Chia-Fu Chou

Electrodeless Dielectrophoresis of Single- and Double-Stranded DNA

Dielectrophoretic trapping of molecules is typically carried out using metal electrodes to provide high field gradients. In this paper we demonstrate dielectrophoretic trapping using insulating constrictions at far lower frequencies than are feasible with metallic trapping structures because of water electrolysis. We demonstrate that electrodeless dielectrophoresis (EDEP) can be used for concentration and patterning of both single-strand and double-strand DNA. A possible mechanism for DNA polarization in ionic solution is discussed based on the frequency, viscosity, and field dependence of the observed trapping force.

Electrokinetic Preconcentration and Detection of Neuropeptides at Patterned Graphene-Modified Electrodes in a Nanochannel

Neuropeptides are vital to the transmission and modulation of neurological signals, with Neuropeptide Y (NPY) and Orexin A (OXA) offering diagnostic information on stress, depression, and neurotrauma. NPY is an especially significant biomarker, since it can be noninvasively collected from sweat, but its detection has been limited by poor sensitivity, long assay times, and the inability to scale-down sample volumes. Herein, we apply electrokinetic preconcentration of the neuropeptide onto patterned graphene-modified electrodes in a nanochannel by frequency-selective dielectrophoresis for 10 s or by electrochemical adsorptive accumulation for 300 s, to enable the electrochemical detection of NPY and OXA at picomolar levels from subnanoliter samples, with sufficient signal sensitivity to avoid interferences from high levels of dopamine and ascorbic acid within biological matrices. Given the high sensitivity of the methodology within small volume samples, we envision its utility toward off-line detection from droplets collected by microdialysis for the eventual measurement of neuropeptides at high spatial and temporal resolutions.

Electrodeless dielectrophoresis for micro total analysis systems

Several examples where a sample-to-answer, lab-on-a-chip chemical analysis system may benefit from EDEP technology in upstream sample preparation (cell sorting, cell lysing, DNA concentration, and purification) and in a back-end detection platform for hybridization and sensitivity enhancement were presented. The simplicity of the device and the lack of metallic electrodes at the trap, which cause electrochemical reactions involving gas evolution, made possible the investigation of the response of biological objects in a wide range of frequencies, especially the low-frequency regime. Above all, EDEP may be seamlessly integrated with the metallic DEP layout, this increasing the flexibility in custom-tailored chip design.

Thermal management of BioMEMS: temperature control for ceramic-based PCR and DNA detection devices

Integrated microfluidic devices for amplification and detection of biological samples that employ closed-loop temperature monitoring and control have been demonstrated within a multilayer low temperature co-fired ceramics (LTCC) platform. Devices designed within this platform demonstrate a high level of integration including integrated microfluidic channels, thick-film screen-printed Ag-Pd heaters, surface mounted temperature sensors, and air-gaps for thermal isolation. In addition, thermal-fluidic finite element models have been developed using CFDRC ACE+ software which allows for optimization of such parameters as heater input power, fluid flow rate, sensor placement, and air-gap size and placement. Two examples of devices that make use of these concepts are provided. The first is a continuous flow polymerase chain reaction (PCR) device that requires three thermally isolated zones of 94/spl deg/C, 65/spl deg/C, and 72/spl deg/C, and the second is an electronic DNA detection chip which requires hybridization at 35/spl deg/C. Both devices contain integrated heaters and surface mount silicon transistors which function as temperature sensors. Closed loop feedback control is provided by an external PI controller that monitors the temperature dependant I-V relationship of the sensor and adjusts heater power accordingly. Experimental data confirms that better than /spl plusmn/0.5/spl deg/C can be maintained for these devices irrespective of changing ambient conditions. In addition, good matching with model predictions has been achieved, thus providing a powerful design tool for thermal-fluidic microsystems.

Surface modification of nanofibrous polycaprolactone/gelatin composite scaffold by collagen type I grafting for skin tissue engineering

In the present study, a tri-polymer polycaprolactone (PCL)/gelatin/collagen type I composite nanofibrous scaffold has been fabricated by electrospinning for skin tissue engineering and wound healing applications. Firstly, PCL/gelatin nanofibrous scaffold was fabricated by electrospinning using a low cost solvent mixture [chloroform/methanol for PCL and acetic acid (80% v/v) for gelatin], and then the nanofibrous PCL/gelatin scaffold was modified by collagen type I (0.2-1.5wt.%) grafting. Morphology of the collagen type I-modified PCL/gelatin composite scaffold that was analyzed by field emission scanning electron microscopy (FE-SEM), showed that the fiber diameter was increased and pore size was decreased by increasing the concentration of collagen type I. Fourier transform infrared (FT-IR) spectroscopy and thermogravimetric (TG) analysis indicated the surface modification of PCL/gelatin scaffold by collagen type I immobilization on the surface of the scaffold. MTT assay demonstrated the viability and high proliferation rate of L929 mouse fibroblast cells on the collagen type I-modified composite scaffold. FE-SEM analysis of cell-scaffold construct illustrated the cell adhesion of L929 mouse fibroblasts on the surface of scaffold. Characteristic cell morphology of L929 was also observed on the nanofiber mesh of the collagen type I-modified scaffold. Above results suggest that the collagen type I-modified PCL/gelatin scaffold was successful in maintaining characteristic shape of fibroblasts, besides good cell proliferation. Therefore, the fibroblast seeded PCL/gelatin/collagen type I composite nanofibrous scaffold might be a potential candidate for wound healing and skin tissue engineering applications.

A miniaturized cyclic PCR device—modeling and experiments

Abstract With the aid of thermal and fluidic modeling using CFDRC ACE+™, we designed and fabricated the first miniaturized cyclic polymerase chain reaction (PCR) device in low-temperature cofired ceramics. The device comprises of a serpentine channel with different cross-sectional areas in different reactor zones to provide adequate residence time for the melting, annealing, and extension reaction to take place. This is in contrary to the thermal cycling in the batch PCR system. With a flow rate of 15 μl/min, the designed time to complete 30 PCR cycles is less than 40 min, given the total volume of the device 19 μl, provided an internal pump may be implemented to reduce the dead volume. We have demonstrated DNA amplification in this device, using an external peristaltic pump, and the PCR product was used with a DNA bioelectronic sensor chip (Motorola e-Sensors™) for genotyping experiment.

Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device.

Monitoring the periodic diurnal variations in cortisol from small volume samples of serum or saliva is of great interest, due to the regulatory role of cortisol within various physiological functions and stress symptoms. Current detection assays are immunologically based and require cumbersome antibody immobilization chemistries, thereby limiting the assay versatility, kinetics, and reproducibility. We present a quantitative aptamer-based detection methodology for cortisol that does not require target labeling, capture probe immobilization on the detection surface or wash steps prior to readout. Using a recognition system of aptamer functionalized gold nanoparticles pre-bound with electro-active triamcinolone, the cortisol level is detected based on its competitive binding to the aptamer by following signal from the displaced triamcinolone using square wave voltammetry at patterned graphene-modified electrodes in a microfluidic or nanoslit device. Due to the 3D analyte diffusion profile at the aptamer interface and the ability to enhance the surface area for cortisol capture, this assay shows signal linearity over a five-log analyte concentration range (10 μg/mL to 30 pg/mL) and exhibits rapid binding kinetics with cortisol versus other glucocorticoids, as apparent from the absence of interferences from estradiol, testosterone and progesterone. The assay is carried out within the biologically relevant range for glucocorticoids in serum and saliva matrices, and benchmarked versus ELISA and radioimmunoassays. Based on absence of cumbersome surface immobilization and wash steps for carrying out this assay, its quantitative signal characteristics and its ability to resist interferences from other glucocorticoids, we envision its application towards routine monitoring of cortisol within bio-fluids.

Nanoscale molecular traps and dams for ultrafast protein enrichment in high-conductivity buffers.

We report a new approach, molecular dam, to enhance mass transport for protein enrichment in nanofluidic channels by nanoscale electrodeless dielectrophoresis under physiological buffer conditions. Dielectric nanoconstrictions down to 30 nm embedded in nanofluidic devices serve as field-focusing lenses capable of magnifying the applied field to 10(5)-fold when combined with a micro- to nanofluidic step interface. With this strong field and the associated field gradient at the nanoconstrictions, proteins are enriched by the molecular damming effect faster than the trapping effect, to >10(5)-fold in 20 s, orders of magnitude faster than most reported methods. Our study opens further possibilities of using nanoscale molecular dams in miniaturized sensing platforms for rapid and sensitive protein analysis and biomarker discovery, with potential applications in precipitation studies and protein crystallization and possible extensions to small-molecules enrichment or screening.

Entropy-Driven Single Molecule Tug-of-War of DNA at Micro−Nanofluidic Interfaces

Entropy-driven polymer dynamics at the nanoscale is fundamentally important in biological systems but the dependence of the entropic force on the nanoconfinement remains elusive. Here, we established an entropy-driven single molecule tug-of-war (TOW) at two micro-nanofluidic interfaces bridged by a nanoslit, performed the force analysis from a modified wormlike chain in the TOW scenario and the entropic recoiling process, and determined the associated scalings on the nanoconfinement. Our results provide a direct experimental evidence that the entropic forces in these two regimes, though unequal, are essentially constant at defined slit heights, irrespective of the slit lengths and the DNA segments within. Our findings have the implications to polymer transport at the nanoscale, device design for single molecule analysis, and biotechnological applications.

Fabrication and characterization of novel nano-biocomposite scaffold of chitosan-gelatin-alginate-hydroxyapatite for bone tissue engineering.

A novel nano-biocomposite scaffold was fabricated in bead form by applying simple foaming method, using a combination of natural polymers-chitosan, gelatin, alginate and a bioceramic-nano-hydroxyapatite (nHAp). This approach of combining nHAp with natural polymers to fabricate the composite scaffold, can provide good mechanical strength and biological property mimicking natural bone. Environmental scanning electron microscopy (ESEM) images of the nano-biocomposite scaffold revealed the presence of interconnected pores, mostly spread over the whole surface of the scaffold. The nHAp particulates have covered the surface of the composite matrix and made the surface of the scaffold rougher. The scaffold has a porosity of 82% with a mean pore size of 112±19.0μm. Swelling and degradation studies of the scaffold showed that the scaffold possesses excellent properties of hydrophilicity and biodegradability. Short term mechanical testing of the scaffold does not reveal any rupturing after agitation under physiological conditions, which is an indicative of good mechanical stability of the scaffold. In vitro cell culture studies by seeding osteoblast cells over the composite scaffold showed good cell viability, proliferation rate, adhesion and maintenance of osteoblastic phenotype as indicated by MTT assay, ESEM of cell-scaffold construct, histological staining and gene expression studies, respectively. Thus, it could be stated that the nano-biocomposite scaffold of chitosan-gelatin-alginate-nHAp has the paramount importance for applications in bone tissue-engineering in future regenerative therapies.