Małgorzata A. Witek

High-throughput selection, enumeration, electrokinetic manipulation, and molecular profiling of low-abundance circulating tumor cells using a microfluidic system.

A circulating tumor cell (CTC) selection microfluidic device was integrated to an electrokinetic enrichment device for preconcentrating CTCs directly from whole blood to allow for the detection of mutations contained within the genomic DNA of the CTCs. Molecular profiling of CTCs can provide important clinical information that cannot be garnered simply by enumerating the selected CTCs. We evaluated our approach using SW620 and HT29 cells (colorectal cancer cell lines) seeded into whole blood as a model system. Because SW620 and HT29 cells overexpress the integral membrane protein EpCAM, they could be immunospecifically selected using a microfluidic device containing anti-EpCAM antibodies immobilized to the walls of a selection bed. The microfluidic device was operated at an optimized flow rate of 2 mm s(-1), which allowed for the ability to process 1 mL of whole blood in <40 min. The selected CTCs were then enzymatically released from the antibody selection surface and hydrodynamically transported through a pair of Pt electrodes for conductivity-based enumeration. The efficiency of CTC selection was found to be 96% ± 4%. Following enumeration, the CTCs were hydrodynamically transported at a flow rate of 1 μL min(-1) to an on-chip electromanipulation unit, where they were electrophoretically withdrawn from the bulk hydrodynamic flow and directed into a receiving reservoir. Using an electric field of 100 V cm(-1), the negatively charged CTCs were enriched into an anodic receiving reservoir to a final volume of 2 μL, providing an enrichment factor of 500. The collected CTCs could then be searched for point mutations using a PCR/LDR/capillary electrophoresis assay. The DNA extracted from the CTCs was subjected to a primary polymerase chain reaction (PCR) with the amplicons used for a ligase detection reaction (LDR) to probe for KRAS oncogenic point mutations. Point mutations in codon 12 of the KRAS gene were successfully detected in the SW620 CTCs for samples containing <10 CTCs in 1 mL of whole blood. However, the HT29 cells did not contain these mutations, consistent with their known genotype.

Microsystems for the Capture of Low-Abundance Cells

Efficient selection and enumeration of low-abundance biological cells are highly important in a variety of applications. For example, the clinical utility of circulating tumor cells (CTCs) in peripheral blood is recognized as a viable biomarker for the management of various cancers, in which the clinically relevant number of CTCs per 7.5 ml of blood is two to five. Although there are several methods for isolating rare cells from a variety of heterogeneous samples, such as immunomagnetic-assisted cell sorting and fluorescence-activated cell sorting, they are fraught with challenges. Microsystem-based technologies are providing new opportunities for selecting and isolating rare cells from complex, heterogeneous samples. Such approaches involve reductions in target-cell loss, process automation, and minimization of contamination issues. In this review, we introduce different application areas requiring rare cell analysis, conventional techniques for their selection, and finally microsystem approaches for low-abundance-cell isolation and enumeration.

Purification and preconcentration of genomic DNA from whole cell lysates using photoactivated polycarbonate (PPC) microfluidic chips

We discuss the use of a photoactivated polycarbonate (PPC) microfluidic chip for the solid-phase, reversible immobilization (SPRI) and purification of genomic DNA (gDNA) from whole cell lysates. The surface of polycarbonate was activated by UV radiation resulting in a photo-oxidation reaction, which produced a channel surface containing carboxylate groups. The gDNA was selectively captured on this photoactivated surface in an immobilization buffer, which consisted of 3% polyethylene glycol, 0.4 M NaCl and 70% ethanol. The methodology reported herein is similar to conventional SPRI in that surface-confined carboxylate groups are used for the selective immobilization of DNA; however, no magnetic beads or a magnetic field are required. As observed by UV spectroscopy, a load of ∼7.6 ± 1.6 µg/ml of gDNA was immobilized onto the PPC bed. The recovery of DNA following purification was estimated to be 85 ± 5%. The immobilization and purification assay using this PPC microchip could be performed within ∼25 min as follows: (i) DNA immobilization ∼6 min, (ii) chip washout with ethanol 10 min, and (iii) drying and gDNA desorption ∼6 min. The PPC microchip could also be used for subsequent assays with no substantial loss in recovery, no observable carryover and no need for ‘reactivation’ of the PC surface with UV light.

Modular microsystem for the isolation, enumeration, and phenotyping of circulating tumor cells in patients with pancreatic cancer.

In this manuscript, we discuss the development and clinical use of a thermoplastic modular microsystem for the high-throughput analysis of CTCs directly from whole blood. The modular system offers some innovative features that address challenges currently associated with many CTC platforms; it can exhaustively process 7.5 mL of blood in less than 45 min with recoveries >90%. In addition, the system automates the postselection CTC processing steps and thus, significantly reduces assay turnaround time (from selection to enumeration <1.5 h as compared to >8 h for many reported CTC platforms). The system is composed of 3 functional modules including (i) a thermoplastic CTC selection module composed of high aspect ratio (30 μm × 150 μm) channels containing anti-EpCAM antibodies that is scalable in terms of throughput by employing channel numbers ranging from 50 to 320; the channel number is user selected to accommodate the volume of blood that must be processed; (ii) an impedance sensor module for label-less CTC counting; and (iii) a staining and imaging module for the placement of released cells into a 2D array within a common imaging plane for phenotypic identification. To demonstrate the utility of this system, blood samples from patients with local resectable and metastatic pancreatic ductal adenocarcinoma (PDAC) were analyzed. We demonstrate the ability to select EpCAM positive CTCs from PDAC patients in high purity (>86%) and with excellent yields (mean = 53 CTCs per mL for metastatic PDAC patients) using our modular system. In addition, we demonstrate the ability to detect CTCs in PDAC patients with local resectable disease (mean = 11 CTCs per mL).

Enrichment and Detection of Escherichia coli O157:H7 from Water Samples Using an Antibody Modified Microfluidic Chip

Low abundant (<100 cells mL(-1)) E. coli O157:H7 cells were isolated and enriched from environmental water samples using a microfluidic chip. The poly(methylmethacrylate), PMMA, chip contained 8 devices, each equipped with 16 curvilinear high aspect ratio channels that were covalently decorated with polyclonal anti-O157 antibodies (pAb) and could search for rare cells through a pAb mediated process. The chip could process independently 8 different samples or one sample using 8 different parallel inputs to increase volume processing throughput. After cell enrichment, cells were released and enumerated using benchtop real-time quantitative polymerase chain reaction (PCR), targeting genes which effectively discriminated the O157:H7 serotype from other nonpathogenic bacteria. The recovery of target cells from water samples was determined to be approximately 72%, and the limit-of-detection was found to be 6 colony forming units (cfu) using the slt1 gene as a reporter. We subsequently performed analysis of lake and wastewater samples. The simplicity in manufacturing and ease of operation makes this device attractive for the selection of pathogenic species from a variety of water supplies suspected of containing bacterial pathogens at extremely low frequencies.

UV activation of polymeric high aspect ratio microstructures: Ramifications in antibody surface loading for circulating tumor cell selection

The need to activate thermoplastic surfaces using robust and efficient methods has been driven by the fact that replication techniques can be used to produce microfluidic devices in a high production mode and at low cost, making polymer microfluidics invaluable for in vitro diagnostics, such as circulating tumor cell (CTC) analysis, where device disposability is critical to mitigate artifacts associated with sample carryover. Modifying the surface chemistry of thermoplastic devices through activation techniques can be used to increase the wettability of the surface or to produce functional scaffolds to allow for the covalent attachment of biologics, such as antibodies for CTC recognition. Extensive surface characterization tools were used to investigate UV activation of various surfaces to produce uniform and high surface coverage of functional groups, such as carboxylic acids in microchannels of different aspect ratios. We found that the efficiency of the UV activation process is highly dependent on the microchannel aspect ratio and the identity of the thermoplastic substrate. Colorimetric assays and fluorescence imaging of UV-activated microchannels following EDC/NHS coupling of Cy3-labeled oligonucleotides indicated that UV-activation of a PMMA microchannel with an aspect ratio of ~3 was significantly less efficient toward the bottom of the channel compared to the upper sections. This effect was a consequence of the bulk polymers damping of the modifying UV radiation due to absorption artifacts. In contrast, this effect was less pronounced for COC. Moreover, we observed that after thermal fusion bonding of the devices cover plate to the substrate, many of the generated functional groups buried into the bulk rendering them inaccessible. The propensity of this surface reorganization was found to be higher for PMMA compared to COC. As an example of the effects of material and microchannel aspect ratios on device functionality, thermoplastic devices for the selection of CTCs from whole blood were evaluated, which required the immobilization of monoclonal antibodies to channel walls. From our results, we concluded the CTC yield and purity of isolated CTCs were dependent on the substrate material with COC producing the highest clinical yields for CTCs as well as better purities compared to PMMA.

EndoV/DNA ligase mutation scanning assay using microchip capillary electrophoresis and dual-color laser-induced fluorescence detection

We report the ability to detect with high sensitivity sporadic mutations using a mutation scanning assay, which employs thermostable endonuclease V (EndoV) and DNA ligase. The products of the mutation scanning assay were separated using microchip capillary electrophoresis (μCE) and detected with a dual-color laser-induced fluorescence (LIF) detector. PCR products from mutant and wild-type DNA of p53 exon 8 were generated using Cy3-labeled forward and Cy5-labeled reverse primers to allow LIF detection with μCE. EndoV recognizes and primarily cleaves heteroduplexed DNA one base 3′ to a mismatch and can nick matched sites at low levels as well. DNA ligase is used to reseal nicks generated at matched sites, which creates a highly sensitive and specific assay for analyzing sporadic mutations in genomic DNA. Heteroduplexed DNA samples were treated with EndoV alone and with both EndoV and DNA ligase and separated using a 4% (w/v) linear polyacrylamide gel constituted in 1x TTE buffer, 7 M urea, and 0.05% (w/v) methyl hydroxyethyl cellulose, which was used to suppress the EOF in the microchip. Sizing of the bands appearing in the electropherogram revealed the approximate position of the mutation. In this study, mutations present in p53 exon 8 generated Cy3-labeled cleavage products of 158 nt and Cy5-labeled cleavage products of 195 nt. The DNA fragments were simultaneously monitored at their respective color using a dual-color LIF system with the 158 and 195 nt fragments detected along with heteroduplexed fragments of 350 nt. The microchip separation was completed within 7 min, almost ten-fold shorter time compared to conventional capillary gel electrophoresis.

Parallel Affinity-Based Isolation of Leukocyte Subsets Using Microfluidics: Application for Stroke Diagnosis

We report the design and performance of a polymer microfluidic device that can affinity select multiple types of biological cells simultaneously with sufficient recovery and purity to allow for the expression profiling of mRNA isolated from these cells. The microfluidic device consisted of four independent selection beds with curvilinear channels that were 25 μm wide and 80 μm deep and were modified with antibodies targeting antigens specifically expressed by two different cell types. Bifurcated and Z-configured device geometries were evaluated for cell selection. As an example of the performance of these devices, CD4+ T-cells and neutrophils were selected from whole blood as these cells are known to express genes found in stroke-related expression profiles that can be used for the diagnosis of this disease. CD4+ T-cells and neutrophils were simultaneously isolated with purities >90% using affinity-based capture in cyclic olefin copolymer (COC) devices with a processing time of ∼3 min. In addition, sufficient quantities of the cells could be recovered from a 50 μL whole blood input to allow for reverse transcription-polymerase chain reaction (RT-PCR) following cell lysis. The expression of genes from isolated T-cells and neutrophils, such as S100A9, TCRB, and FPR1, was evaluated using RT-PCR. The modification and isolation procedures demonstrated here can also be used to analyze other cell types as well where multiple subsets must be interrogated.

Polymer-based microfluidic devices for biomedical applications

Two types of Microfluidic bioanalytical systems were designed and fabricated in polymer substrates using the LIGA process. A continuous flow polymerase chain reaction (CFPCR) Microfluidic device was fabricated in polycarbonate (PC), which utilized isothermal zone and shuttling the sample through each zone to achieve amplification. A 20-cycle PCR amplification of a fragment of a plasmid DNA template was achieved in 5.3 min. The results were comparable to those obtained in commercial laboratory-scale PCR system. The second system consisted of a microchip contating a low-density array assembled into the Microfluidic channel, which was hot-embossed in poly(methyl methacrylate) (PMMA). The detection of low-abundant mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancer was successfully performed. The array accessed microfluidics in order to enhance the kinetic associated with hybridization.

Solid-phase extraction and purification of membrane proteins using a UV-modified PMMA microfluidic bioaffinity μSPE device

We present a novel microfluidic solid-phase extraction (μSPE) device for the affinity enrichment of biotinylated membrane proteins from whole cell lysates. The device offers features that address challenges currently associated with the extraction and purification of membrane proteins from whole cell lysates, including the ability to release the enriched membrane protein fraction from the extraction surface so that they are available for downstream processing. The extraction bed was fabricated in PMMA using hot embossing and was comprised of 3600 micropillars. Activation of the PMMA micropillars by UV/O3 treatment permitted generation of surface-confined carboxylic acid groups and the covalent attachment of NeutrAvidin onto the μSPE device surfaces, which was used to affinity select biotinylated MCF-7 membrane proteins directly from whole cell lysates. The inclusion of a disulfide linker within the biotin moiety permitted release of the isolated membrane proteins via DTT incubation. Very low levels (∼20 fmol) of membrane proteins could be isolated and recovered with ∼89% efficiency with a bed capacity of 1.7 pmol. Western blotting indicated no traces of cytosolic proteins in the membrane protein fraction as compared to significant contamination using a commercial detergent-based method. We highlight future avenues for enhanced extraction efficiency and increased dynamic range of the μSPE device using computational simulations of different micropillar geometries to guide future device designs.