Ultrasensitive Detection of Cancer Prognostic miRNA Biomarkers Based on Surface Plasmon Enhanced Light Scattering

The development of simple yet ultrasensitive biosensing approaches for the detection of cancer prognostic microRNA is an important step toward their successful clinical implementation. We demonstrate the relevance for the detection of circulating miRNA of a novel signal amplification scheme based on surface plasmon resonance enhanced light scattering (SP-LS). In addition to experimental optimization carried out using gold nanoparticle (AuNP) tags conjugated with a monoclonal antibody with high affinity for RNA*DNA hybrid duplexes, simulation modeling was conducted to obtain insights about SP-LS biosensing. SP-LS enabled the detection of miRNA-122 at subpicomolar concentrations within 30 min, and a limit of detection of 2 attomoles (60 fM, 50 μL) was determined. MiRNA-122 could also be reliably detected in a high concentration background of nontarget miRNA. The proposed SP-LS miRNA detection approach could be readily applied to other miRNA targets of diagnostic importance and further developed to allow for multiplex measurements of miRNA panels. The promising results obtained in this study and advantageous features of SP-LS warrant further development and its application to clinical samples

Robust and Flexible Fabrication of Chemical Micropatterns for Tumor Spheroid Preparation

A robust and flexible approach is described for the straightforward preparation of multicellular tumor spheroids of controllable dimensions. The approach is based on a one-step plasma polymerization of the monomer allylamine carried out through conformal micropatterning physical masks that is used to deposit amine-rich (PolyAA) micrometer-scale features that promote cellular attachment and initiate the formation of multicellular spheroids. A simple backfilling step of the nonpolymerized poly­(dimethylsiloxane) background with Pluronic F127 significantly reduced background cellular adhesion on the untreated substrate and, in turn, improved the quality of the spheroid formed. Tumor cells grown on the PolyAA/F127 patterned surfaces reliably formed multicellular spheroids within 24–48 h depending on the cell type. The dimension of the spheroids could be readily controlled by the dimension of the amine-rich micropatterns. This simple approach is compatible with the long-term culture of multicellular spheroids and their characterization with high-resolution optical microscopy. These features facilitate the development of on-chip assays, as demonstrated here for the study of the binding of transferrin-functionalized gold nanoparticles to multicellular tumor spheroids

Cellular Micromotion Monitored by Long-Range Surface Plasmon Resonance with Optical Fluctuation Analysis

Long-range surface plasmon resonance (LRSPR) is a powerful biosensing technology due to a substantially larger probing depth into the medium and sensitivity, compared with conventional SPR. We demonstrate here that LRSPR can provide sensitive noninvasive measurement of the dynamic fluctuation of adherent cells, often referred to as the cellular micromotion. Proof of concept was achieved using confluent layers of 3T3 fibroblast cells and MDA-MB-231 cancer cells. The slope of the power spectral density (PSD) of the optical fluctuations was calculated to determine the micromotion index, and significant differences were measured between live and fixed cell layers. Furthermore, the performances of LRSPR and conventional surface plasmon resonance (cSPR) were compared with respect to micromotion monitoring. Our study showed that the micromotion index of cells measured by LRSPR sensors was higher than when measured with cSPR, suggesting a higher sensitivity of LRSPR to the micromotion of cells. To investigate further this finding, simulations were conducted to establish the relative sensitivities of LRSPR and cSPR to membrane fluctuations. Increased signal intensity was predicted for LRSPR in comparison to cSPR, suggesting that membrane fluctuations play a significant role in the optical micromotion measured in LRSPR. Analogous to cellular micromotion measured using impedance techniques, LRSPR micromotion has the potential to provide important biological information on the metabolic activity and viability of adherent cells

Exploiting Surface-Plasmon-Enhanced Light Scattering for the Design of Ultrasensitive Biosensing Modality

Development of new detection methodologies and amplification schemes is indispensable for plasmonic biosensors to improve the sensitivity for the detection of trace amounts of analytes. Herein, an ultrasensitive scheme for signal enhancement based on the concept of surface-plasmon-resonance-enhanced light scattering (SP-LS) was validated experimentally and theoretically. The SP-LS of gold nanoparticles’ (AuNPs) tags was employed in a sandwich assay for the detection of cardiac troponin I and provided up to 2 orders of magnitude improved sensitivity over conventional AuNPs-enhanced refractometric measurements and 3 orders of magnitude improvement over label-free SPR. Simulations were also performed to provide insights into the physical mechanisms

Sensitive and Specific Biomimetic Lipid Coated Microfluidics to Isolate Viable Circulating Tumor Cells and Microemboli for Cancer Detection

<div><p>Here we presented a simple and effective membrane mimetic microfluidic device with antibody conjugated supported lipid bilayer (SLB) “smart coating” to capture viable circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) directly from whole blood of all stage clinical cancer patients. The non-covalently bound SLB was able to promote dynamic clustering of lipid-tethered antibodies to CTC antigens and minimized non-specific blood cells retention through its non-fouling nature. A gentle flow further flushed away loosely-bound blood cells to achieve high purity of CTCs, and a stream of air foam injected disintegrate the SLB assemblies to release intact and viable CTCs from the chip. Human blood spiked cancer cell line test showed the ~95% overall efficiency to recover both CTCs and CTMs. Live/dead assay showed that at least 86% of recovered cells maintain viability. By using 2 mL of peripheral blood, the CTCs and CTMs counts of 63 healthy and colorectal cancer donors were positively correlated with the cancer progression. In summary, a simple and effective strategy utilizing biomimetic principle was developed to retrieve viable CTCs for enumeration, molecular analysis, as well as <i>ex vivo</i> culture over weeks. Due to the high sensitivity and specificity, it is the first time to show the high detection rates and quantity of CTCs in non-metastatic cancer patients. This work offers the values in both early cancer detection and prognosis of CTC and provides an accurate non-invasive strategy for routine clinical investigation on CTCs.</p></div

Overview of the CMx platform.

<p>(A) Strategies to capture, purify and release CTCs of the CMx platform. In the top row, blood flows through a microfluidic channel coated with anti-EpCAM conjugated to NeutrAvidin which is adhered to a SLB layer on substrate. Selective binding of CTCs to anti-EpCAM is reinforce by the antibody clustering effects through the mobility of the fluidic SLB layer, while other blood cells are easily flush away from the fluidic surface. The bottom row showed the release process, in which introduced air bubbles disrupt the weakest links between substrate and the SLB layer allowing elute intact CTCs for the collection outside of microfluidic chip. (B) Overview of CMx platform and summary of the CMx platform workflow. About 2 mL of the whole blood samples obtained freshly from the CRC patients were loaded equally into each CTC capturing devices. All chips went through cell capture, purification, and released for various downstream applications, including immunofluorescence staining, cell counting, molecular analysis, <i>ex vivo</i> cell culture and/or cryobanking.</p

Release of captured cells by air foams from CMx platform.

<p>(A) The florescent images of the HCT116 cancer cell released from the Texas Red-SLB coated chip. The cell was wrapped with Texas Red-SLB lipid molecules around the membrane (blue: DAPI; green: pre-stained CellTracker Green CMFDA; red: Texas Red conjugated lipid molecule). (B and C) Overlaid fluorescent images of the released single CTC and CTM for cell characterization. Eluted cells from clinical samples were categorized as CTC by size, morphology and immunostainning (DAPI+/CK20+/CD45-). WBCs were identified by DAPI+/CK20-/CD45+ immunostaining (blue: DAPI; green: CD45; red: CK20).</p

Enumeration and correlation of single CTCs and cluster CTCs with disease progression.

<p>(A) Correlation and statistical analysis of single CTC enumeration and clinical stage of CRC patients and healthy donors. (B) Correlation and statistical analysis of CTM enumeration and clinical stage of CRC patients and healthy donors. Correlation between (C) CTCs and (D) CTM and lymphnode metastasis of CRC patients were shown with mean±SEM. (* <i>p</i> < 0.05, ** <i>p</i> < 0.01)</p

Cultivation of viable eluted CTCs.

<p>Capture and release of HCT116 cancer cell from CMx platform for further cultivation under (A) complete DMEM medium, (B) SPH culture medium, (C) suspension culture with SPH medium. (D) CTCs isolated from a stage III CRC patient. The cells gathered and attached to the bottom of culture plate at day 7 and became more spread out at day 9. Cells exhibited CK20+ (red) and nuclei+ (blue) with a size around 25μm. To prove these cells have a propensity to re-attach, they were treated with 0.1% trypsin and re-seeded at day 9. After 1 day, these cells re-attached to the substrate firmly (day 10). (E and F) Immunocytochemistry stain for the confirmation of colorectal origin of patient-derived CTC primary cultured cells. The skin-derived fibroblast cell line HS68 was used as negative staining control (blue: DAPI; red: CK20; green: α-SMA or FGFR).</p

Capture performance and purification by Ab-SLB coated microfluidics.

<p>(A) The geometry and patterns of 6 different microfluidic channel designs (left) and the capture efficiency of these microfluidic platforms (right) as defined by dividing captured cells over total spiked cells. (B) The cropped fluorescent images (5.5 mm x 5.5mm) inside the flow channel and the enumeration of HCT116 (green) and WBCs (blue) on Ab-SLB or Ab-silane coated Type E chips. (C) Cell detachment efficiency (%, Y-Axis) vs. flow rates (ml/h, lower X-axis) and the corresponding shear stress (upper X-axis). Flow rates are generally maintained below 4 ml/h to avoid any potential loss of captured CTCs. (D) Highly CTM capture and recovery efficiency of the Ab-SLB coated chip. The capture efficiency and recovery rate of HCT116-RFP generated tumor microemboli were showed in right panel. The released HCT116-RFP CTC clusters with DAPI staining were showed in left panel. *: <i>p</i> < 0.05; **: <i>p</i> < 0.01.</p