 to 500 pg mL−1 with a limit of detection of 0.23 pg mL−1, making the detection of PSA serum samples from prostate cancer patients satisfactory, demonstrating that the sensitive enzyme-assisted dissolved AgNPs SERS immunoassay of PSA has potential applications in clinical diagnosis.
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