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Sensors 2020, 20, 6890 14 of 23 thrombin detection was from 55.6 pM to 13.5 nM, with the limit of 6.2 pM. PDGF-BB was detected with the range of concentration from 625 pM to 20 nM and the detection limit was 156 pM. They showed that by using AgNPs probes for Cy3 and Cy5 fluorescence enhancement, the detection limit could be improved 80-fold for thrombin and 8-fold for PDGF-BB, when compared to aptamers without enhancement [80]. Liu et al. developed a multiplexed antibody microarray for circulating biomarkers associated with lung cancer detection [81]. Microarray plates were modified with gold nanostructures to enhance the fluorescence of IRDye800, which was used to label detection antibodies in sandwich immunoassay. Lung cancer biomarkers CEA, CYFRA 21-1 and NSE were detected directly from human serum. Simultaneous detection of three biomarkers was achieved by printing capture antibodies onto 3 × 3 spot matrices. This approach enables a high-throughput testing of lung cancer biomarkers and improved specificity and sensitivity compared with convenient methods such as ELISA and Luminex assay [81]. Similar studies published by the same group demonstrated detection of diabetes biomarkers [82] as well as zika and dengue viruses [83]. Min et al. demonstrated a new and simple technique that allows the multiplexed detection of biomarkers in extracellular vesicles [84]. In this study, plasmon-enhancement was achieved by using Au nanoholes as a substrate. Firstly, vesicles were captured on Au nanoholes by a biotin-avidin reaction. Then, vesicles were stained with antibodies conjugated with fluorescent labels (Alexa Fluor 488, Cy3, Cy5, Cy5.5). Au nanoholes operated as amplifiers for fluorophores under excitation. They applied the designed assay to detect glioblastoma biomarkers CD-pan (CD9, CD63, and CD81), with EGFR, EGFRvIII and GAPDH as control markers, from supernatants of glioblastoma cells. Fluorescence signals of multiple fluorophores were amplified by one order, and both transmembrane and intravesicular biomarkers were detected at the single extracellular vesicle level. However, the proposed assay still has limited multiplexing capability, which is dependent on fluorescent microscope systems, allowing the visualisation of up to three or four fluorophores in one sample. Additionally, this system was not tested with clinical samples, thus its real applicability still is unknown [84]. Liu et al. developed an assay for the detection of SARS-CoV-2 antibodies by using nanostructured plasmonic gold substrate as a near-infrared fluorescence amplifier [85]. In this study, IgG and IgM antibodies against SARS-CoV-2 were detected directly from human serum and saliva using a sandwich immunoassay in microarray plate. They demonstrated high specificity and sensitivity for the detection of antibodies against SARS-CoV-2. Additionally, antibodies to viruses associated with common colds did not cross-react with SARS-CoV-2 or SARS, or reaction was low. Application of this fast and sensitive testing method in daily clinical practice would allow population-based diagnostic mass screening of COVID-19 [85]. 4. Upconverting Nanoparticles Low background signal and high specific signals from molecules under investigation are two main criteria for precise and effective spectral encoding for multiplexed analysis of various biosamples [86]. The main drawback of previously described methods is interference between coded signals and reporter signals at low concentrations, which leads to inaccurate test results. Nanoparticles are mostly optically active in UV–VIS region, where many biomolecules also show strong autofluorescence (Figure 6a). In this case, upconverting nanoparticles (UCNPs) could be useful for in vivo biosensing. UCNPs are inorganic nanocrystals, commonly rare-earth-doped nanoparticles (RENPs), that allow for the generation of anti-Stokes emission under near-infrared (NIR) excitation. Upconversion (UC) emission itself is a unique process, where low energy NIR light is converted into higher energy light through the sequential absorption of multiple photons or energy transfer. UCNPs exhibit great properties with sharp emission bands, large anti-Stokes shift, high resistance to photobleaching, long lifetimes (~ms), high detection sensitivity, low toxicity, and no interference from biomolecular autofluorescence [87] (Figure 6b). The popularity of UCNP application in various biomedical research is constantly growing, including the development of nanobiosensors.PDF Image | Multiplexed Nanobiosensors: Current Trends in Early Diagnostics
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