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Sensors 2020, 20, 6890 5 of 23 applications. However, additional studies are clearly necessary to optimise assay conditions and antibody reagents to produce a reliable and robust multiplexed assay [18]. Song et al. (2015) demonstrated the application of QLISA for the detection of multiplexed residues of antibiotics in milk [19]. A CdTe QDs detection probe conjugated with different antibodies against antibiotics was used. The limit of detection of this assay is 5 pg/mL, thus the developed method showed excellent sensitivity and specificity for the target analytes. Moreover, this assay is quicker than standard ELISA and can be performed in 90 min. Although the authors focused on developing multiplexed nanobiosensors for food safety control, Song et al. developed a method that can easily be adapted for the purpose of various biomarkers detection. 2.2. Magnetic Bead-Quantum Dot Assay Magnetic beads are usually used for the extraction or purification of biomolecules such as proteins, antibodies, and nucleic acids. Bio-functionalised magnetic beads are widely used in cell sorting, bioseparation, and immunoassays. Functionalisation of magnetic beads with specific antibodies leads to decent recognition, separation, and collection of biomarkers in liquid biopsies without additional separation procedures. High surface-to-volume ratios of magnetic beads enables the capture of analytes in a low concentration solutions [20]. The first report of a multiplex magnetic bead assay was in 1977 [21]. This was an immunoassay that used magnetic beads to simultaneously measure multiple analytes in a clinical sample. A multiplex magnetic bead assay is a derivative of a traditional ELISA, using beads for binding the capture antibody, which are available in several different formats based on the utilisation of flow cytometry, chemiluminescence, or electrochemiluminescence technology [14,22]. As mentioned earlier, a traditional ELISA detects only one analyte in a sample, whereas this method was developed with the purpose of measuring multiple analytes in the same sample at the same time. Confusion can be caused by the fact that this method has been described in various ways in the literature, as multiplex bead array assays [14], Luminex multi-analyte or Luminex assay (trademark of Luminex company), multiplex ELISA [23], magnetic immunoassay [24], flow cytometric multiplex arrays or bead-based multiplex assays [22], or a cytometric bead array system (product of BD Biosciences). All the methods mentioned in this context use different antibodies for efficient capture of analytes, while magnetic beads separate target biomolecules from other molecules in the clinical specimens. Nonetheless, bioassays differ according to the optical detection systems used and the different terminologies used in the scientific publications. There are several multiplex magnetic bead assays, which are different from each other only by virtue of small modifications. For clarity, we will shortly explain the most commonly used method—the Luminex assay. This immunoassay technique is the ELISA with flow cytometry [25]. For this assay, paramagnetic microspheres or beads that are internally labelled with different concentrations of fluorophores (red and infrared) and conjugated to analyte-specific capture antibodies are used. Each fluorophore combination corresponds to one analyte. A mixture of colour-coded beads is added to the sample (or vice versa) and the antibodies bind to the analytes of interest. By completion of this procedure, the additional complementation of biotinylated detection antibodies leads to the formation of an antibody–antigen sandwich. At this point, fluorophore (commonly phycoerythrin (PE)) labelled streptavidin is added, which binds to the biotinylated detection antibodies. For detection, a dual-laser flow-based detection instrument is used for bead classification, analyte determination, and amount of analyte evaluation. This assay can identify multiple biomarkers in biofluid samples and precisely quantify them [25]. Magnetic bead–quantum dot assay is a similar detection method, except instead of organic dyes, quantum dots are used (Figure 3). There are several articles that have established magnetic bead–quantum dot assays to detect one specific analyte [20,26–28]. The most sensitive method using a magnetic bead–quantum dot sandwich assay for the capture and detection of human S100B protein (most extensively studied biomarker for mild traumatic brain injury) in serum was demonstratedPDF Image | Multiplexed Nanobiosensors: Current Trends in Early Diagnostics
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