Z. Hugh Fan

Aptamer-Based Microfluidic Device for Enrichment, Sorting, and Detection of Multiple Cancer Cells

The ability to diagnose cancer based on the detection of rare cancer cells in blood or other bodily fluids is a significant challenge. To address this challenge, we have developed a microfluidic device that can simultaneously sort, enrich, and then detect multiple types of cancer cells from a complex sample. The device, which is made from poly(dimethylsiloxane) (PDMS), implements cell-affinity chromatography based on the selective cell-capture of immobilized DNA-aptamers and yields a 135-fold enrichment of rare cells in a single run. This enrichment is achieved because the height of the channel is on the order of a cell diameter. The sorted cells grow at the comparable rate as cultured cells and are 96% pure based on flow cytometry determination. Thus, by using our aptamer based device, cell capture is achieved simply and inexpensively, with no sample pretreatment before cell analysis. Enrichment and detection of multiple rare cancer cells can be used to detect cancers at the early stages, diagnose metastatic relapse, stratify patients for therapeutic purposes, monitor response to drugs and therapies, track tumor progression, and gain a deeper understanding of the biology of circulating tumor cells (CTCs).

Macro-to-micro interfaces for microfluidic devices

Since the concept of miniaturized total analysis systems (microTAS) was invented, a great number of microfluidic devices have been demonstrated for a variety of applications. However, an important hurdle that still needs to be cleared is the connection of a microfluidic device with the rest of the world, which is often referred to as the macro-to-micro interface, interconnect, or world-to-chip interface. In this review, we will examine the methods used by pioneers in the field and other investigators, review the approaches for capillary electrophoresis-based devices and those using pneumatic pumping, and present additional discussion on interface standardization and choosing and designing interconnects for your applications.

Enrichment of Cancer Cells Using Aptamers Immobilized on a Microfluidic Channel

This work describes the development and investigation of an aptamer modified microfluidic device that captures rare cells to achieve a rapid assay without pretreatment of cells. To accomplish this, aptamers are first immobilized on the surface of a poly(dimethylsiloxane) microchannel, followed by pumping a mixture of cells through the device. This process permits the use of optical microscopy to measure the cell-surface density from which we calculate the percentage of cells captured as a function of cell and aptamer concentration, flow velocity, and incubation time. This aptamer-based device was demonstrated to capture target cells with >97% purity and >80% efficiency. Since the cell capture assay is completed within minutes and requires no pretreatment of cells, the device promises to play a key role in the early detection and diagnosis of cancer where rare diseased cells can first be enriched and then captured for detection.

Aptamer-Enabled Efficient Isolation of Cancer Cells from Whole Blood Using a Microfluidic Device

Circulating tumor cells (CTC) in the peripheral blood could provide important information for diagnosis of cancer metastasis and monitoring treatment progress. However, CTC are extremely rare in the bloodstream, making their detection and characterization technically challenging. We report here the development of an aptamer-mediated, micropillar-based microfluidic device that is able to efficiently isolate tumor cells from unprocessed whole blood. High-affinity aptamers were used as an alternative to antibodies for cancer cell isolation. The microscope-slide-sized device consists of >59,000 micropillars, which enhanced the probability of the interactions between aptamers and target cancer cells. The device geometry and the flow rate were investigated and optimized by studying their effects on the isolation of target leukemia cells from a cell mixture. The device yielded a capture efficiency of ~95% with purity of ~81% at the optimum flow rate of 600 nL/s. Further, we exploited the device for isolating colorectal tumor cells from unprocessed whole blood; as few as 10 tumor cells were captured from 1 mL of whole blood. We also addressed the question of low throughput of a typical microfluidic device by processing 1 mL of blood within 28 min. In addition, we found that ~93% of the captured cells were viable, making them suitable for subsequent molecular and cellular studies.

Multivalent DNA nanospheres for enhanced capture of cancer cells in microfluidic devices.

Isolation of circulating tumor cells (CTCs) from peripheral blood or cancer cells from bone marrow has significant applications in cancer diagnosis, therapy monitoring, and drug development. CTCs are cancer cells shed from primary tumors; they circulate in the bloodstream, leading to metastasis. The extraordinary rarity of CTCs in the bloodstream makes their isolation a significant technological challenge. Herein, we report the development of a platform combining multivalent DNA aptamer nanospheres with microfluidic devices for efficient isolation of cancer cells from blood. Gold nanoparticles (AuNPs) were used as an efficient platform for assembling a number of aptamers for high-efficiency cell capture. Up to 95 aptamers were attached onto each AuNP, resulting in enhanced molecular recognition capability. An increase of 39-fold in binding affinity was confirmed by flow cytometry for AuNP-aptamer conjugates (AuNP-aptamer) when compared with aptamer alone. With a laminar flow flat channel microfluidic device, the capture efficiency of human acute leukemia cells from a cell mixture in buffer increased from 49% using aptamer alone to 92% using AuNP-aptamer. We also employed AuNP-aptamer in a microfluidic device with herringbone mixing microstructures for isolation of leukemia cells in whole blood. The cell capture efficiency was also significantly increased with the AuNP-aptamer over aptamer alone, especially at high flow rates. Our results show that the platform combining DNA nanostructures with microfluidics has a great potential for sensitive isolation of CTCs and is promising for cancer diagnosis and prognosis.

Miniaturized capillary isoelectric focusing in plastic microfluidic devices

We report the demonstration of miniaturized capillary isoelectric focusing (CIEF) in plastic microfluidic devices. Conventional CIEF technique was adapted to the microfluidic devices to separate proteins and to detect protein‐protein interactions. Both acidic and basic proteins with isoelectric points (pI) ranging from 5.4 to 11.0 were rapidly focused, mobilized, and detected in a 1.2 cm long channel (50 νm deep×120 νm wide) with a total analysis time of 150 s. In a device with a focusing distance of 4.7 cm, the separation efficiency for a basic protein, lysozyme, was achieved as high as 1.5×105 plates, corresponding to 3.2 million plates per meter. We also experimentally confirmed that IEF resolution is essentially independent of focusing length when the applied voltage is kept the same and within a range that it does not cause Joule heating. Further, we demonstrated the use of miniaturized CIEF to study the interactions between two pairs of proteins, immunoglobulin G (IgG) with protein G and anti‐six histidine (anti‐6xHis) with 6xHis‐tagged green fluorescent protein (GFP). Using this approach, protein‐protein interactions can be detected for as little as 50 fmol of protein. We believe miniaturized CIEF is useful for studying protein‐protein interactions when there is a difference in pI between a protein‐protein complex and its constitutent proteins.

Laminated paper-based analytical devices (LPAD) with origami-enabled chemiluminescence immunoassay for cotinine detection in mouse serum.

Laminated paper-based analytical devices (LPAD) with origami-enabled chemiluminescence immunoassay have been developed for the detection of cotinine, a secondhand smoke (SHS) biomarker. The devices were fabricated by a craft-cutter to define flow channels, followed by lamination. This approach of cutting/lamination to fabricate LPAD is very similar to making an identification card, offering advantages in simplicity and rugged backing when compared to the common method of patterning paper using SU-8 or wax. We also developed a protocol of localized incision and paper-folding to isolate the detection zone from flow channels; the simple origami step eliminated possible reagent diffusion and flow during antibody immobilization steps and numerous washings. By incorporating luminol-based chemiluminescence for detecting horseradish peroxidase-conjugated cotinine, we employed origami-enabled LPAD to detect cotinine in mouse serum using competitive immunoassay. The detection limit was determined to be 5 ng/mL, a clinically relevant concentration. We believe that LPAD with chemiluminescence detection provides a new platform of low cost and sensitive assays for cotinine detection.

Mixing in microfluidic devices and enhancement methods.

Mixing in microfluidic devices presents a challenge due to laminar flows in microchannels, which result from low Reynolds numbers determined by the channels hydraulic diameter, flow velocity, and solutions kinetic viscosity. To address this challenge, novel methods of mixing enhancement within microfluidic devices have been explored for a variety of applications. Passive mixing methods have been created, including those using ridges or slanted wells within the microchannels, as well as their variations with improved performance by varying geometry and patterns, by changing the properties of channel surfaces, and by optimization via simulations. In addition, active mixing methods including microstirrers, acoustic mixers, and flow pulsation have been investigated and integrated into microfluidic devices to enhance mixing in a more controllable manner. In general, passive mixers are easy to integrate, but difficult to control externally by users after fabrication. Active mixers usually take efforts to integrate within a device and they require external components (e.g. power sources) to operate. However, they can be controlled by users to a certain degree for tuned mixing. In this article, we provide a general overview of a number of passive and active mixers, discuss their advantages and disadvantages, and make suggestions on choosing a mixing method for a specific need as well as advocate possible integration of key elements of passive and active mixers to harness the advantages of both types.

Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves

Two-dimensional (2D) protein separation is achieved in a plastic microfluidic device by integrating isoelectric focusing (IEF) with multi-channel polyacrylamide gel electrophoresis (PAGE). IEF (the first dimension) is carried out in a 15 mm-long channel while PAGE (the second dimension) is in 29 parallel channels of 65 mm length that are orthogonal to the IEF channel. An array of microfluidic pseudo-valves is created for introducing different separation media, without cross-contamination, in both dimensions; it also allows transfer of proteins from the first to the second dimension. Fabrication of pseudo-valves is achieved by photo-initiated, in situ gel polymerization; acrylamide and methylenebisacrylamide monomers are polymerized only in the PAGE channels whereas polymerization does not take place in the IEF channel where a mask is placed to block the UV light. IEF separation medium, carrier ampholytes, can then be introduced into the IEF channel. The presence of gel pseudo-valves does not affect the performance of IEF or PAGE when they are investigated separately. Detection in the device is achieved by using a laser induced fluorescence imaging system. Four fluorescently-labeled proteins with either similar pI values or close molecular weight are well separated, demonstrating the potential of the 2D electrophoresis device. The total separation time is less than 10 minutes for IEF and PAGE, an improvement of 2 orders of magnitude over the conventional 2D slab gel electrophoresis.

Cell-Free Protein Synthesis in Microfluidic Array Devices

We report the development of a microfluidic array device for continuous‐exchange, cell‐free protein synthesis. The advantages of protein expression in the microfluidic array include (1) the potential to achieve high‐throughput protein expression, matching the throughput of gene discovery; (2) more than 2 orders of magnitude reduction in reagent consumption, decreasing the cost of protein synthesis; and (3) the possibility to integrate with detection for rapid protein analysis, eliminating the need to harvest proteins. The device consists of an array of units, and each unit can be used for production of an individual protein. The unit comprises a tray chamber for in vitro protein expression and a well chamber as a nutrient reservoir. The tray is nested in the well, and they are separated by a dialysis membrane and connected through a microfluidic connection that provides a means to supply nutrients and remove the reaction byproducts. The device is demonstrated by synthesis of green fluorescent protein, chloramphenicol acetyl‐transferase, and luciferase. Protein expression in the device lasts 5–10 times longer and the production yield is 13–22 times higher than in a microcentrifuge tube. In addition, we studied the effects of the operation temperature and hydrostatic flow on the protein production yield.