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Sensors 2020, 20, 6890 16 of 23 and fluorescence of dyes (FITC, PE), which was used as a reporter. Thus, the authors demonstrated the advantage of upconverting nanomaterials’ utilisation as barcodes in multiplexed immunoassays compared with traditional down-conversion materials (fluorescent dyes or QDs). The detection limit of mouse IgG in this model immunoassay was 0.01 ng/mL. Thus, their proposed detection system has huge potential applications in multiplexed immunoassays [90]. In order to improve the diagnostic detection of several viruses such as influenza (A and B), respiratory syncytial virus, and adenovirus, Kazakova et al. developed a multiplex serological microarray immunoassay for the simultaneous detection of serum IgG antibodies against these viruses [91]. Microarray plates were coated with streptavidin and the serum antigens as well as negative and positive controls for the samples. Different locations of each of the spotted antigens allowed them to implement specificity of this immunoassay. For detection purposes, anti-human IgG-coated NaYF4:Yb,Er upconverting nanoparticles were used. UCNPs were chosen to avoid autofluorescence signals from the sample and to increase detection sensitivity. The nanobiosensor was effectively used for the simultaneous detection of antibodies against seven different viruses. The authors suggest that their multiplexed immunoassay is a promising tool for the diagnostic detection of viral infections in serum samples, and can be utilised in epidemiological and seroprevalence studies [91]. Li et al. demonstrated multiplexed upconversion imaging in vivo by registering distinct lifetimes of UCNPs after excitation at 808 nm [92]. The authors used an NaYF4@NaYbF4@NaYF4:Yb/Tm@NaYF4 UCNP to control the NIR upconversion luminescence (UCL) lifetime in the range 78–2157 μs detected at 808 nm. They conducted in vivo multiplexing experiments using these UCNPs with tuneable lifetimes for in vivo imaging. For the temporal multiplexed imaging, UCNPs with three different lifetimes were injected into a Kunming mouse through tail vein injection and implemented subcutaneous injection into the left and right of the abdomen. UCL signals with two different lifetimes were observed in liver and abdomen subcutis, which corresponded well with UCL imaging. The authors suggest that their temporal upconversion application has the potential for use as a new optical multiplexed imaging technique [92]. Several studies have demonstrated application of RENPs for multiplexed in vivo imaging by utilising only down-conversion emission and lifetime imaging [93–95]. Fan et al. (2018) designed RENPs barcodes with different lifetime characteristics for multiplexed breast cancer biomarkers (ER, PR, HER2) detection in tumour bearing mice [93]. The expression of tumour biomarkers was quantified by lifetime imaging, and a recognition algorithm was used to resolve subtype of tumour, depending on the different expression of biomarkers. 5. Conclusions In this review article, we have provided a comprehensive overview of the current state-of-the-art development of multiplexed nanobiosensors. We have paid special attention to how different nanomaterials could help to achieve better sensitivity and specificity in classical bioassays. The advantages are anticipated in the development of ELISA, flow cytometric, electrochemical and immunofluorescence techniques, with the combination of nanomaterials such as quantum dots, magnetic beads, gold, silver and upconverting nanoparticles. These nanomaterials applied in rapid and cost-effective multiplexed nanobiosensors show promise for the specific and highly sensitive detection of clinically relevant biomarkers. However, there are still some challenges that remain before nanobiosensors can be applied for daily diagnostic purposes. Firstly, colloidal stability of nanoparticles is usually not sufficient for longer shelf life. This disadvantage could be eliminated when nanoparticles are dispersed on substrates by chemical entrapment or by growing them directly on substrates. Another big issue is the biofunctionalisation of nanomaterials. Despite the availability of a broad variety of chemical methods of biomolecule attachment to the surface of nanoparticles, this process usually requires a controlled environment for long-term stability. Repeatability and sensitivity are also key factors for translation from laboratory to clinic. Laboratories are equipped with exclusive and specific equipment, which allows investigations to reach greater sensitivity inPDF Image | Multiplexed Nanobiosensors: Current Trends in Early Diagnostics
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