Hongbo Zhou

A microfluidic droplet digital PCR for simultaneous detection of pathogenic Escherichia coli O157 and Listeria monocytogenes

Sensitive and rapid identification of pathogenic bacterial is extremely important due to the serious threat of pathogens to human health. In this study, we demonstrate the simultaneous and sensitive detection of pathogenic Escherichia coli O157 and Listeria monocytogenes using a novel duplex droplet digital PCR (ddPCR) platform. The ddPCR platform, which uses a mineral oil-saturated polydimethylsiloxane (OSP) chip to overcome the problem of droplet evaporation, integrates the functions of droplet generation, on-chip amplification and end-point fluorescence readout. Simultaneous detection of two kinds of bacterial is achieved by the design of differentially labeled TaqMan-MGB fluorescent probes. Compared with a quantitative real-time PCR approach, the OSP chip-based duplex ddPCR platform exhibits high sensitivity, which is at the level of single molecule resolution without significant cross-assay interference. Moreover, the applicability of the proposed method is also evaluated in artificially contaminated drinking water sample, which displays a low detection limit down to 10 CFU/mL for both pathogenic bacterial within 2 h.

Bead-based microarray immunoassay for lung cancer biomarkers using quantum dots as labels.

In this study, we developed a multiplex immunoassay system that combines the suspension and planar microarray formats within a single layer of polydimethylsiloxane (PDMS) using soft lithography technology. The suspension format was based on the target proteins forming a sandwich structure between the magnetic beads and the quantum dot (QD) probes through specific antibody-antigen interactions. The planar microarray format was produced by fabricating an array of micro-wells in PDMS. Each micro-well was designed to trap a single microbead and eventually generated a microbead array within the PDMS chamber. The resultant bead-based on-chip assay could be used for simultaneously detecting three lung cancer biomarkers-carcinoembryonic antigen (CEA), fragments of cytokeratin 19 (CYFRA21-1) and neuron-specific enolase (NSE)-in 10 μl of human serum, with a wide linear dynamic range (1.03-111 ng/mL for CEA and CYFRA21-1; 9.26-1000 ng/ml for NSE) and a low detection limit (CEA: 0.19 ng/ml; CYFRA21-1: 0.97 ng/ml; NSE: 0.37 ng/ml; S/N=3). Our micro-well chip does not require complex e-beam lithography or the reactive ion etching process as with existing micro-well systems, which rely on expensive focused ion beam (FIB) milling or optical fiber bundles. Furthermore, the current approach is easy to operate without extra driving equipment such as pumps, and can make parallel detection for multiplexing with rapid binding kinetics, small reagent consumption and low cost. This work has demonstrated the importance of the successful application of on-chip multiplexing sandwich assays for the detection of biomarker proteins.

A facile microfluidic strategy for measuring interfacial tension

We report a facile method for measuring interfacial tension (IFT, γ) of immiscible fluids using a microfluidic device. The IFT is determined by monitoring the pressure drop across a microchannel, where a pair of modified Laplace sensors (formed by tapered channels) are connected, and the curvatures of the interfaces in the tapered channels. The method was tested with the model oil/water systems, and the results agreed well with a commercial tensiometry. We expect this method to be easily implemented in common microfluidic laboratories and supply a low-cost and fast way for interfacial tension measurement.

Analysis of circulating tumor cells from lung cancer patients with multiple biomarkers using high-performance size-based microfluidic chip

Circulating tumor cells (CTCs) have attracted pretty much attention from scientists because of their important relationship with the process of metastasis. Here, we developed a size-based microfluidic chip containing triangular pillar array and filter channel array for detecting single CTCs and CTC clusters independent of tumor-specific markers. The cell populations in chip were characterized by immune-fluorescent staining combining an epithelial marker and a mesenchymal marker. We largely decreased the whole time of detection process to nearly 1.5h with this microfluidic device. The CTCs were subsequently measured in 77 patients with lung cancer and 39 healthy persons. The microfluidic device allowed for the detection of CTCs with apparent high sensitivity and specificity (82.7% sensitivity and 100% specificity). Furthermore, the total CTC counts were found to be elevated in advanced patients with metastases when compared with those without (20.89±14.57 vs 8.428±5.858 cells/mL blood; P<0.01). Combined epithelial marker and mesenchymal marker analysis of CTCs could provide more information about metastasis in patients than only usage of epithelial marker. In conclusion, the development of the size-based microfluidic device for efficient capture of CTCs will enable detailed characterization of their biological properties and values in cancer diagnosis.

Fabrication of dome-shaped flexible electrode arrays for retinal prostheses

In order to enable electrode sites to get closer to target neurons, obtain a better stimulation and minimize the electrochemical erosion of electrode, we proposed dome-shaped flexible neural microelectrode arrays (MEAs) for neural applications. With the use of photosensitive polyimide (Durimide 7510, PI) as substrate, a flexible microelectrode with 6 × 6 array of dome-shaped electrode sites was fabricated by combining photolithography and metal patterning with electroplating process. An evaluation of the dome-shaped electrode was also performed by simulation, SEM and impedance test. Experimental results showed, compared with conventional planar microelectrodes with the same base area, the 3D dome-shaped microelectrodes exhibited an 80% decrease in electrode impedance. The dome-shaped electrodes produce more uniform current distribution than the 3D pyramid-shaped microelectrode, which is helpful for its long-term safety of stimulation.

High aspect ratio induced spontaneous generation of monodisperse picolitre droplets for digital PCR

Droplet microfluidics, which involves micrometer-sized emulsion droplets on a microfabricated platform, has been demonstrated as a unique system for many biological and chemical applications. Robust and scalable generation of monodisperse droplets at high throughput is of fundamental importance for droplet microfluidics. Classic designs for droplet generation employ shear fluid dynamics to induce the breakup of droplets in a two-phase flow and the droplet size is sensitive to flow rate fluctuations, often resulting in polydispersity. In this paper, we show spontaneous emulsification by a high aspect ratio (>3.5) rectangular nozzle structure. Due to the confinement and abrupt change of the structure, a Laplace pressure difference is generated between the dispersed and continuous phases, and causes the thread thinning and droplet pinch-off without the need to precisely control external flow conditions. A high-throughput droplet generator was developed by parallelization of a massive number of the basic structures. This device enabled facile and rapid partition of aqueous samples into millions of uniform picolitre droplets in oil. Using this device, on-chip droplet-based digital polymerase chain reaction (PCR) was performed for absolute quantification of rare genes with a wide dynamic range.

An emulsion digital PCR quantitative method based on microbeads and micropillar array chip

BEAMing (beads, emulsion, amplification, and magnetics) is a high sensitive emulsion digital PCR (polymerase chain reaction) technology. Target beads obtained via BEAMing can be used to determine the copy number of target DNA population. However, the existing beads counting process requires specialized instrument, which has increased the detection costs. Here we developed an easy and low cost beads counting method combined with microspheres and micropillar array chips. Through counting the chip-trapped polystyrene microspheres which have captured the target magnetic beads, we could easily infer the number of target magnetic beads and the copy number of target DNA. The trapping mechanism was based on the difference of microsphere diameter and pillar intervals. Fabrication of the chip was simple and low-cost. This method has reduced the difficulty of beads counting process in BEAMing and may promote the applications of BEAMing in clinical filed.

A new picoinjector in microfluidics

In droplet-based microfluidics, adding reagents into drops is one of the most important functions. In this paper, we develop a new picoinjector to add controlled volumes of reagent into droplets at kilohertz rates. In the new picoinjector, the adding process is triggered by the coming droplet itself, and the dosing volume can be precisely controlled. Meanwhile, the trigger voltage is lowered to several volts. These features make the system more practical and reliable. We expect the new picoinjector will find important applications of droplet-based microfluidics in automated biological assay, directed evolution, enzyme assay, and so on.