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Sensors 2020, 20, 6890 11 of 23 Huang et al. (2012) developed a multiplexed bioanalytical assay which allows simultaneous detection of human serum specimens infected by Schistosoma japonicum and tuberculosis pathogens without sample pre-treatment. Two different populations of gold nanorods (AuNRs) were modified with antibodies of different antigens. Detection of biomarkers was achieved by registering a shift in the LSPR, using a standard visible/NIR spectrometer. This nanobiosensor was able to identify infected and uninfected samples and provide a semi-quantitative readout [57]. Utilising the LSPR properties of plasmonic metal nanoparticles, a new variation of the conventional ELISA was established, known as plasmonic ELISA (pELISA) [10]. Several studies have demonstrated the application of pELISA for diagnostics of diseases such as prostate cancer [58–60], syphilis [61] tuberculosis [62], hepatitis B [63] and HIV [64]. However, all pELISA systems are designed for one analyte, and despite its great potential there are no multiplexed pELISAs thus far. 3.2. Multiplexed Colorimetric Detection The nanoparticle-based colorimetric assays are one of the most attractive methods for the detection of various biomolecules such as DNA, RNA, enzymes, proteins, and other small molecules. The key parameter for colorimetric sensing is the ability to change colour of colloidal solution due to changes of noble metal nanoparticles size or distance between them. Such colorimetric sensors can be divided into four types: aggregation, etching, growth and nanoenzyme [65]. The most commonly used gold or silver nanoparticle-based sensors are of the aggregation type, in which optical features of solutions change depending on their level of aggregation. During aggregation of nanoparticles, the surface plasmons of the particles couple and their LSPR profile changes. The main advantages of colorimetric assays are that results can be monitored with the naked eye without the need for any instrumentation. Additionally, this method is simple, convenient, and low cost. Most studies have developed colorimetric detection tools for one analyte [66], however, some multiplexed colorimetric assays have been published. Mancuso et al. used AuNPs and AgNPs for Kaposi’s sarcoma associated herpesvirus and Bartonella DNA simultaneous detection [67]. Specific DNA primers, which recognise targeted oligonucleotide sequences, were attached to AuNPs and AgNPs. When targeted oligonucleotides appear in solution, nanoparticles conjugated with primers bond with them and with each other. Consequently, aggregates of AuNPs and AgNPs are formed and the solution changes colour. Changes were identified visually and by measuring the absorption of the solutions. Authors have demonstrated that the proposed method works when AuNPs and AgNPs are mixed in the same solution—both colour change reactions can be seen independently of each other. In this assay, the limit of DNA detection for the AuNPs is 2 nM and for the AgNPs is 1 nM [67]. Heo et al. demonstrated two-plexed colorimetric assay using three types of nanoparticles, functionalised with DNA: gold nanoparticles (AuNP-DNA), silver nanoparticles (AgNP-DNA), and gold nanorods (AuNR-DNA) [68]. The mixture of these nanoparticles is black-coloured, but after targeted analytes are introduced, the colour of the solution changes, based on which type of nanoparticles (or combination) have aggregated. The authors used a modified CMYK (cyan, magenta, yellow, and key (black)) colour model: when one type of nanoparticle aggregates, corresponding colours are detracted from the mixture. For example, when red-coloured AuNPs aggregate due to interaction with targeted analytes, the mixture turns light-green (cyan and yellow combination). Using this approach, the authors successfully demonstrated the detection of thrombin and platelet-derived growth factor (PDGF) in human blood plasma. The sensitivity of this method was not high (the limit of detection of PDGF was 19 nM), however, it was possible to visually detect the detection limit and concentration (20 nM) with the naked eye [68]. Different approaches for the multiplexed colorimetric diagnostic detection of cancer was demonstrated by Di et al. [69]. The authors used decorated AuNPs and antibody conjugated exosomes for a nanozyme-assisted immunosorbent assay to detect CD63, CEA, GPC-3, PD-L1 and HER2 exosomal proteins from four cell lines as well as from clinical serum samples. This method allowsPDF Image | Multiplexed Nanobiosensors: Current Trends in Early Diagnostics
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