Such efforts have started to unveiling tumor-associated exosomal fingerprints, particularly in RNA profiles (both coding and noncoding). Exosomal protein analysis, on the other hand, still remains challenging. inside a 200 nm-thick Au film on a glass substrate. We laid out a 12 3 array of sensing devices with multi-channel microfluidics placed on top (Number 6b). Each channel spanned over three sensing devices for triplicate measurements. The sample volume per sensing unit was ~1 L. For parallel measurements of nPLEX arrays, an intensity-based detection system integrated with miniaturized optics consisting of a laser diode and an image sensor was also developed (Number 6c). This system can simultaneously monitor changes in the transmitted light intensities of 36 arrays for high-throughput parallel measurements. Open in a separate windowpane Fig. 6 First nPLEX prototype(a) A photograph of the nanohole device integrated with microfluidics. A 12-channel fluidic cell was placed on top of a glass slide comprising nanohole arrays. (b) A total of 36 measurement sites were arranged into a 12 3 array. Each measurement site had periodic nanoholes (right). The structure was patterned inside a gold film (200 nm solid) deposited on a glass substrate. (c) A photograph of the miniaturized nPLEX imaging system. The nPLEX chip was located directly on an image sensor, which measured transmitted light intensities of the 36 sites simultaneously. Reproduced from Im H, Shao H, Park YI et al. Label-free detection and molecular profiling of exosomes having a nano-plasmonic sensor. Nat Biotechnol 2014;32:490C495 with permission from Nature Publishing Group, copyright 2014 [18]. Analytical nPLEX assay for molecular profiling To impart molecular specificity, the nanohole surface was coated with different antibodies in each channel. Following antibody conjugation, exosomes were launched and spectral shifts are measured before and after exosome binding. An IgG control channel was integrated to measure the contribution from non-specific binding and its transmission was subtracted from each target channel. To determine the detection level of sensitivity, we functionalized the sensor surface with antibodies against CD63, a type III lysosomal membrane protein enriched in exosomes. On the (R)-MIK665 other hand, additional exosome-specific lysosomal membrane proteins (e.g. CD9, CD81) were also used[49]. Samples were prepared from CaOV3 (human being ovarian carcinoma) cell lines, and their initial exosome concentrations were estimated by nanoparticle-tracking analysis (NTA). A pair of nPLEX detectors, functionalized with CD63 and control IgG antibodies respectively, were used to measure the relative spectral (CD63) or intensity (nPLEX screening showed good agreement of protein manifestation between exosomes and their parental cells across different ovarian malignancy cell lines (Number 8a). Such close coordinating of molecular profiles between exosome and cells was previously recognized in glioblastoma multiforme (GBM) cell lines using NMR[17]. In addition, our nPLEX screening showed that EpCAM and CD24 were highly indicated in tested ovarian malignancy cell lines. Open in a separate windowpane Fig. 8 Molecular profiling of ovarian malignancy exosomes(a) study. Ovarian cancer connected markers (EpCAM, CD24, CA-125, CA19-9, HER2, MUC18, EGFR, Claudin3), immune sponsor cell markers (CD41, CD45) and a mesothelial marker (D2-40) were profiled on both parental ovarian cells (right, using circulation cytometry) and their derived exosomes (R)-MIK665 (remaining, using nPLEX sensor). Exosomal protein profiles showed an excellent match with those of originating cells. A two-marker combination comprising EpCAM and CD24 could efficiently distinguish tumor exosomes from benign exosomes. MFI, mean fluorescence intensity. (b) Ascites exosomes from ovarian malignancy and non-cancer individuals were evaluated from the nPLEX sensor. Malignancy exosomes were captured on EpCAM and CD24-specific sensor sites, (R)-MIK665 and the exosomal manifestation levels of these markers were measured. Ovarian cancer patients (= 20) were associated with elevated EpCAM and CD24 expression, while non-cancer patients (= 10) showed negligible signals. (c) Longitudinal nPLEX assays. Ascites samples were collected sequentially from ovarian cancer patients undergoing chemotherapy (= 8). Measuring temporal changes in exosomal expressions of EpCAM and Rabbit Polyclonal to 4E-BP1 CD24 could distinguish treatment response. Reproduced from Im H, Shao (R)-MIK665 H, Park YI et al. Label-free detection and molecular profiling of exosomes with a nano-plasmonic sensor. Nat Biotechnol 2014;32:490C495 with permission from Nature Publishing Group, copyright 2014 [18]. Based on these results, the nPLEX system was applied to detect ovarian cancer exosomes in patient-derived ascites (Physique 8b). Thirty ascites samples were obtained: 20 patients were diagnosed with Stage 3 (= 10) and 4 (= 10) ovarian cancer and 10 control ascites patients were diagnosed with liver cirrhosis[18]. The study.