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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/45484

Title: Pulse Laser-Induced Generation of Cluster Codes from Metal Nanoparticles for Immunoassay Applications.
Authors: Chia-Yin Chang;Han-Wei Chu;Binesh Unnikrishnan;Lung-Hsiang Peng;Jinshun Cang;Pang-Hung Hsu;Chih-Ching Huang
Contributors: 國立臺灣海洋大學:生命科學系
Date: 2017
Issue Date: 2018-03-21T06:51:13Z
Publisher: APL Materials
Abstract: Abstract: In this work, we have developed an assay for the detection of proteins by functionalized nanomaterials coupled with laser-induced desorption/ionization mass spectrometry (LDI-MS) by monitoring the generation of metal cluster ions. We achieved selective detection of three proteins [thrombin, vascular endothelial growth factor-A165 (VEGF-A165), and platelet-derived growth factor-BB (PDGF-BB)] by modifying nanoparticles (NPs) of three different metals (Au, Ag, and Pt) with the corresponding aptamer or antibody in one assay. The Au, Ag, and Pt acted as metal bio-codes for the analysis of thrombin, VEGF-A165, and PDGF-BB, respectively, and a microporous cellulose acetate membrane (CAM) served as a medium for an in situ separation of target protein-bound and -unbound NPs. The functionalized metal nanoparticles bound to their specific proteins were subjected to LDI-MS on the CAM. The functional nanoparticles/CAM system can function as a signal transducer and amplifier by transforming the protein concentration into an intense metal cluster ion signal during LDI-MS analysis. This system can selectively detect proteins at picomolar concentrations. Most importantly, the system has great potential for the detection of multiple proteins without any pre-concentration, separation, or purification process because LDI-MS coupled with CAM effectively removes all signals except for those from the metal cluster ions.
The greatest challenge in the detection of specific proteins or tumor markers for the diagnosis of cancer is their low concentrations in human plasma.1,2 Interferences due to other proteins with similar properties also cause difficulties in their selective detection by conventional methods.2 Therefore, to achieve high selectivity for the identification of specific proteins in biological fluids, such as plasma, immunoassays based on aptamer (Apt)-protein-specific and antigen-antibody (Ab)-specific interactions are widely used in both clinical and medical research.3 Currently, the enzyme-linked immunosorbent assay (ELISA) has demonstrated reasonable sensitivity and specificity; however, it fails in the analysis of multiple proteins in a single well, which limits its application.4
Matrix-assisted laser-induced desorption/ionization (MALDI) time-of-flight mass spectrometry (MS) is an effective tool for identifying biomacromolecules such as proteins, nucleic acids, and polysaccharides with great selectivity.5 However, non-uniform matrix-analyte co-crystallization and interference signals from organic matrices significantly decrease the reproducibility and sensitivity of this method.6 Recent reports show that surface-assisted laser-induced desorption/ionization mass spectrometry (SALDI-MS) using nanomaterials, such as a matrix (substrate), can improve reproducibility and reduce matrix interference.7–10 SALDI-MS has been successfully used in the analysis of a wide variety of analytes such as proteins, DNA, microbes, and tumor cells. However, in the case of multiple-target analysis and quantitation by SALDI-MS, the fragmentation of several analytes, background molecules, and their adducts is unpredictable. Thus, the comprehensive detection of complex samples such as plasma containing complicated proteins, small molecules, and salts, is difficult. To resolve the interference from background proteins and the unpredictable fragmentation of analytes encountered in SALDI-MS, we have developed a simple immunoassay that exhibits significant potential for the simultaneous detection of different proteins in a single analysis by monitoring the cluster ion signals generated from the metal nanoparticles (NPs) under pulse laser irradiation.
In this work, instead of observing the various intact or fragmented protein ions, we measure the metal-codes (specific metal cluster ions) from the NPs itself for the quantitative detection of the three analytes: thrombin, vascular endothelial growth factor-A165 (VEGF-A165), and platelet-derived growth factor-BB (PDGF-BB). These proteins play critical roles in angiogenesis and tumor progression.11–13 Thrombin promotes angiogenesis by activating PAR1 receptors in platelet and endothelial cells.11 VEGF and PDGF are signal proteins, which are highly expressed by tumor cells to stimulate tumor angiogenesis and vascular remodeling by binding to specific receptors on endothelial cells.12,13 Therefore, the determination of the concentration of these three cytokines in plasma and in tumor environments is very important for the diagnosis of tumor growth and metastasis.11–13
The functionalization of different metal NPs with their respective aptamers or antibodies enables specific targeting of thrombin, VEGF-A165, and PDGF-BB (Fig. 1). Here, three metal (Au, Ag, and Pt) NPs are used as mass tags for the proteins, rather than a SALDI matrix, to enhance the ionization of the analyte molecules. The metal NPs absorb pulsed laser energy and undergo photothermal evaporation and/or Coulombic explosion, which produces a substantial amount of cluster ions useful for signal amplification.14–17 We used cellulose acetate membrane (CAM), which can be directly mounted onto a plate for LDI-MS analysis, to serve as a medium (substrate) to separate the antibody (Ab)- or aptamer (Apt)-modified NPs and their conjugates formed with their targeting proteins in situ. This assay does not require any additional processing steps such as separation, preconcentration, or washing. Because of the increased particle weight or decreased affinity towards CAM upon interaction with the target proteins, target-bound nanoparticles penetrate deeper into the CAM. Consistent with our hypothesis, the intensity of metal cluster ions ([Mn]+; M = Au and Ag, n = 1–3) is observed to decrease with increasing concentration of target protein. Platinum ions ([Pt]+) exhibit the opposite trend: their intensity increases as the concentration of target protein increases. The target protein-induced aggregation of Pt NPs leads to the deposition of NPs onto the upper layer of the CAM.
URI: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/45484
Appears in Collections:[生命科學系] 期刊論文

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