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Sensors 2020, 20, 6890 7 of 23 2.3. Multiplex Flow Cytometric Immunoassay Magnetic beads are convenient for immunoassays, due to the possibility of controlling them with a magnetic field and separating them from the mixture when it is needed. However, non-magnetic beads are more frequently used for multiplexed flow cytometric immunoassays. Yu et al. (2012) have developed a competitive microbead-based flow cytometric immunoassay for the simultaneous detection of two biomarkers using QDs luminescent labels [35]. The purpose of their study was the development of a duplex detection system for the detection of toxins (microcystin-LR, benzo[a]pyrene), which can be found in drinking water as contaminants. These two molecules are too small to apply a sandwich immunoassay, thus a competitive immunoassay was designed to determine molecules using a single antibody. During competitive reaction, antigen competitors on polystyrene beads are changed with target molecules. Then, QDs conjugated with antibodies against the target molecules are added to the solution. The final stage of the assay is photoluminescent measurement of the sample without any wash step, using a flow cytometer. QD–antibody conjugates label both free and polystyrene bead attached antigens, although during flow cytometer analysis the groups are easily separated. This method allows not only qualitative, but also quantitative analysis. Microcystin-LR detection dynamic range was 0.52–30 μg L−1 and for benzo[a]pyrene it was 0.13–10 μg L−1. The proposed assay was performed within 30 min, thus this is a relatively fast method for multiple analyte detection [35]. Bilan et al. (2017) demonstrated the detection of multiple lung cancer biomarkers using microspheres encoded with QDs (QDEMs) in clinical samples of bronchoalveolar lavage fluid [36]. In their study, 4.08, 6.1 and 8.24 μm carboxylated melamine formaldehyde resin microspheres as matrix cores and CdSe/ZnS QDs (λem = 515 nm) as optical codes for the preparation of QDEMs were used. Antibodies against three lung cancer biomarkers (AMBP, PRDX2, and PARK7) were conjugated with different sized QDEMs. Using QDEM–antibody conjugates, all three lung cancer biomarkers were successfully and simultaneously detected in clinical samples using a flow cytometer. The authors compared their method’s reproducibility and reliability with the commonly used Luminex xMAP® bead-based immunoassay. Their results showed that QDEM technology may be considered as an alternative to Luminex xMAP® for the diagnostic purpose using conventional flow cytometers. However, the authors did not achieve a high analytical sensitivity at low concentrations of the biomarkers, thus the sensitivity of this method remains unknown [36]. The main issue with the flow cytometry method is difficulties in achieving nanoparticles of homogeneous size. Heterogeneity of nanoparticles leads to interference due to morphology dependant on Rayleigh scattering signals. 2.4. Electrochemical Immunoassay Electrochemical immunoassay is based on a solid phase system where an antibody–antigen reaction occurs and an electrochemical detection system is integrated in the same device [37]. In this method, electrochemical sensors are physically attached to the detection probe surface. When the detection probe interacts with the target analyte, a measurable electrochemical signal appears. These biosensors have impressive detection characteristics and are recognised as one the best sensing systems [37]. The first utilisation of semiconductor nanoparticle labels for this method was demonstrated for the electrochemical DNA hybridisation assay [38,39]. Since then, only a few papers have described how quantum dots could be used in multiplexed electrochemical immunoassay. A multiplex electrochemiluminescence immunoassay has been developed for the simultaneous determination of two different tumour biomarkers (alpha-fetoprotein (AFP) and CEA) in human serum and saliva, using multicolour QD labels and graphene as a conducting bridge [40]. Streptavidin-coated CdSe/ZnS (525 nm and 625 nm) were conjugated with biotin-labelled secondary antibodies (anti-AFP2 and anti-CEA2, respectively). QDs conjugated with secondary antibodies produced electrochemiluminescence reactions after the immunoreaction and the intensity of electrochemiluminescence was amplified using graphene as a conducting bridge. The quantity of AFP and CEA was indicated by the electrochemiluminescencePDF Image | Multiplexed Nanobiosensors: Current Trends in Early Diagnostics
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