Steven A. Soper

Highly Efficient Circulating Tumor Cell Isolation from Whole Blood and Label-Free Enumeration Using Polymer-Based Microfluidics with an Integrated Conductivity Sensor

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (>/=1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 mum width x 150 mum depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation.

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.

Surface modification of polymer-based microfluidic devices

Abstract We report the chemical modification of poly(methyl methacrylate) (PMMA), and poly(carbonate) (PC) surfaces for applications in microfluidic systems. For PMMA, a reaction of the surface methyl ester groups with a monoanion of α,ω-diaminoalkanes (aminolysis reaction) to yield amine-terminated PMMA surfaces will be described. Furthermore, it was found that the amine functionalities were tethered to the PMMA backbone through an alkane bridge to amide bonds formed during the aminolysis of the surface ester functionalities. The electro-osmotic flow (EOF) in aminated-PMMA microchannels was reversed when compared to that in unmodified channels. Finally, the availability of the surface amine groups was further demonstrated by their reaction with n -octadecane–1-isocyanate to form PMMA surfaces terminated with well ordered and highly crystalline octadecane chains, appropriate for performing reverse-phase separations. Examples of reverse-phase separations of ion-paired double-stranded DNAs in electric fields (capillary electrochromatography (CEC)) will be demonstrated using a PMMA-based fluidic chip. For PC, sulfonation of the surface with SO 3 will be described; this sulfonation makes the surface very hydrophilic. EOF studies of the sulfonated-PC surfaces indicated changes in the pH-dependent profile when compared to unmodified PC.

Highly efficient capture and enumeration of low abundance prostate cancer cells using prostate-specific membrane antigen aptamers immobilized to a polymeric microfluidic device

Prostate tumor cells over‐express a prostate‐specific membrane antigen (PSMA) that can be used as a marker to select these cells from highly heterogeneous clinical samples, even when found in low abundance. Antibodies and aptamers have been developed that specifically bind to PSMA. In this study, anti‐PSMA aptamers were immobilized onto the surface of a capture bed poised within a PMMA, microchip, which was fabricated into a high‐throughput micro‐sampling unit (HTMSU) used for the selective isolation of rare circulating prostate tumor cells resident in a peripheral blood matrix. The HTMSU capture bed consisted of 51 ultra‐high‐aspect ratio parallel curvilinear channels with a width similar to the prostate cancer cell dimensions. The surface density of the PSMA‐specific aptamers on an ultraviolet‐modified PMMA microfluidic capture bed surface was determined to be 8.4×1012 molecules/cm2. Using a linear velocity for optimal cell capture in the aptamer‐tethered HTMSU (2.5 mm/s), a recovery of 90% of LNCaP cells (prostate cancer cell line; used as a model in this example) was found. Due to the low abundance of these cells, the input volume required was 1 mL and this could be processed in ∼29 min using an optimized linear flow rate of 2.5 mm/s. Captured cells were subsequently released intact from the affinity surface using 0.25% w/w trypsin followed by counting individual cells using a contact conductivity sensor integrated into the HTMSU that provided high detection and sampling efficiency (∼100%) and did not require staining of the cells for enumeration.

THE PHOTOPHYSICAL CONSTANTS OF SEVERAL FLUORESCENT DYES PERTAINING TO ULTRASENSITIVE FLUORESCENCE SPECTROSCOPY

Abstract— The successful implementation of ultrasensitive fluorescence spectroscopy of biological and chemical species depends upon certain photophysical parameters associated with the fluorescent dye used in the investigation. These parameters include the fluorescence quantum efficiency, photodestruction quantum efficiency, absorption cross section and fluorescence lifetime. These photophysical constants were measured for several fluorescent dyes that are used for the tagging of biological species. Three different solvents, ethanol, water and a cationic surfactant used above its critical micelle concentration, were studied. The effective photon yield (ratio of the fluorescence quantum yield to the photodestruction quantum efficiency) for the dyes is nearly 100 times greater in ethanol than it is in water because of the superior photostabilities of these dyes in ethanol solvents. The implications of these parameters for the design of an ultrasensitive fluorescence experiment are discussed.

Microfluidic chips for mass spectrometry-based proteomics

Microfluidic devices coupled to mass spectrometers have emerged as excellent tools for solving the complex analytical challenges associated with the field of proteomics. Current proteome identification procedures are accomplished through a series of steps that require many hours of labor-intensive work. Microfluidics can play an important role in proteomic sample preparation steps prior to mass spectral identification such as sample cleanup, digestion, and separations due to its ability to handle small sample quantities with the potential for high-throughput parallel analysis. To utilize microfluidic devices for proteomic analysis, an efficient interface between the microchip and the mass spectrometer is required. This tutorial provides an overview of the technologies and applications of microfluidic chips coupled to mass spectrometry for proteome analysis. Various approaches for combining microfluidic devices with electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) are summarized and applications of chip-based separations and digestion technologies to proteomic analysis are presented.

Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices

Single photon burst techniques were used to detect double‐stranded DNA molecules in poly(methylmethacrylate) (PMMA) and polycarbonate (PC) microfluidic devices. A confocal epi‐illumination detection system was constructed to monitor the fluorescence signature from single DNA molecules that were multiply labeled with the mono‐intercalating dye, TOPRO‐5, which possessed an absorption maximum at 765 nm allowing excitation with a solid‐state diode laser and fluorescence monitoring in the near‐infrared (IR). Near‐IR excitation minimized autofluorescence produced from the polymer substrate, which was found to be significantly greater when excitation was provided in the visible range (488 nm). A solution containing ‐DNA (48.5 kbp) was electrokinetically transported through the microfluidic devices at different applied voltages and solution pH values to investigate the effects of polymer substrate on the transport rate and detection efficiency of single molecular events. By applying an autocorrelation analysis to the data, we were able to obtain the molecular transit time of the individual molecules as they passed through the 7 νm laser beam. It was observed that the applied voltage for both devices affected the transport rate. However, solution pH did not alter the transit time for PMMA‐based devices since the electroosmotic flow of PMMA was independent of solution pH. In addition, efforts were directed toward optimizing the sampling efficiency (number of molecules passing through the probe volume) by using either hydrodynamically focused flows from a sheath generated by electrokinetic pumping from side channels or reducing the channel width of the microfluidic device. Due to the low electroosmotic flows generated by both PMMA and PC, tight focusing of the sample stream was not possible. However, in PMMA devices, flow gating was observed by applying field strengths >–120 V/cm to the sheath flow channels. By narrowing the microchannel width, the number of molecular events detected per unit time was found to be four times higher in channels with 10 νm widths compared to those of 50 νm, indicating improved sampling efficiency for the narrower channels without significantly deteriorating detection efficiency. Attempts were made to do single molecule sizing of ‐DNA, M13 (7.2 kbp) and pUC19 (2.7 kbp) using photon burst detection. While the average number of photons for each DNA type were different, the standard deviations were large due to the Gaussian intensity profile of the excitation beam. To demonstrate the sensitivity of single molecule analysis in the near‐IR using polymer microfluidic devices, the near‐IR chromophore, NN382, was analyzed using our confocal imager. A detection efficiency of 94% for single NN382 molecules was observed in the PC devices.

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.

Detection and identification of single molecules in solution

We have extended our recent experiments in the detection of single fluorescent molecules in solution to the exploration of spectroscopy at the single-molecule level. As a first step we have developed a technique that can efficiently distinguish between two species of dye molecules on the basis of differences in their emission spectra. We have also demonstrated that another spectroscopic property, fluorescence lifetime, can be accurately determined at the single-molecule level. Spectroscopic properties can be used to identify fluorescent molecules and to reveal static or slowly varying aspects of the microenvironment of each molecule, thereby yielding information unavailable from bulk studies.

Rapid DNA sequencing based upon single molecule detection

We are developing a laser-based technique for the rapid sequencing of 40-kb or larger fragments of DNA at a rate of 100 to 1000 bases per second. The approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA fragment into a flowing sample stream, and detection of individual fluorescently labeled bases as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will significantly reduce the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.