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Sensors 2020, 20, 6890 6 of 23 by Kim et al. in 2015 [20]. Kim et al. have demonstrated that their method provides highly specific detection of S100B protein—with a detection limit of 10 pg mL−1—and a 3 order of magnitude of the detection range up to 10 ng mL−1. The overall assay time is 1 h, making this method relatively fast and sensitive [20]. Sensors 2020, 20, x FOR PEER REVIEW 6 of 23 Figure 3. Schematic illustration of a magnetic bead–quantum dot (MB–QD) sandwich assay for Figure 3. Schematic illustration of a magnetic bead–quantum dot (MB–QD) sandwich assay for biomarker capture and detection; (a) magnetic bead–biomarker–quantum dot conjugate; (b) simplified biomarker capture and detection; (a) magnetic bead–biomarker–quantum dot conjugate; (b) flow cytometer scheme. simplified flow cytometer scheme. In 2017, the same group developed a droplet-based device for simple, fast, and cheap malaria In 2017, the same group developed a droplet-based device for simple, fast, and cheap malaria biomarker PfHRP2 detection [29]. This method was designed by adapting a magnetic bead–quantum biomarker PfHRP2 detection [29]. This method was designed by adapting a magnetic bead–quantum dot assay to vial-based assay. This detection tool allows for quantitative and sensitive evaluation of the dot assay to vial-based assay. This detection tool allows for quantitative and sensitive evaluation of target biomarker [29]. the target biomarker [29]. Liu et al. (2016) proposed multiplex magnetic bead–quantum dot assay in microarray format Liu et al. (2016) proposed multiplex magnetic bead–quantum dot assay in microarray format to to detect lung cancer biomarkers [30]. The 6.5 μm diameter magnetic beads and QDs (525 nm, detect lung cancer biomarkers [30]. The 6.5 μm diameter magnetic beads and QDs (525 nm, 585 nm 585 nm and 625 nm) both conjugated with antibodies against cytokeratin-19 fragment (CYRFA 21-1), and 625 nm) both conjugated with antibodies against cytokeratin-19 fragment (CYRFA 21-1), neuron neuron specific enolase (NSE) or carcinoembryonic antigen (CEA) lung cancer biomarkers were added specific enolase (NSE) or carcinoembryonic antigen (CEA) lung cancer biomarkers were added into into human serum samples and mixed in a test tube, and were introduced onto the array of the human serum samples and mixed in a test tube, and were introduced onto the array of the chip. chip. Microbead–antigen–QD conjugates were detected by fluorescent microscope, equipped with a Microbead–antigen–QD conjugates were detected by fluorescent microscope, equipped with a charge-coupled device (CCD) camera. Results showed that this sandwiched immunoassay successfully charge-coupled device (CCD) camera. Results showed that this sandwiched immunoassay detects the presence of different lung cancer associated biomarkers such as CYRFA 21-1, NSE and CEA successfully detects the presence of different lung cancer associated biomarkers such as CYRFA 21- in the given biological fluid at low concentrations (detection limit 0.97 ng/mL; 0.37 ng/mL; 0.19 ng/mL, 1, NSE and CEA in the given biological fluid at low concentrations (detection limit 0.97 ng/mL; 0.37 respectively). The authors suggest that this method could be a low cost tool for disease diagnosis [30]. ng/mL; 0.19 ng/mL, respectively). The authors suggest that this method could be a low cost tool for In later work, the same group demonstrated a similar approach focusing on the same three lung disease diagnosis [30]. In later work, the same group demonstrated a similar approach focusing on cancer biomarkers, using the multiplexed detection and micro-magnetic beads as immune carriers the same three lung cancer biomarkers, using the multiplexed detection and micro-magnetic beads and QDs as detection probes [31]. Immunocomplexes were visualised using fluorescence microscopy as immune carriers and QDs as detection probes [31]. Immunocomplexes were visualised using and the micrographs were analysed by image analysis software to quantify the luminescence of QDs. fluorescence microscopy and the micrographs were analysed by image analysis software to quantify Using this method, the authors managed to reach lower than 1 ng/mL detection limit (364 pg/mL for the luminescence of QDs. Using this method, the authors managed to reach lower than 1 ng/mL CYRFA 21-1, 38 pg/mL for CEA, 370 pg/mL for NSE) [31]. detection limit (364 pg/mL for CYRFA 21-1, 38 pg/mL for CEA, 370 pg/mL for NSE) [31]. Bai et al. (2019) used bead-based microarray to detect three lung cancer biomarkers (CEA, CYFRA Bai et al. (2019) used bead-based microarray to detect three lung cancer biomarkers (CEA, 21-1 and ProGRP) from exosomes [32]. By using a microfluidic system, exosomes were collected from CYFRA 21-1 and ProGRP) from exosomes [32]. By using a microfluidic system, exosomes were cell culture supernatant or plasma samples from lung cancer patients. After the isolation of exosomes, collected from cell culture supernatant or plasma samples from lung cancer patients. After the tumour biomarkers were detected by using QDs conjugated with detection antibodies. The difference isolation of exosomes, tumour biomarkers were detected by using QDs conjugated with detection between experimental result and clinical data was minimal, thus this method could be applied for antibodies. The difference between experimental result and clinical data was minimal, thus this clinical testing [32]. method could be applied for clinical testing [32]. Li and co-workers developed barcodes for multiplexed detection using magnetic beads and Li and co-workers developed barcodes for multiplexed detection using magnetic beads and QDs QDs [33,34]. In 2016 they introduced barcodes for the multiplexed detection of five different tumour [33,34]. In 2016 they introduced barcodes for the multiplexed detection of five different tumour biomarkers in serum (AFP, CEA, CA199, CA125, and CA242). Cadmium-free NIR-emitting CuInS2/ZnS biomarkers in serum (AFP, CEA, CA199, CA125, and CA242). Cadmium-free NIR-emitting QDs and superparamagnetic Fe3O4 in PSMA microspheres were used for simultaneous biomarkers CuInS2/ZnS QDs and superparamagnetic Fe3O4 in PSMA microspheres were used for simultaneous detection. Using these barcodes, they detected sub-ng/mL concentrations of analytes [33]. biomarkers detection. Using these barcodes, they detected sub-ng/mL concentrations of analytes [33]. 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.PDF Image | Multiplexed Nanobiosensors: Current Trends in Early Diagnostics
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