John X. J. Zhang

Ciliated micropillars for the microfluidic-based isolation of nanoscale lipid vesicles

We fabricated a microfluidic device consisting of ciliated micropillars, forming a porous silicon nanowire-on-micropillar structure. We demonstrated that the prototype device can preferentially trap exosome-like lipid vesicles, while simultaneously filtering out proteins and cell debris. Trapped lipid vesicles can be recovered intact by dissolving the porous nanowires in PBS buffer.

Versatile immunomagnetic nanocarrier platform for capturing cancer cells

Sensitive and quantitative assessment of changes in circulating tumor cells (CTCs) can help in cancer prognosis and in the evaluation of therapeutics efficacy. However, extremely low occurrence of CTCs in the peripheral blood (approximately one CTC per billion blood cells) and potential changes in molecular biomarkers during the process of epithelial to mesenchymal transition create technical hurdles to the enrichment and enumeration of CTCs. Recently, efforts have been directed toward development of antibody-capture assays based on the expression of the common biomarker-the epithelial cell adhesion molecule (EpCAM) of epithelium-derived cancer cells. Despite some promising results, the assays relying on EpCAM capture have shown inconsistent sensitivity in clinical settings and often fail to detect CTCs in patients with metastatic cancer. We have addressed this problem by the development of an assay based on hybrid magnetic/plasmonic nanocarriers and a microfluidic channel. In this assay, cancer cells are specifically targeted by antibody-conjugated magnetic nanocarriers and are separated from normal blood cells by a magnetic force in a microfluidic chamber. Subsequently, immunofluorescence staining is used to differentiate CTCs from normal blood cells. We demonstrated in cell models of colon, breast, and skin cancers that this platform can be easily adapted to a variety of biomarkers, targeting both surface receptor molecules and intracellular biomarkers of epithelial-derived cancer cells. Experiments in whole blood showed capture efficiency greater than 90% when two cancer biomarkers are used for cell capture. Thus, the combination of immunotargeted magnetic nanocarriers with microfluidics provides an important platform that can improve the effectiveness of current CTC assays by overcoming the problem of heterogeneity of tumor cells in the circulation.

Immunomagnetic nanoscreening of circulating tumor cells with a motion controlled microfluidic system

Combining the power of immunomagnetic assay and microfluidic microchip operations, we successfully detected rare CTCs from clinical blood samples. The microfluidic system is operated in a flip-flop mode, where a computer-controlled rotational holder with an array of microfluidic chips inverts the microchannels. We have demonstrated both theoretically and experimentally that the direction of red blood cell (RBC) sedimentation with regards to the magnetic force required for cell separation is important for capture efficiency, throughput, and purity. The flip-flop operation reduces the stagnation of RBCs and non-specific binding on the capture surface by alternating the direction of the magnetic field with respect to gravity. The developed immunomagnetic microchip-based screening system exhibits high capture rates (more than 90%) for SkBr3, PC3, and Colo205 cell lines in spiked screening experiments and successfully isolates CTCs from patient blood samples. The proposed motion controlled microchip-based immunomagnetic system shows great promise as a clinical tool for cancer diagnosis and prognosis.

Microscale Magnetic Field Modulation for Enhanced Capture and Distribution of Rare Circulating Tumor Cells

Immunomagnetic assay combines the powers of the magnetic separation and biomarker recognition and has been an effective tool to perform rare Circulating Tumor Cells detection. Key factors associated with immunomagnetic assay include the capture rate, which indicates the sensitivity of the system, and distributions of target cells after capture, which impact the cell integrity and other biological properties that are critical to downstream analyses. Here we present a theoretical framework and technical approach to implement a microscale magnetic immunoassay through modulating local magnetic field towards enhanced capture and distribution of rare cancer cells. Through the design of a two-dimensional micromagnet array, we characterize the magnetic field generation and quantify the impact of the micromagnets on rare cell separation. Good agreement is achieved between the theory and experiments using a human colon cancer cell line (COLO205) as the capture targets.

Enhanced microcontact printing of proteins on nanoporous silica surface

We demonstrate porous silica surface modification, combined with microcontact printing, as an effective method for enhanced protein patterning and adsorption on arbitrary surfaces. Compared to conventional chemical treatments, this approach offers scalability and long-term device stability without requiring complex chemical activation. Two chemical surface treatments using functionalization with the commonly used 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were compared with the nanoporous silica surface on the basis of protein adsorption. The deposited thickness and uniformity of porous silica films were evaluated for fluorescein isothiocyanate (FITC)-labeled rabbit immunoglobulin G (R-IgG) protein printed onto the substrates via patterned polydimethlysiloxane (PDMS) stamps. A more complete transfer of proteins was observed on porous silica substrates compared to chemically functionalized substrates. A comparison of different pore sizes (4-6 nm) and porous silica thicknesses (96-200 nm) indicates that porous silica with 4 nm diameter, 57% porosity and a thickness of 96 nm provided a suitable environment for complete transfer of R-IgG proteins. Both fluorescence microscopy and atomic force microscopy (AFM) were used for protein layer characterizations. A porous silica layer is biocompatible, providing a favorable transfer medium with minimal damage to the proteins. A patterned immunoassay microchip was developed to demonstrate the retained protein function after printing on nanoporous surfaces, which enables printable and robust immunoassay detection for point-of-care applications.

Screening and Molecular Analysis of Single Circulating Tumor Cells Using Micromagnet Array.

Immunomagnetic assay has been developed to detect rare circulating tumor cells (CTCs), which shows clinical significance in cancer diagnosis and prognosis. The generation and fine-tuning of the magnetic field play essential roles in such assay toward effective single-cell-based analyses of target cells. However, the current assay has a limited range of field gradient, potentially leading to aggregation of cells and nanoparticles. Consequently, quenching of the fluorescence signal and mechanical damage to the cells may occur, which lower the system sensitivity and specificity. We develop a micromagnet-integrated microfluidic system for enhanced CTC detection. The ferromagnetic micromagnets, after being magnetized, generate localized magnetic field up to 8-fold stronger than that without the micromagnets, and strengthen the interactions between CTCs and the magnetic field. The system is demonstrated with four cancer cell lines with over 97% capture rate, as well as with clinical samples from breast, prostate, lung, and colorectal cancer patients. The system captures target CTCs from patient blood samples on a standard glass slide that can be examined using the fluorescence in-situ hybridization method for the single-cell profiling. All cells showed clear hybridization signals, indicating the efficacy of the compact system in providing retrievable cells for molecular studies.

Aligned PVDF-TrFE Nanofibers With High-Density PVDF Nanofibers and PVDF Core–Shell Structures for Endovascular Pressure Sensing

Nanostructures of polyvinyledenedifluoride-tetrafluoroethylene (PVDF-TrFE), a semicrystalline polymer with high piezoelectricity, results in significant enhancement of crystallinity and better device performance as sensors, actuators, and energy harvesters. Using electrospinning of PVDF to manufacture nanofibers, we demonstrate a new method to pattern high-density, highly aligned nanofibers. To further boost the charge transfer from such a bundle of nanofibers, we fabricated novel core-shell structures. Finally, we developed pressure sensors utilizing these fiber structures for endovascular applications. The sensors were tested in vitro under simulated physiological conditions. We observed significant improvements using core-shell electrospun fibers (4.5 times gain in signal intensity, 4000 μV/mmHg sensitivity) over PVDF nanofibers (280 μV/mmHg). The preliminary results showed that core-shell fiber-based devices exhibit nearly 40-fold higher sensitivity, compared to the thin-film structures demonstrated earlier.

PVDF-Nafion nanomembranes coated microneedles for in vivo transcutaneous implantable glucose sensing

We demonstrate that microporous PVDF membranes sandwiched between multiple layers of nanomaterials can be used for continuous monitoring of glucose level in vivo. This is achieved by coating needle electrodes with Polyaniline nanofiber, Platinum nanoparticles, glucose oxidase enzyme and porous layers, successfully fabricated with layer-by-layer deposition. Nanoparticles incorporated into conductive Polyaniline nanofibers resulted in high surface to volume ratio and electrocatalytic activity for glucose enzyme. A composite coating membrane of porous PVDF and nano-sphere Nafion limited the glucose transportation and increased the lifetime of in vivo measurements. The glucose biosensor exhibited a sub-microamperometric output current, fast response time of less than 30s and a sensitivity of 0.23 μA/mM. The linear sensing range in terms of glucose concentration was from 0 to 20mM. Implantable experiments using mice models showed excellent response to the variation of blood glucose concentration while maintaining biocompatibility with the surrounding tissues. The sensitivity was shown to remain within 10% close to initial sensitivity within the 7 days of continuous monitoring, and maintain at 70% of the initial sensitivity within 21 days.

Efficient apertureless scanning probes using patterned plasmonic surfaces

We present a novel concept to design apertureless plasmonic probes for near-field scanning optical microscopy (NSOM) with enhanced optical power throughput and near-field enhancement. Specifically, we combine unidirectional surface plasmon polariton (SPP) generation along the tip lateral walls with nanofocusing of SPPs through adiabatic propagation towards an apertureless tip. Three key design parameters are considered: the nanoslit width, the pitch period of nanogrooves for unidirectional plasmonic excitation and the pyramidal geometry of the NSOM probe for SPP focusing. Optimal design parameters are obtained with 2D analysis and two realistic probe geometries with patterned plasmonic surfaces are proposed using the optimized designs. The electromagnetic properties of the designed probes are characterized in the near-field and compared to those of a conventional single-aperture probe with same pyramidal shape. The optimized probes feature FWHM around 150nm, comparable with conventional NSOM designs, but over 3 orders of magnitude larger field enhancement, without degrading its spatial resolution. Our ideas effectively combine the resolution of apertureless probes with throughput levels much larger than those available even in aperture-based devices.

Handheld Diffuse Reflectance Spectral Imaging (DRSi) for in-vivo characterization of skin.

Diffuse reflectance spectroscopy provides a noninvasive means to measure optical and physiological properties of tissues. To expand on these measurements, we have developed a handheld diffuse reflectance spectral imaging (DRSi) system capable of acquiring wide field hyperspectral images of tissue. The image acquisition time was approximately 50 seconds for a 50x50 pixel image. A transport model was used to fit each spectra for reduced scattering coefficient, hemoglobin concentration and melanin concentration resulting in optical property maps. The system was validated across biologically relevant levels of reduced scattering (5.14% error) and absorption (8.34% error) using tissue simulating phantoms. DRSi optical property maps of a pigmented skin lesion were acquired in vivo. These trends in optical properties were consistent with previous observations using point probe devices.