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Sensors 2020, 20, 6890 4 of 23 Sensors 2020, 20, x FOR PEER REVIEW 4 of 23 Moreover, QDs have a high photoluminescence quantum yield, which allows acquisition of high signal intensities from relatively low concentrations [16]. Therefore, QDs could work as amplifiers when of high signal intensities from relatively low concentrations [16]. Therefore, QDs could work as low concentrations of analyte need to be detected. Finally, QDs, due to their optical and chemical amplifiers when low concentrations of analyte need to be detected. Finally, QDs, due to their optical properties, are good candidates for use in multiplexing (Figure 2). and chemical properties, are good candidates for use in multiplexing (Figure 2). Figure 2. Multiplexed diagnostics using quantum dot-linked immunosorbent assay (QLISA). Figure 2. Multiplexed diagnostics using quantum dot-linked immunosorbent assay (QLISA). Firstly, Firstly, the surface of the quantum dots (QDs) is modified with specific antibodies using the surface of the quantum dots (QDs) is modified with specific antibodies using 1-Ethyl-3-(3- 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling, succinimidyl-4-(N-maleimidomethyl) dimethylaminopropyl)carbodiimide (EDC) coupling, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) conjugation, or streptavidin–biotin interaction. Clinical samples cyclohexane-1-carboxylate (SMCC) conjugation, or streptavidin–biotin interaction. Clinical samples from patients are processed and loaded to a QLISA plate, which is coated with capture antibodies. from patients are processed and loaded to a QLISA plate, which is coated with capture antibodies. Then, modified QDs are loaded and the plate is scanned using a microplate reader. Different QDs in Then, modified QDs are loaded and the plate is scanned using a microplate reader. Different QDs in the sample are excited with the same wavelength and several bands of photoluminescence are detected. the sample are excited with the same wavelength and several bands of photoluminescence are The final step is the analysis of the acquired data. detected. The final step is the analysis of the acquired data. Mansur et al. (2015) successfully demonstrated the detection of the cluster of differentiation 20 Mansur et al. (2015) successfully demonstrated the detection of the cluster of differentiation 20 (CD20) antigen, which is a biomarker overexpressed by B-lymphocyte cancer cells, using QLISA [17]. (CD20) antigen, which is a biomarker overexpressed by B-lymphocyte cancer cells, using QLISA [17]. Suzuki et al. (2017) reported the application of QLISA for the detection of the multifunctional Suzuki et al. (2017) reported the application of QLISA for the detection of the multifunctional cytokine cytokine interleukin 6 (IL-6) which is involved in many cellular processes, such as receptor synthesis, interleukin 6 (IL-6) which is involved in many cellular processes, such as receptor synthesis, inflammation, cell proliferation, and cancer cell signalling [10]. Functionalised QDs were used in inflammation, cell proliferation, and cancer cell signalling [10]. Functionalised QDs were used in QLISA to detect and quantify IL-6. Results demonstrated that the lower limit of IL-6 detection would QLISA to detect and quantify IL-6. Results demonstrated that the lower limit of IL-6 detection would be approximately 50 pg/mL, which is undetectable using a standard ELISA method [10]. However, be approximately 50 pg/mL, which is undetectable using a standard ELISA method [10]. However, these two studies demonstrated working QLISA for the detection of only one biomarker. Nevertheless, these two studies demonstrated working QLISA for the detection of only one biomarker. the strategies that they described could also be applied for multiplexed QLISA by increasing the Nevertheless, the strategies that they described could also be applied for multiplexed QLISA by number of different QDs-antibody conjugates to detect more than one biomarker. The first multiplexed QLISA was described by Goldman et al., published in 2004 [18]. In this study, The first multiplexed QLISA was described by Goldman et al., published in 2004 [18]. In this four different toxins (cholera toxin, ricin, shiga-like toxin 1, and staphylococcal enterotoxin B) were study, four different toxins (cholera toxin, ricin, shiga-like toxin 1, and staphylococcal enterotoxin B) simultaneously detected using CdSe/ZnS QDs (510, 555, 590, and 610 nm, respectively) bioconjugates. were simultaneously detected using CdSe/ZnS QDs (510, 555, 590, and 610 nm, respectively) The detection of the four analytes was demonstrated at 1000 and 30 ng/mL of each toxin in the mixture. bioconjugates. The detection of the four analytes was demonstrated at 1000 and 30 ng/mL of each increasing the number of different QDs-antibody conjugates to detect more than one biomarker. In their study, Goldman et al. demonstrated the principle of multiplexed QLISA and proposed that toxin in the mixture. In their study, Goldman et al. demonstrated the principle of multiplexed QLISA their work highlights the advantages of QDs versus conventional organic dyes for use in multiplexing and proposed that their work highlights the advantages of QDs versus conventional organic dyes for use in multiplexing applications. However, additional studies are clearly necessary to optimise assay conditions and antibody reagents to produce a reliable and robust multiplexed assay [18].PDF Image | Multiplexed Nanobiosensors: Current Trends in Early Diagnostics
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