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Sensors 2020, 20, 6890 3 of 23 high resistance to photobleaching, and can provide excitation of several different emission colours (quantum dots) using a single wavelength for excitation. In this review, we summarize progress in nanomaterial-based multiplexed bioassays, by focusing on their applications in medicine. 2. Quantum Dots Since their first description in a biological context [6] quantum dots (QDs) have been considered one of the most attractive luminescence label in biomedicine. Quantum dots are inorganic semiconductor crystals with a diameter of 1 nm to 10 nm (typically 2–6 nm), with optical properties which arise from interactions between electrons, holes, and their local environments [7,8]. QDs absorb photons whose energy exceeds the photoluminescence excitation bandgap. In this process, electrons are transferred from the valence band to the conduction band. Due to many electronic states, QD photoluminescence excitation spectra are wide, but on the other hand, QD emission bands are narrow and symmetrical. These unique properties allow different QDs to be excited by the same light source and obtain different coloured visualisations in one biological sample. For comparison, the majority of organic fluorescent dyes have relatively narrow excitation bands and wide fluorescent band. Therefore, in order to perform multicolour imaging, several different light sources are required [8]. By varying the size and composition of QDs, the emission wavelength can be tuned from blue to near-infrared [8]. Additionally, because the surface of QDs are easily modifiable, it is easy to attach various antibodies and other biomolecules to them [9]. Thus, QDs are highly applicable for multiplexed immunoassays. Here, we briefly review how QDs could be used for various multicolour biosensing applications. 2.1. QLISA Quantum dot-linked immunosorbent assay (QLISA) is an approach similar to the enzyme-linked immunosorbent assay (ELISA), except QDs are used instead of enzymes [10]. The principle of these two methods is the same. For both methods, the first step is antibody coating—the specific capture antibody is immobilised on high protein-binding plates. When the capture antibody is immobilised, the uncovered space on the plate is blocked with an irrelevant blocking protein, e.g., serum albumin. After that, samples and standard dilutions are added to the wells. In this step, the analyte adheres to the capture antibodies. In some ELISA cases, analytes can be attached directly on the plate surface, without using a capture antibody. The excess sample is removed, and then specific biotinylated detection antibody is added to the wells to enable detection of the captured protein. In the case of QLISA, QDs with conjugated detection antibody are used. After detection, the antibody binds to the analyte, and streptavidin conjugated with alkaline phosphatase/horseradish peroxidase (ELISA) or streptavidinated QDs (QLISA) is added to the wells which binds to the biotinylated antibody. For the ELISA method one addition step is required before analysis—addition of colorimetric substrate which form a coloured solution when catalysed by the enzyme. Samples are then analysed with microplate readers. The optical density in each well of the multi-well plate is measured, whereas for QLISA, photoluminescence of the QDs is registered. ELISA is an established standard biomarker detection method that is widely used in biomedical research and in the clinical diagnostic setting [2,11], whereas QLISA is still in the development stage. Nevertheless, the ELISA method still has some important disadvantages, which hopefully QLISA could solve. The main drawback of the conventional ELISA method is that the detection limit is barely less than the nanomolar concentration level, which is usually not enough to reach the clinical threshold of many protein biomarkers, especially in the early stages of disease [12]. This disadvantage is due to low signal-to-noise ratio and limited signal amplification [13]. An additional drawback is that traditional ELISA detects only one analyte in a sample, and multiplexed detections require several measurements simultaneously carried out [14]. These disadvantages can be solved by using luminescent QDs instead of enzymatic colorimetric reactions for identification of the detection antibody. First of all, photoluminescence-based assays are more sensitive than absorbance-based assays [15], thus the signal-to-noise ratio increases by measuring photoluminescence instead of absorption.PDF Image | Multiplexed Nanobiosensors: Current Trends in Early Diagnostics
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