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Sensors 2020, 20, 6890 13 of 23 the surface of prostate cancer cells were evaluated visually and three prostate cancer cell lines had been distinguished from each other. The authors suggest that usage of the multiplexed SERS imaging technique could improve the identification of cancer cell phenotypes [74]. Li et al. (2018) developed a multiplexed nanobiosensor by adapting the sandwich-type immunoassay and SERS methods for the detection of cancer biomarkers. Three soluble cancer protein biomarkers (soluble programmed death 1 (sPD-1), soluble programmed death-ligand 1 (sPD-L1) and soluble epithermal growth factor receptor (sEGFR)) were analysed directly from human serum [75]. In this study, for Raman signal improvement, gold-silver alloy nanoboxes were used as SERS tags to facilitate highly sensitive detection. The limit of detection for sPD-1, sPD-L1, and sEGFR achieved by this platform was 6.17 pg/mL, 0.68 pg/mL, and 69.86 pg/mL, respectively. They proposed that their nanobiosensor has huge potential in the clinic, because it can accurately and specifically detect several cancer biomarkers in human serum at once [75]. Another study demonstrated multiplex profiling of oestrogen receptor (ER), progesterone receptor (PR), and epidermal growth factor receptor (EGFR) expression in breast cancer tissue and normal tissue sections, using AuNP-based SERS tags [76]. 60 nm AuNPs with incorporated alkyne and nitrile groups and conjugated to primary antibodies against the growth factors were used as SERS tags. They demonstrated that their SERS nanotags have individual and definite bands in cellular regions without any Raman signal. The method was tested in vitro using the MCF-7 cell line, which expresses ER, EGFR, and PR at high levels and the normal cell line 3T3 in which expression of the biomarkers is downregulated. The results corresponded with data obtained with other methods. Additionally, the authors examined the use of multiple SERS tags for ER, EGFR, and PR imaging in human breast cancer tissue specimens. The results showed higher expression levels of these three biomarkers in breast cancer tissues compared to healthy samples, consistent with the diagnostic and prognostic classification of these tissues. Thus, the authors suggest that their proposed method has potential for multiplex imaging in clinical cancer diagnosis [76]. SERS is a reliable analytical method suitable for the detection of multiple analytes in biological samples even if there are extremely low quantities of target biomarker. By using SERS-nanotags, assays can be conducted directly in biopsy samples as well as in tissues or live cells, if necessary. However, utilisation of noble metal nanoparticles in multiplexed SERS biosensors is not yet cost effective and has limitations, such as high-cost fabrication of nanoparticles, low batch-to-batch consistency, high complexity, and relatively low specificity. Reproducible methodologies suitable for the mass production of testing kits based on gold or silver SERS nanotags also remain a challenge. Additionally, the majority of published multiplexed SERS imaging techniques require specific equipment, thus it would be complicated to adapt these methods for clinical needs. Despite these challenges, the field of multiplex SERS has great potential for innovation in diagnostic applications. 3.4. Plasmon-Enhanced Multiplexed Biosensing Plasmon-enhanced fluorescence (PEF) is a phenomenon by which the fluorescence intensity of a nearby fluorophore can be remarkably enhanced by a plasmonic nanostructure [77]. By utilising PEF, the quantum efficiency and photostability of fluorophores are increased. Additionally, extremely low amounts of fluorophore could be detected. Thus, these features enable sensitive detection for very low abundance biomarkers. For the past few years, PEF-based biosensors have received a great deal of attention for the ultrasensitive detection of single analytes. Ventura et al. used the surface of patterned AuNPs for FITC fluorescence enhancement to detect immunoglobulins in real urine samples and showed that their method works in a 10–100 μg/L detection range with a limit of detection of 8 μg/L [78]. Zhang et al. used an Au nanohole array for prostate-specific antigen detection and demonstrated a limit of detection of 140 fM [79]. Wang et al. demonstrated thrombin and platelet-derived growth factor-BB (PDGF-BB) simultaneous detection using aptamer-modified AgNPs as a capture substrate, and fluorescent dye-modified aptamers as detection probes in a sandwich immunoassay [80]. The linear range ofPDF Image | Multiplexed Nanobiosensors: Current Trends in Early Diagnostics
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