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Sensors 2020, 20, 6890 8 of 23 responses of QDs525 and QDs625, respectively. The working range of the proposed method was 0.001–0.1 pg/mL and the detection limit was 0.4 fg/mL for both analytes [40]. SensorsU20lt2r0a,s2e0n,xsiFtOivRePeEleEcRtrRoEcVhIEemWiluminescenceimmunoassayshavebeendevelopedforthesimulta8noefo2u3s determination of two biomarkers, the cancer antigens CA 125 and CA 15-3. The immunosensor designed immunosensor designed in this study can be used for the sequential detection of CA 125 and CA 15- in this study can be used for the sequential detection of CA 125 and CA 15-3 biomarkers with the wide 3 biomarkers with the wide detection ranges of 1 μU/mL–1 U/mL and 0.1 mU/mL–100 U/mL with detection ranges of 1 μU/mL–1 U/mL and 0.1 mU/mL–100 U/mL with very low detection limits of very low detection limits of 0.1 μU/mL and 10 μU/mL, respectively [41]. 0.1 μU/mL and 10 μU/mL, respectively [41]. 2..5..MuultltipiplelexxAnnttigigeennIImaaggininggininCeellslsaannddTiisssueess Conveenttiionall iimmunohiisttochemiisttry ((IIHC)) iis wiidelly used ass adiiagnossttiicctteecchniiqueiinttheeffiieelld offpathology,,esepspeceicailalyllyinicnanccaenrcderiagdniaogsntiocs.tiHcso.wHeovwere,vtheer,stahnedastradnIdHaCrdmIeHthCodmaelltohwodsthalelolawbselltihneg loabf eallsiingloefmaasriknegrlepemratriskseurepseerctiossnu,ewsheicthioins,inwshuiffichciesnitnisnufsfoicmienctaisnes,otmhuescmasueslt,ipthleusemctuioltnisploef sseacmtioentisssoufesamreestiasisnueedarnedsteaxianmedinaendd,wexhaimchinisedti,mwehainchdirsestiomurecaencdonrseusomuirncge.cTohnesumseinogf.seTvheraulsQeDofs sceovnejruaglaQteDdswcoitnhjuspgeacteifidcwanitthibsopdeiceisfiecnaanbtliebsotdhieseaesnyablalebseltlhinegeaosfymluablteipllliengbiofmmaruklteirpsleinboionme saprkeecirms ien oanedstphecidmisetinagnudishthinegdoisfticnagnuceisrhcienlglsoffrocmancheracltehlylscferollms (Fhiegaultrhey4)c.ells (Figure 4). Figure 4. Targeting of cancer cells in tissue samples: multiple immuno-nanoparticles with specific Figure 4. Targeting of cancer cells in tissue samples: multiple immuno-nanoparticles with specific antigens against cancer biomarkers label cancer cells. Each antibody is conjugated with different antigens against cancer biomarkers label cancer cells. Each antibody is conjugated with different coloured luminescent nanoparticles, which allows the simultaneous detection of several biomarkers in coloured luminescent nanoparticles, which allows the simultaneous detection of several biomarkers one sample. in one sample. The first report of QD-based imaging of biomarkers was published by Wu et al., 2002 [42]. The first report of QD-based imaging of biomarkers was published by Wu et al., 2002 [42]. The The authors used CdSe/ZnS QDs (λex 535 nm and 630 nm) to simultaneously label two breast cancer authors used CdSe/ZnS QDs (λex 535 nm and 630 nm) to simultaneously label two breast cancer biomarkers in SK-BR-3 cells (cell surface antigen Her2 and nuclear antigens). They demonstrated that biomarkers in SK-BR-3 cells (cell surface antigen Her2 and nuclear antigens). They demonstrated that QDs were effective for the two-colour fluorescence labelling of distinct cellular components [42]. QDs were effective for the two-colour fluorescence labelling of distinct cellular components [42]. Yezhelyev et al. published a breakthrough multiplex cell imaging article in 2007 [43]. Yezhelyev et al. published a breakthrough multiplex cell imaging article in 2007 [43]. They They demonstrated the quantitative and simultaneous profiling of five biomarkers in breast cancer demonstrated the quantitative and simultaneous profiling of five biomarkers in breast cancer cells cells and clinical tissue specimens, using QDs for labelling. Five different QDs (QD525, QD565, QD605, and clinical tissue specimens, using QDs for labelling. Five different QDs (QD525, QD565, QD605, QD655, QD705) conjugated with antibodies against HER2, ER, PR, EGFR, and mTOR biomarkers, QD655, QD705) conjugated with antibodies against HER2, ER, PR, EGFR, and mTOR biomarkers, respectively, were used for multiplexed detection of these antigens in breast cancer cells (MCF-7 and respectively, were used for multiplexed detection of these antigens in breast cancer cells (MCF-7 and BT474 cell lines) and in tumour biopsy specimens. All samples were visualised using confocal BT474 cell lines) and in tumour biopsy specimens. All samples were visualised using confocal microscopy and, additionally, spectroscopic measurements were performed for quantification purposes. microscopy and, additionally, spectroscopic measurements were performed for quantification The authors concluded that multiplexed detection and quantification of ER, PR, and Her2 biomarkers purposes. The authors concluded that multiplexed detection and quantification of ER, PR, and Her2 correlated firmly with the results gained from other traditional methods such as IHC, Western blotting, biomarkers correlated firmly with the results gained from other traditional methods such as IHC, Western blotting, and fluorescence in situ hybridisation, implying that the QD-based sensing technology is applicable for the molecular profiling of tumour biomarkers in vitro [43]. Approaching clinical application, Liu et al. (2010) reported the use of QDs for the detection and characterisation of a class of low-abundant tumour cells in Hodgkin’s lymphoma [44]. They developed multiplexed detection of four protein biomarkers (CD15, CD30, CD45, and Pax5) on human tissue biopsies. Immunostaining of tissues was performed in two steps repeating them twice: firstly, mouse and rabbit primary antibodies against Pax5 and CD30 antigens, respectively, were usedPDF Image | Multiplexed Nanobiosensors: Current Trends in Early Diagnostics
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