Frederic Zenhausern
University of Arizona, College of Medicine, Faculty Member
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A major hindrance to advances in the care of patients with malignant gliomas is the presence of the blood brain barrier (BBB) and blood-brain tumor barrier (BBTB) that greatly restricts drug access from the plasma to the tumor cells.... more
A major hindrance to advances in the care of patients with malignant gliomas is the presence of the blood brain barrier (BBB) and blood-brain tumor barrier (BBTB) that greatly restricts drug access from the plasma to the tumor cells. Bubble-assisted Focused Ultrasound (BAFUS) has proven effective in opening the BBB for treatment of glial tumors in adults and pediatric cases. BAFUS has been previously shown to disrupt noninvasively, selectively, and transiently the BBB in small animals in vivo. However, there is a lack of an in vitro preclinical model suitable for testing the genetic determinants of endothelial cell tight junction integrity and vulnerability to the physical disruption. Our BBB organ-on-chip platform will enable precision medicine of brain cancers through identifying patient-specific parameters by which to open the BBB allowing use of drugs and drug combinations otherwise unsuitable. We intend to sequence these in vitro models to verify that the genotype (alleles/SNPs) of tight junction proteins contribute to BBB structure and integrity. To initiate this effort, we report the development of an ultrasound transparent organ-on-chip model populated by iPSC-derived endothelial cells (iPSC-EC) co-cultured with astrocytes. Western blot, immunocytochemistry, permeability, and transelectrical endothelial resistance (TEER) studies all convey expression of key EC proteins and marked barrier integrity. Further benchmarking of device-ultrasound interactions, successful iPSC differentiation, tight junction formation, and fabrication of nanobubbles and their assistance in ultrasound BBB disruption will be presented. Efforts are underway to analyze nine characteristic BBB tight junction genes from WGS data to determine associations between iPSC-EC genotype and phenotype. Citation Format: Jayashree Iyer, Adam Akkad, Nanyun Tang, Sen Peng, Michael Berens, Frederic Zenhausern, Jian Gu. A focused ultrasound blood brain barrier disruption model to test the influence of tight junction genes to treat brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 195.
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Treating primary or metastatic tumors in the brain (glioblastomas, melanoma, lung cancer, breast cancer) proves challenging by virtue of the protective function of the blood brain barrier (BBB). Recently, it has been shown that low... more
Treating primary or metastatic tumors in the brain (glioblastomas, melanoma, lung cancer, breast cancer) proves challenging by virtue of the protective function of the blood brain barrier (BBB). Recently, it has been shown that low intensity focused ultrasonic (LIFU) waves stably cavitate infused microbubbles which then mechanically disrupt the tight junctions of the BBB. This leads to temporary, recoverable opening of the BBB, and passage of otherwise disqualified cancer-therapeutic drugs at precise locations targeted by the focused ultrasound. To date, potential genetic influences on the durability and vulnerability of tight junctions to LIFU have not been elucidated, nor have the determinants of tight junction repair post LIFU been thoroughly investigated. We report the development of an ultrasound transparent organ-on-chip model to test LIFU with microbubble infusion treatment on a cell-engineered BBB. The BBB is developed using brain-specific endothelial cells derived from genomically characterized immortalized pluripotent stem cells (iPSC). Furthermore, to test genetic variation effects we propose that alleles coding for the proteins involved in tight junction assembly contribute to LIFU disruption variability. Developing preclinical models of the BBB to accommodate cell sources with tight junction genes of different allele makeup will shed light on how individuals will respond to different ultrasound frequencies. The in vitro BBB device is composed of two orthogonally stacked fluidic channels formed by top and bottom polydimethylsiloxane (PDMS) membranes and a middle polyester membrane with 3 µm pores. An ultrasound system is constructed with a waveform generator, amplifier, and 1MHz ultrasound transducer. A 0.5 MHz receiving transducer and a digital storage oscilloscope are used for stable cavitation monitoring. Nanobubbles (FUS Instruments) transduce the LIFU into a mechanical vibration force to disrupt the BBB. To deliver the ultrasonic waves, the device is submerged in degassed DI water in a custom tank. Fluid flow was achieved, and subharmonic ultrasound signal is observed using the digital oscilloscope with Fast Fourier Transform (FFT). Preliminary results convey stable cavitation with LIFU and the formation of tight junctions in a brain microvascular endothelial cell monolayer in the device, eventually leading to a versatile platform to evaluate genetic-based vulnerability of the BBB. Citation Format: Jayashree Iyer, Adam Akkad, Nanyun Tang, Michael E. Berens, Frederic Zenhausern, Jian Gu. Building an in vitro blood brain barrier model to test the influence of tight junction gene alleles on disruption by focused ultrasound to treat brain tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2984.
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Interferometric near-field optical microscopy achieving a resolution of 10 angstroms is demonstrated. The scattered electric field variation caused by a vibrating probe tip in close proximity to a sample surface is measured by encoding it... more
Interferometric near-field optical microscopy achieving a resolution of 10 angstroms is demonstrated. The scattered electric field variation caused by a vibrating probe tip in close proximity to a sample surface is measured by encoding it as a modulation in the optical phase of one arm of an interferometer. Unlike in regular near-field optical microscopes, where the contrast results from a weak source (or aperture) dipole interacting with the polarizability of the sample, the present form of imaging relies on a fundamentally different contrast mechanism: sensing the dipole-dipole coupling of two externally driven dipoles (the tip and sample dipoles) as their spacing is modulated.
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Yersinia pestis is a Gram-negative bacterium that is the causative agent of plague and is widely recognized as a potential biological weapon. Due to the high fatality rate of plague when diagnosis is delayed, the development of rapid,... more
Yersinia pestis is a Gram-negative bacterium that is the causative agent of plague and is widely recognized as a potential biological weapon. Due to the high fatality rate of plague when diagnosis is delayed, the development of rapid, sensitive, specific, and cost-effective methods is needed for its diagnosis. The Y. pestis low calcium response V (LcrV) protein has been identified as a potential microbial biomarker for the diagnosis of plague. In this paper, we present a highly sensitive, paper-based, vertical flow immunoassay (VFI) prototype for the detection of LcrV and the diagnosis of plague. An antigen-capture assay using monoclonal antibodies is employed to capture and detect the LcrV protein, using a colorimetric approach. In addition, the effect of miniaturizing the VFI device is explored based on two different sizes of VFI platforms, denoted as "large VFI" and "mini VFI." Also, a comparative analysis is performed between the VFI platform and a lateral flow immunoassay (LFI) platform to exhibit the improved assay sensitivity suitable for point-of-care (POC) diagnostics. The analytical sensitivity or limit of detection (LOD) in the mini VFI is approximately 0.025 ng/mL, that is, 10 times better than that of the large VFI platform or 80 times over a standard lateral flow configuration. The low LOD of the LcrV VFI appears to be highly suitable for testing clinical samples and potentially diagnosing plague at earlier time points. In addition, optimization of the gold nanoparticle (AuNP) concentration, nanomaterial plasmonic properties, and flow velocity analysis could improve the performance of the VFI. Furthermore, we developed automated image analysis software that shows potential for integrating the diagnostic system into a smartphone. These methods and findings demonstrate that the VFI platform is a highly sensitive device for detecting the LcrV and potentially many other biomarkers.
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The scanning probe microscopies applied to the sequencing of DNA is a challenging goal attempted by several groups. But one limitant parameter has been the sample preparation of DNA molecules. Here we report how to hold DNA molecules... more
The scanning probe microscopies applied to the sequencing of DNA is a challenging goal attempted by several groups. But one limitant parameter has been the sample preparation of DNA molecules. Here we report how to hold DNA molecules fixed on mica substrate and we show the three-dimensional configuration of double-stranded DNA obtained with our scanning force microscope. We can image DNA under negative supercoiling, a feature of general importance controlling the activities of DNA. We compared the electron micrographs of a carbon replica of the same DNA specimen with scanning force images which demonstrates well the feasibility and accuracy of our scanning probe measurements.
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4471 Pancreatic cancer is the fourth leading cause of death from cancer in the United States. More than 33,000 people nation wide will die from pancreatic cancer this year. The overall five-year survival is less than 4%. This dismal... more
4471 Pancreatic cancer is the fourth leading cause of death from cancer in the United States. More than 33,000 people nation wide will die from pancreatic cancer this year. The overall five-year survival is less than 4%. This dismal picture of pancreatic cancer is mainly due to the lack of early diagnosis and effective treatment for patients with advanced disease. The best way to increase the survival rate is early detection of the disease by screening for “markers”. Analytical methodology using solid phase micro-extraction (SPME) in combination with gas chromatography and mass spectrometry (GC/MS) will be described to find new prognostic and diagnostic volatile organic compounds related to pancreatic cancer. By comparing the GC/MS profiles of normal healthy SPME samples to the profiles resulting from SPME samples of pancreatic cancer cells or patients with pancreatic cancer, a quantifiable differentiation in the component profiles could become apparent.
We will report some preliminary data resulting from the SPME and GC/MS analysis of pancreatic serum samples obtained on 12 samples, 6 of which were obtained from patients with pancreatic cancer and the remain 6 from healthy volunteers. The volatile compound profiles of this limited sample set of serum samples were very similar in chromatographic appearance (as to be expected for the variation in human population) and showed, quantitative subtleties, even in this well known A vs. B biological sample set. However, normalized peak area ratios of peak 2 to peak 7 and peak 6 to peak 7 suggested a differentiation between the normal vs. pancreatic cancer serum samples. Preliminary identifications for the peaks were cyclopentanone (2), m-xylene (6), and cyclohexanone (7). Data analyses for identifying target metabolic pathways and further experimental development will be reported.
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Current in vitro tumor models have issues in accuracy in that the 2D structures and (often rare) cell co-culture technologies that exist, lack many features or characteristics found in vivo. The use of decellularized plant structures... more
Current in vitro tumor models have issues in accuracy in that the 2D structures and (often rare) cell co-culture technologies that exist, lack many features or characteristics found in vivo. The use of decellularized plant structures recellularized with human cells, aims to overcome these issues by taking advantage of their natural 3D structure. By using this approach on spinach leaves as a 3D scaffold, we have developed a new model that may be used as a new tumor model for radiobiology research. Spinach leaves were decellularized following serial chemical treatments with hexanes, SDS, Triton-X100 and bleach. In order to characterize the efficiency of the decellularization process, the rigidity of the leaves was assessed by Atomic Force Microscope (AFM) and DNA and protein quantification. Human prostate (PC3) and breast (MCF7) cancer cells were then seeded onto leaf. Seeding efficiency was assessed by optical microscopy and viability and proliferation ability were tested by MTT assay. In order to evaluate if the cells were biologically active, we then assessed radiation response. Extra cell-seeded leaves were irradiated and the expression of radiation-responsive genes were assessed in MCF7 cells. Additionally, DNA damage levels in PC3 cells were evaluated by γ-H2AX foci measurement using fluorescence microscopy. The decellularization process was successful, showing a protein content of 0.31 μg/mg tissue compared to the fresh leaf at 14.4 μg/mg tissue. The DNA quantity was similarly disparate between fresh and decellularized leaves. Microscopy showed that PC3 and MCF7 cells were well attached to the decellularized leaf surface after 24h incubation. Mechanical testing with AFM confirmed attachment by measuring Young's modulus values of 2.81, 88 and 197 MPa for decellularized, recellularized and fresh leaves respectively. Viability assays confirmed that cells were alive and able to proliferate. The gene expression assay showed changes in expression levels between 2D cell culture and cells seeded on leaves both at basal state and after 5Gy-irradiation in MCF7 cells. Finally, γ-H2AX immunofluorescent imaging showed DNA damage repairs are induced 1 hour after 5Gy of X-ray irradiation in PC3 cells and are effective up to 24h. Plant can be decellularized in order to create a 3D scaffold that may act as a support for cell seeding. Interestingly, radiation response can be measured on this new model and even show significant difference with standard 2D cell culture. Together, these results suggest that this approach is a new promising 3D cellular model for radiation research. However, additional studies are required to compare this model with in vivo response in order to clearly assess if this model is more suitable to mimick in vivo tumor/microenvironment than 2D standard model. Citation Format: Jerome Lacombe, Ryan Zenhausern, Frederic Zenhausern. Vegetal scaffold as radiobiology model to study radiation cancer response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1316.
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We have adapted an existing emerging method to characterize elastic properties of thin (30–150 nm) polystyrene (PS) films based on the observation of the wrinkling patterns of the film (J. Huang et al, Science 2007, 317, 650–653). Our... more
We have adapted an existing emerging method to characterize elastic properties of thin (30–150 nm) polystyrene (PS) films based on the observation of the wrinkling patterns of the film (J. Huang et al, Science 2007, 317, 650–653). Our goal is to evaluate the stiffness of cell layers cultured on top of such polystyrene films when they enter metastasis as compared to healthy epithelial cells. Recent reports using atomic force microscopy (AFM) suggest differences in cell stiffness at the local single cell level (J.K. Gimzewski et al, Nature Nanotechnology 2007, 2, 780–783). We plan to measure similar properties when cells are arranged in a sheet and compare them to AFM measurements. A relevant system is the melanoma cell lines such as A 375 and SK-Mel28, for instance. We characterized the wrinkling patterns obtained using a stereomicroscope to provide a first straightforward assessment of the elastic properties by counting the number of wrinkles and measuring their average lengths. By comparing the wrinkling pattern when either healthy or melanoma cells are grown on top of the thin PS films to that obtained when bare thin PS film floats on water and developing a theoretical model based on 2 films of different but close Young moduli associated in parallel, one can extract quantities of interest to characterize the elastic properties of normal vs. cancer cells. In addition, we propose to use digital holographic microscopy (DHM) to improve the observation of the wrinkling patterns and allow access to other relevant viscoelastic information such as relaxation of the films. Because we propose an innovative technique coupled with an emerging form of microscopy yet unexplored for cancer research, i.e. DHM, to study the mechanical elastic forces within an interconnected assembly of metastatic cells with the goal of complementing current methods of clinical diagnostic, we hope to answer the current need for new ways of studying the mechanisms of cancer. Citation Information: Cancer Res 2009;69(23 Suppl):C69.
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Introduction Analysis of biological samples for volatile organic compound (VOC) profiles or fingerprints can potentially be utilized as a non-invasive, early stage diagnostic tool for disease related to metabolic processes. A hypothesis... more
Introduction Analysis of biological samples for volatile organic compound (VOC) profiles or fingerprints can potentially be utilized as a non-invasive, early stage diagnostic tool for disease related to metabolic processes. A hypothesis was presented in 1989 that dogs could detect malignant tumors by scent and several studies of canine scent detection for cancer followed. Our team has taken an analogous approach using solid phase microextraction in combination with capillary gas chromatography time of flight mass spectrometry (SPME GC-TOFMS) in place of canine scent detection to discover indicators for pancreatic cancer, a deadly cancer difficult to detect in its early development stage. VOCs are not commonly considered in “biomarker” analysis of clinical samples which usually require extensive pre-processing. The VOC fingerprint from a biological sample presumes many such substances are products, by-products, and waste resulting from human metabolism (and/or symbiotic microbiom) of nutrients and intermediates from endogenous processes, absorbed environmental contaminants, and endo/exogenous bacterial metabolism. Methods IRB approval was obtained to enroll patients with advanced pancreatic cancer and healthy volunteers. Blood was collected in pre-conditioned vacutainers from 11 patients and 5 healthy volunteers, all non-smokers, no chemotherapy in preceding 5 days, and analyzed within 2 weeks. SPME GC-TOFMS is a means for eliminating the biological matrix and pre-concentrating specific ranges of complex volatile fraction polarities at low concentrations and a very sensitive analytical instrumental platform for the characterization of VOC chromatographic profiles extracted from the 3ml blood samples. Multivariate statistical methods were used to analysis retention time exact mass observations and integrated peak areas. Results Even with the numerous variables contributing to an already diverse underlying metabolism of each participant, our chemometric approach did discriminate between “healthy” volunteers and the cancer patients with a 5% prediction error using a “leave one out” statistical analysis for 37 samplesb from the 16 subjects. Key retention time and exact mass observations from a fiber specific SPME were associated to preliminary molecule identifications via NIST data base searches. Our procedures for system tune, calibration, and monitoring achieved a response stability over a 1 month period of ∼10% rsd and accurate mass measurement of at least 5 mDa (30 - 300 Da) using benzene-d6, BFB, and naphthalene-d8. Conclusions VOC fingerprints from a SPME GC-TOFMS analysis of blood can be successful in discovering differentiating indicators related to APC. Future work will involve other SPME fibers and more samples. (Supported by the IBIS & Scottsdale Health Care Foundations) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1264. doi:1538-7445.AM2012-1264
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Tip/sample interaction on the scanning force microscope (SFM) is a particularly difficult problem with biological materials. One major factor affecting image quality is the tip shape. Improved electron beam induced deposition technique... more
Tip/sample interaction on the scanning force microscope (SFM) is a particularly difficult problem with biological materials. One major factor affecting image quality is the tip shape. Improved electron beam induced deposition technique with a scanning electron microscope (SEM) was used for the reproducible fabrication of carbon sharp tips on the end of commercially available silicon nitride cantilevers for scanning force microscopy. By aligning a fine focused beam of 20 nm diameter directly down the axis of the pyramidal tip at electron energy of 20 kV, carbon deposits grow with full cone angle of about 25°, cone length of 2 μm, and radii of curvature down to 10 nm, making these e-beam tips suitable for biomolecules imaging. The tip dimensions also were controlled by adjusting the beam parameters. Three different types of SFM tips were used to image tobacco mosaic virus (TMV). Conventional pyramidal tips appeared generally worse for imaging helical particles of TMV than SEM-deposited tips which were found more robust than commercially available conical tips. The use of sharper tips for SFM imaging of protein DNA revealed a 25% improvement in lateral resolution.
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Research Interests: Materials Science, Electron Microscopy, Microscopy, Scanning Electron Microscopy, Titanium, and 10 moreTransmission Electron Microscopy, Cryopreservation, Medicine, Mica, Ultramicroscopy, Scanning Force Microscopy, Optical physics, Scanning Electron Microscope, Cryo Electron Microscopy, and Phosphotungstic Acid
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Nano optical biosensors employ the interaction between biomolecules and light confined in nanometer scale structures to report the bio-recognition events. This small scale sensing area/volume can ensure that small amount of biorecognition... more
Nano optical biosensors employ the interaction between biomolecules and light confined in nanometer scale structures to report the bio-recognition events. This small scale sensing area/volume can ensure that small amount of biorecognition events could be detected. The exceptional sensitivity and high spatial density of nano optical biosensors make them unique in practical applications in nucleic acid detection. Lab-on-a-Chip systems provide the capabilities of separation, cell lysing, polymerase chain reaction (PCR), allowing finishing bio agent detection processes on a chip. In this paper, we present our recent efforts on integrating some novel nanooptical biosensors into Lab-on-a-Chip systems and some preliminary test results.
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Research Interests: Medicine and Biodosimetry
We report our fabrication of nanoscale devices using electron beam and nanoimprint lithography (NIL). We focus our study in the emerging fields of NIL, nanophotonics and nanobiotechnology and give a few examples as to how these... more
We report our fabrication of nanoscale devices using electron beam and nanoimprint lithography (NIL). We focus our study in the emerging fields of NIL, nanophotonics and nanobiotechnology and give a few examples as to how these nanodevices may be applied toward genomic ...
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The purpose of this research is to evaluate the supercritical carbon dioxide (scCO2) sterilization-based NovaClean process for decontamination and reprocessing of personal protective equipment (PPE) such as surgical masks, cloth masks,... more
The purpose of this research is to evaluate the supercritical carbon dioxide (scCO2) sterilization-based NovaClean process for decontamination and reprocessing of personal protective equipment (PPE) such as surgical masks, cloth masks, and N95 respirators. Preliminarily, Bacillus atrophaeus were inoculated into different environments (dry, hydrated, and saliva) to imitate coughing and sneezing and serve as a “worst-case” regarding challenged PPE. The inactivation of the microbes by scCO2 sterilization with NovaKill or H2O2 sterilant was investigated as a function of exposure times ranging from 5 to 90 minutes with a goal of elucidating possible mechanisms. Also, human coronavirus SARS-CoV-2 and HCoV-NL63 were inoculated on the respirator material, and viral activity was determined post-treatment. Moreover, we investigated the reprocessing ability of scCO2-based decontamination using wettability testing and surface mapping. Different inactivation mechanisms have been identified in scCO2 sanitization, such as membrane damage, germination defect, and dipicolinic acid leaks. Moreover, the viral sanitization results showed a complete inactivation of both coronavirus HCoV-NL63 and SARS-CoV-2. We did not observe changes in PPE morphology, topographical structure, or material integrity, and in accordance with the WHO recommendation, maintained wettability post-processing. These experiments establish a foundational understanding of critical elements for the decontamination and reuse of PPE in any setting and provide a direction for future research in the field.
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All polymer field effect transistors have been fabricated combining nanoimprint lithography and inkjet printing. Trenches with hydrophilic bottoms confined by hydrophobic walls with considerable height are patterned by nanoimprint... more
All polymer field effect transistors have been fabricated combining nanoimprint lithography and inkjet printing. Trenches with hydrophilic bottoms confined by hydrophobic walls with considerable height are patterned by nanoimprint lithography. Conducting polymer solutions were then delivered into these trench liquid containers by inkjet printing. Dried conducting polymer in nearby trenches forms source-drain electrodes with the channel length accurately defined by the gap in between the designed two trenches. Top-gate all polymer field effect transistors with submicron channel lengths were successfully realized by such low-cost process.
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Interferometric near-field optical microscopy achieving a resolution of 10 angstroms is demonstrated. The scattered electric field variation caused by a vibrating probe tip in close proximity to a sample surface is measured by encoding it... more
Interferometric near-field optical microscopy achieving a resolution of 10 angstroms is demonstrated. The scattered electric field variation caused by a vibrating probe tip in close proximity to a sample surface is measured by encoding it as a modulation in the optical phase of one arm of an interferometer. Unlike in regular near-field optical microscopes, where the contrast results from a weak source (or aperture) dipole interacting with the polarizability of the sample, the present form of imaging relies on a fundamentally different contrast mechanism: sensing the dipole-dipole coupling of two externally driven dipoles (the tip and sample dipoles) as their spacing is modulated.
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We have constructed nanoscale molecular traps using electrodeless, or insulator-based, dielectrophoresis [1, 2]. The molecular traps consist an array of nanoscale dielectric constrictions defined using electron-beam lithography on... more
We have constructed nanoscale molecular traps using electrodeless, or insulator-based, dielectrophoresis [1, 2]. The molecular traps consist an array of nanoscale dielectric constrictions defined using electron-beam lithography on nanofluidic passages. The device was then sealed using an extremely simple room-temperature sealing process with virtually no pressure applied. Upon the application of an external ac electric field, the field will be