I-Fang Cheng

Rapid (<5 min) identification of pathogen in human blood by electrokinetic concentration and surface-enhanced Raman spectroscopy.

This study reports a novel microfluidic platform for rapid and long-ranged concentration of rare-pathogen from human blood for subsequent on-chip surface-enhanced Raman spectroscopy (SERS) identification/discrimination of bacteria based on their detected fingerprints. Using a hybrid electrokinetic mechanism, bacteria can be concentrated at the stagnation area on the SERS-active roughened electrode, while blood cells were excluded away from this region at the center of concentric circular electrodes. This electrokinetic approach performs isolation and concentration of bacteria in about three minutes; the density factor is increased approximately a thousand fold in a local area of ~5000 μm2 from a low bacteria concentration of 5 × 103 CFU/ml. Besides, three genera of bacteria, S. aureus, E. coli, and P. aeruginosa that are found in most of the isolated infections in bacteremia were successfully identified in less than one minute on-chip without the use of any antibody/chemical immobilization and reaction processes.

A dielectrophoretic chip with a roughened metal surface for on-chip surface-enhanced Raman scattering analysis of bacteria

We present an analysis of the results of in situ surface-enhanced Raman scattering (SERS) of bacteria using a microfluidic chip capable of continuously sorting and concentrating bacteria via three-dimensional dielectrophoresis (DEP). Microchannels were made by sandwiching DEP microelectrodes between two glass slides. Avoiding the use of a metal nanoparticle suspension, a roughened metal surface is integrated into the DEP-based microfluidic chip for on-chip SERS detection of bacteria. On the upper surface of the slide, a roughened metal shelter was settled in front of the DEP concentrator to enhance Raman scattering. Similarly, an electrode-patterned bottom layer fabricated on a thin cover-slip was used to reduce fluorescence noise from the glass substrate. Gram positive (Staphylococcus aureus) and Gram negative (Pseudomonas aeruginosa) bacteria were effectively distinguished in the SERS spectral data. Staphylococcus aureus (concentration of 10(6) CFUml) was continuously separated and concentrated via DEP out of a sample of blood cells. At a flow rate of 1 mulmin, the bacteria were highly concentrated at the roughened surface and ready for on-chip SERS analysis within 3 min. The SERS data were successfully amplified by one order of magnitude and analyzed within a few minutes, resulting in the detection of signature peaks of the respective bacteria.

Screening of Antibiotic Susceptibility to β-Lactam-Induced Elongation of Gram-Negative Bacteria Based on Dielectrophoresis

We demonstrate a rapid antibiotic susceptibility test (AST) based on the changes in dielectrophoretic (DEP) behaviors related to the β-lactam-induced elongation of Gram-negative bacteria (GNB) on a quadruple electrode array (QEA). The minimum inhibitory concentration (MIC) can be determined within 2 h by observing the changes in the positive-DEP frequency (pdf) and cell length of GNB under the cefazolin (CEZ) treatment. Escherichia coli and Klebsiella pneumoniae and the CEZ are used as the sample bacteria and antibiotic respectively. The bacteria became filamentous due to the inhibition of cell wall synthesis and cell division and cell lysis occurred for the higher antibiotic dose. According to the results, the pdfs of wild type bacteria decrease to hundreds of kHz and the cell length is more than 10 μm when the bacterial growth is inhibited by the CEZ treatment. In addition, the growth of wild type bacteria and drug resistant bacteria differ significantly. There is an obvious decrease in the number of wild type bacteria but not in the number of drug resistant bacteria. Thus, the drug resistance of GNB to β-lactam antibiotics can be rapidly assessed. Furthermore, the MIC determined using dielectrophoresis-based AST (d-AST) was consistent with the results of the broth dilution method. Utilizing this approach could reduce the time needed for bacteria growth from days to hours, help physicians to administer appropriate antibiotic dosages, and reduce the possibility of the occurrence of multidrug resistant (MDR) bacteria.

A rapid electrochemical biosensor based on an AC electrokinetics enhanced immuno-reaction

Fluorescent labelling and chromogenic reactions that are commonly used in conventional immunoassays typically utilize diffusion dominated transport of analytes, which is limited by slow reaction rates and long detection times. By integrating alternating current (AC) electrokinetics and electrochemical impedance spectroscopy (EIS), we construct an immunochip for rapid, sensitive, and label-free detection. AC electroosmosis (ACEO) and positive dielectrophoresis (DEP), induced by a biased AC electric field, can rapidly convect and trap the analyte onto an EIS working electrode within a few minutes. This allows the change of electron-transfer resistance (ΔRet) caused by the antibody-antigen (IgG-protein A) binding to be measured and quantified in real time. The measured impedance change achieves a plateau after electrokinetic concentration for only 90 s, and the detection limit is able to reach 200 pg ml⁻¹. Compared to the conventional incubation method, the electrokinetics-enhanced method is approximately 100 times faster in its reaction time, and the detection limit is reduced by 30 times. The ΔRet of the positive response is two orders of magnitude higher than the negative control, demonstrating excellent specificity for practical applications.

Dielectrophoresis and shear-enhanced sensitivity and selectivity of DNA hybridization for the rapid discrimination of Candida species.

We present a dielectrophoresis (DEP)-based microfluidic chip that is capable of enhancing the sensitivity and selectivity of DNA hybridization using an AC electric field and hydrodynamic shear in a continuous through-flow. Molecular DEP was employed to rapidly trap ssDNA molecules in a flowing solution to a cusp-shaped nanocolloid assembly on a microfluidic chip with a locally amplified AC electric field gradient. The detection time can be accelerated to sub-minute periods, and the sensitivity can reach the pico-molar level due to the AC DEP-enhanced molecule concentration (at an optimal AC frequency of 900 kHz) in a small region (∼100 μm(2)) instead of the broad area used in a tank reactor (∼10(6) μm(2)). Continuous flow in a microchannel provides a constant and high shear rate that can shear off most non-specific target-probe binding to promote the discriminating selectivity. On-chip multi-target discrimination of Candida species can be achieved within a few minutes under optimal conditions.

A bead-based immunofluorescence-assay on a microfluidic dielectrophoresis platform for rapid dengue virus detection

The proof of concept of utilizing a microfluidic dielectrophoresis (DEP) chip was conducted to rapidly detect a dengue virus (DENV) in vitro based on the fluorescence immunosensing. The mechanism of detection was that the DEP force was employed to capture the modified beads (mouse anti-flavivirus monoclonal antibody-coated beads) in the microfluidic chip and the DENV modified with fluorescence label, as the detection target, can be then captured on the modified beads by immunoreaction. The fluorescent signal was then obtained through fluorescence microscopy, and then quantified by ImageJ freeware. The platform can accelerate an immuno-reaction time, in which the on-chip detection time was 5min, and demonstrating an ability for DENV detection as low as 104 PFU/mL. Furthermore, the required volume of DENV samples dramatically reduced, from the commonly used ~50µL to ~15µL, and the chip was reusable (>50x). Overall, this platform provides a rapid detection (5min) of the DENV with a low sample volume, compared to conventional methods. This proof of concept with regard to a microfluidic dielectrophoresis chip thus shows the potential of immunofluorescence based-assay applications to meet diagnostic needs.

Ripple structure-generated hybrid electrokinetics for on-chip mixing and separating of functionalized beads

We present an electrokinetics-based microfluidic platform that is capable of on-chip manipulating, mixing, and separating microparticles through adjusting the interrelated magnitudes of dielectrophoresis and AC electroosmosis. Hybrid electrokinetic phenomenon is generated from an electric field-induced micro-ripple structure made of ultraviolet-curable glue. Size-dependent particle separation and selective removal over the ripple structure is demonstrated successfully. Varying the waveform from sine-wave to square-wave allows generating a fluid convection at specific positions to mix the antibody-functionalized beads and antigen. Potential application in the bead-based immunoassay was also demonstrated for immuno-reaction and subsequently separating the bead-bead aggregate and non-binding beads on-chip.

Manipulation of Bioparticles on Electrodeless Dielectrophoretic Chip Based on AC Electrokinetic Control

An electrodeless dielectrophoretic (EDEP) chip was designed and applied to separate the micro-particles in different sizes, and the ability of bio-separation of real samples also be examined. The dielectrophoretic (DEP) force can be principally created and controlled by provide a non-uniform electric field that is geometrically constricted by the insulator-patterned chip in combination with an alternative current (AC) electric field at frequency 10 kHz and 500 Vp-p set on the two sides of channel inlet. The EDEP chip was used to separate the E. coli and red blood cells (RBC), which from human whole blood sample, via well control of DEP force. Our results showed the bacteria and RBC can be separated into the higher and lower electric field regions of the EDEP chip in few seconds, respectively. A rapid, useful diagnosis tool, based on the EDEP method could be applied and used in the various fields of the bio-industry technology, the detection and the identification of clinical infections.

Development and application of dielectrophoretic chip for rapid detection of food bacteria

The purpose of our research is to develop a novel analysis technique. It is used for food safety control system and able to rapid separation, identification and quantification of microorganism. A three dimension dielectrophoresis (DEP) chip is developed with rapid separation and detection functions. Due to the low bacteria concentration (about 103~105/ml) in fresh milk, a novel design is used for trapping the target bacteria in the specific area, which the unique design can easily combined to Raman spectroscopy and bacteria can be identified via its specific fingerprinting. This integrated chip completed bio-particle separation, trapping, concentration and detection in continuous flow. Bacteria strain and population can be detected in our 3D DEP chip within 3 minutes.