Label-free enrichment of rare unconventional circulating neoplastic cells using a microfluidic dielectrophoretic sorting device.

Cellular circulating biomarkers from the primary tumor such as circulating tumor cells (CTCs) and circulating hybrid cells (CHCs) have been described to harbor tumor-like phenotype and genotype. CHCs are present in higher numbers than CTCs supporting their translational potential. Methods for isolation of CHCs do not exist and are restricted to low-throughput, time consuming, and biased methodologies. We report the development of a label-free dielectrophoretic microfluidic platform facilitating enrichment of CHCs in a high-throughput and rapid fashion by depleting healthy peripheral blood mononuclear cells (PBMCs). We demonstrated up to 96.5% depletion of PBMCs resulting in 18.6-fold enrichment of cancer cells. In PBMCs from pancreatic adenocarcinoma patients, the platform enriched neoplastic cells identified by their KRAS mutant status using droplet digital PCR with one hour of processing. Enrichment was achieved in 75% of the clinical samples analyzed, establishing this approach as a promising way to non-invasively analyze tumor cells from patients.

A Caenorhabditis elegans behavioral assay distinguishes early stage prostate cancer patient urine from controls.

Current methods for non-invasive prostate cancer (PrCa) detection have a high false-positive rate and often result in unnecessary biopsies. Previous work has suggested that urinary volatile organic compound (VOC) biomarkers may be able to distinguish PrCa cases from benign disease. The behavior of the nematode Caenorhabditis elegans has been proposed as a tool to take advantage of these potential VOC profiles. To test the ability of C. elegans Bristol N2 to distinguish PrCa cases from controls, we performed chemotaxis assays using human urine samples collected from men screened for PrCa. Behavioral response of nematodes towards diluted urine from PrCa cases was compared to response to samples from cancer-free controls. Overall, we observed a significant attraction of young adult-stage C. elegans nematodes to 1:100 diluted urine from confirmed PrCa cases and repulsion of C. elegans to urine from controls. When C. elegans chemotaxis index was considered alongside prostate-specific antigen levels for distinguishing cancer from cancer-free controls, the accuracy of patient classification was 81%. We also observed behavioral attraction of C. elegans to two previously reported VOCs to be increased in PrCa patient urine. We conclude nematode behavior distinguishes PrCa case urine from controls in a dilution-dependent manner.

Plasmonic Sensor Could Enable Label-Free DNA Sequencing.

We demonstrated a proof-of-principle concept of a label-free platform that enables nucleic acid sequencing by binding methodology. The system utilizes gold surfaces having high fidelity plasmonic nanohole arrays which are very sensitive to minute changes of local refractive indices. Our novel surface chemistry approach ensures accurate identification of correct bases at individual positions along a targeted DNA sequence on the gold surface. Binding of the correct base on the gold sensing surface triggers strong spectral variations within the nanohole optical response, which provides a high signal-to-noise ratio and accurate sequence data. Integrating our label-free sequencing platform with a lens-free imaging-based device, we reliably determined targeted DNA sequences by monitoring the changes within the plasmonic diffraction images. Consequently, this new label-free surface chemistry technique, integrated with plasmonic lens-free imaging platform, will enable monitoring multiple biomolecular binding events, which could initiate new avenues for high-throughput nucleic acid sequencing.

Label-free detection of DNA hybridization using carbon nanotube network field-effect transistors.

We report carbon nanotube network field-effect transistors (NTNFETs) that function as selective detectors of DNA immobilization and hybridization. NTNFETs with immobilized synthetic oligonucleotides have been shown to specifically recognize target DNA sequences, including H63D single-nucleotide polymorphism (SNP) discrimination in the HFE gene, responsible for hereditary hemochromatosis. The electronic responses of NTNFETs upon single-stranded DNA immobilization and subsequent DNA hybridization events were confirmed by using fluorescence-labeled oligonucleotides and then were further explored for label-free DNA detection at picomolar to micromolar concentrations. We have also observed a strong effect of DNA counterions on the electronic response, thus suggesting a charge-based mechanism of DNA detection using NTNFET devices. Implementation of label-free electronic detection assays using NTNFETs constitutes an important step toward low-cost, low-complexity, highly sensitive and accurate molecular diagnostics.

Microfluidic sorting of mammalian cells by optical force switching.

Microfluidic-based devices have allowed miniaturization and increased parallelism of many common functions in biological assays; however, development of a practical technology for microfluidic-based fluorescence-activated cell sorting has proved challenging. Although a variety of different physical on-chip switch mechanisms have been proposed, none has satisfied simultaneously the requirements of high throughput, purity, and recovery of live, unstressed mammalian cells. Here we show that optical forces can be used for the rapid (2-4 ms), active control of cell routing on a microfluidic chip. Optical switch controls reduce the complexity of the chip and simplify connectivity. Using all-optical switching, we have implemented a fluorescence-activated microfluidic cell sorter and evaluated its performance on live, stably transfected HeLa cells expressing a fused histone-green fluorescent protein. Recovered populations were verified to be both viable and unstressed by evaluation of the transcriptional expression of two genes, HSPA6 and FOS, known indicators of cellular stress.

Time-of-flight optophoresis analysis of live whole cells in microfluidic channels.

Optophoresis is a non-invasive cell analysis technique that is based on the interaction of live whole cells with optical gradient fields, typically generated by a near-infrared laser. The magnitude of the interaction depends upon the intrinsic physical properties of the cells, such as their refractive index, composition, size, and morphology. Time-of-flight (TOF) optophoresis is an implementation of this technique in a microfluidic environment. It measures cell travel times through a fixed distance with and without irradiation from a laser beam. The magnitude of the optical force from the laser, and therefore the change in transit time introduced by the presence of the infrared laser provides a signature for the cell. By accumulating such measurements for a population of cells (typically 200-300 cells per population), different cell types, drug treatments, or biological states can be compared quantitatively without the need for external labels or markers. An integrated TOF system has been constructed and characterized. The system typically uses square capillaries with 50-100 microm internal diameter and uses a syringe-pump-based flow system that generates initial bulk flow velocities between 200 and 600 microm/sec. Using this TOF technique, we have been able to consistently detect significant differences between normal skin and melanoma cell lines, CCD-1037 and A375, respectively. We have also been able to measure consistent differences in a cell differentiation model (HL60 cell line with DMSO treatment). These early results indicate the potential biological sensitivity of the TOF measurement technique for cellular analysis and cancer diagnostic applications.

Use of moving optical gradient fields for analysis of apoptotic cellular responses in a chronic myeloid leukemia cell model.

To facilitate quantitation of cellular apoptotic responses to various antineoplastic agents, a laser-based technology, Optophoresis, has been developed to provide analysis of cells without any need for labeling or cell processing. Optophoresis is defined as the analysis of the motion of cells, where the motion is either induced or modified by a moving optical gradient field, which produces radiation pressure forces on the cells in an aqueous suspension. Quantitation of the induced motion provides a basis for distinguishing one population of cells from another. One Optophoretic technique, Fast Scan, measures the distribution of distances traversed by a population of cells when exposed to a fast-moving optical gradient. Fast Scan was validated using a cell-based model of chronic myeloid leukemia treated with Gleevec, a specific inhibitor of aberrant Bcr-Abl protein kinase. The Optophoretic measurements were quantitatively comparable to reference assays with regard to drug selectivity and potency and to target specificity, demonstrating the suitability of this technology for pharmaceutical and clinical applications.

Optical forces for noninvasive cellular analysis.

A novel, noninvasive measurement technique for quantitative cellular analysis is presented that utilizes the forces generated by an optical beam to evaluate the physical properties of live cells in suspension. In this analysis, a focused, near-infrared laser line with a high cross-sectional intensity gradient is rapidly scanned across a field of cells, and the interaction of those cells with the beam is monitored. The response of each cell to the laser depends on its size, structure, morphology, composition, and surface membrane properties; therefore, with this technique, cell populations of different type, treatment, or biological state can be compared. To demonstrate the utility of this cell analysis platform, we evaluated the early stages of apoptosis induced in the U937 cancer cell line by the drug camptothecin and compared the results with established reference assays. Measurements on our platform show detection of cellular changes earlier than either of the fluorescence-based Annexin V or caspase assays. Because no labeling or additional cell processing is required and because accurate assays can be performed with a small number of cells, this measurement technique may find suitable applications in cell research, medical diagnostics, and drug discovery.

Active microelectronic array system for DNA hybridization, genotyping and pharmacogenomic applications.

Microelectronic arrays have been developed for DNA hybridization analysis of point mutations, single nucleotide polymorphisms, short tandem repeats and gene expression. In addition to a variety of molecular biology and genomic research applications, such devices will also be used for infectious disease detection, genetic and cancer diagnostics, and pharmacogenomic applications. These microelectronic array devices are able to produce defined electric fields on their surfaces that allow charged molecules and other entities to be transported to or from any test site or micro-location on the planar surface of the device. These molecules and entities include DNA, RNA, proteins, enzymes, antibodies and cells. Electronic-based molecule addressing and hybridization can then be carried out, where the electric field is now used to greatly accelerate the hybridization reactions that occur on the selected test sites. When reversed, the electric field can be used to provide an additional parameter for improved hybridization. Special low-conductance buffers have been developed that provide for the rapid transport of the DNA molecules and facilitate the electronic hybridization reactions under conditions that do not support hybridization. Important to the device function is the permeation layer that overcoats the underlying microelectrodes. Generally composed of a porous hydrogel material impregnated with attachment chemistry, this permeation layer prevents the destruction of analytes at the active microelectrode surface, ameliorates the adverse effects of electrolysis products on the sensitive hybridization and affinity reactions, and serves as a support structure for attaching DNA probes and other molecules to the array. The microelectronic chip or array device is incorporated into a cartridge package (NanoChip trade mark cartridge) that provides the electronic, optical, and fluidic interfacing. A complete instrument system (NanoChip trade mark Molecular Biology Workstation) provides a chip loader, fluorescent reader, computer control interface and data display screen. The probe loader component allows DNA probes or target molecules (polymerase chain reactions amplicons, genomic DNA, RNA, etc.) to be selectively addressed to the array test sites, providing the end-user with 'make your own chip' capabilities. The electronic hybridization can then be carried out and the chip analyzed using a fluorescent detector system. In addition to carrying out rapid, accurate and highly reliable genotyping (point mutations, single nucleotide polymorphisms, short tandem repeats), other future applications include gene expression analysis, or on-chip amplification, immunoassays and cell separation and selection. Smaller and more compact systems are also being designed for portable sample to answer and point of care diagnostics.

Microelectronic array devices and techniques for electric field enhanced DNA hybridization in low-conductance buffers.

A variety of electronic DNA array devices and techniques have been developed that allow electric field enhanced hybridization to be carried out under special low-conductance conditions. These devices include both planar microelectronic DNA array/chip devices as well as electronic microtiter plate-like devices. Such "active" electronic devices are able to provide controlled electric (electrophoretic) fields that serve as a driving force to move and concentrate nucleic acid molecules (DNA/RNA) to selected microlocation test-sites on the device. In addition to ionic strength, pH, temperature and other agents, the electric field provides another controllable parameter that can affect and enhance DNA hybridization. With regard to the planar microelectronic array devices, special low-conductance buffers were developed in order to maintain rapid transport of DNA molecules and to facilitate hybridization within the constrained low current and voltage ranges for this type of device. With regard to electronic microtiter plate type devices (which do not have the low current/voltage constraints), the use of mixed buffers (low conductance upper chamber/high conductance lower chamber) can be used in a unique fashion to create favorable hybridization conditions in a microzone within the test site location. Both types of devices allow DNA molecules to be rapidly and selectively hybridized at the array test sites under conditions where the DNA in the bulk solution can remain substantially denatured.