Targeted messenger RNA profiling of transfected breast cancer gene in a living cell.
ABSTRACT: Selective messenger RNA (mRNA) profiling of transfected breast cancer gene expression in a living cell is demonstrated. Atomic force microscope (AFM) probe tips are structurally modified to create a dielectrophoretic force that attracts mRNA molecules within the cell nucleus. The tip end is chemically modified to hybridize only to the target mRNA from a pool of molecules within the nucleus. We successfully combined this scheme with standard assay techniques to develop an assay technology that can be used for early disease detection and basic studies in cell biology.
Project description:We report on a selective and nondestructive measurement of mRNA (messenger ribonucleic acid) expression levels within a living cell. We first modify an atomic force microscope tip to create a tapered nanoscale coaxial cable. Application of an ac (alternating potential) between the inner and outer electrodes of this cable creates a dielectrophoretic force attracting mRNA molecules toward the tip-end which is pretreated with gene specific primers. We selectively extracted and analyzed both high ( approximately 2500) and extremely low (11 0) copy number mRNA from a living cell mRNA in less than 10 s.
Project description:This study proposes a capillary dielectrophoretic chip to separate blood cells from a drop of whole blood (approximately 1 μl) sample using negative dielectrophoretic force. The separating efficiency was evaluated by analyzing the image before and after dielectrophoretic force manipulation. Blood samples with various hematocrits (10%-60%) were tested with varied separating voltages and chip designs. In this study, a chip with 50 μm gap design achieved a separation efficiency of approximately 90% within 30 s when the hematocrit was in the range of 10%-50%. Furthermore, glucose concentration was electrochemically measured by separating electrodes following manipulation. The current response increased significantly (8.8-fold) after blood cell separation, which was attributed not only to the blood cell separation but also to sample disturbance by the dielectrophoretic force.
Project description:In this work, we studied the stretching of ? phage DNA molecules immobilized on an optical fiber tip attached to a force sensitive tuning fork under ac electric fields. We designed a two electrodes stretching system in a small chamber: one is a gold-coated optical fiber tip electrode, and the other is a gold-coated flat electrode. By applying a dielectrophoretic (DEP) force, the immobilized ? DNA molecules on the tip are stretched and the stretching process is monitored by a fluorescent microscope. The DNA stretching in three-dimensional space is optimized by varying electrode shape, electrode gap distance, ac frequency, and solution conductivity. By observing the vibrational amplitude change of a quartz tuning fork, we measured the effects due to Joule heating and the DEP force on the tethered DNA molecules in solution. This work demonstrates a method to manipulate and characterize immobilized ? DNA molecules on a probe tip for further study of single DNA molecules.
Project description:Transport of messenger RNA (mRNA) from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. In the cell nucleus, a precursor mRNA undergoes a series of processing steps, including capping at the 5' ends, splicing and cleavage/polyadenylation at the 3' ends. During this process, the mRNA associates with a wide variety of proteins, forming a messenger ribonucleoprotein (mRNP) particle. Association with factors involved in nuclear export also occurs during transcription and processing, and thus nuclear export is fully integrated into mRNA maturation. The coupling between mRNA maturation and nuclear export is an important mechanism for providing only fully functional and competent mRNA to the cytoplasmic translational machinery, thereby ensuring accuracy and swiftness of gene expression. This review describes the molecular mechanism of nuclear mRNA export mediated by the principal transport factors, including Tap-p15 and the TREX complex.
Project description:We experimentally demonstrate dielectrophoretic concentration of biological analytes on the surface of a gold nanohole array, which concurrently acts as a nanoplasmonic sensor and gradient force generator. The combination of nanohole-enhanced dielectrophoresis, electroosmosis, and extraordinary optical transmission through the periodic gold nanohole array enables real-time label-free detection of analyte molecules in a 5 μL droplet using concentrations as low as 1 pM within a few minutes, which is more than 1000 times faster than purely diffusion-based binding. The nanohole-based optofluidic platform demonstrated here is straightforward to construct, applicable to both charged and neutral molecules, and performs a novel function that cannot be accomplished using conventional surface plasmon resonance sensors.
Project description:Chimeric major histocompatibility complex (MHC) molecules supplemented with T cell receptor (TCR) signaling motifs function as activation receptors and can redirect gene-modified T cells against pathogenic CD8 T cells. We have shown that ?2 microglobulin (?2m) operates as a universal signaling component of MHC-I molecules when fused with the CD3-? chain. Linking the H-2Kd-binding insulin B chain peptide insulin B chain, amino acids 15-23 (InsB15-23) to the N terminus of ?2m/CD3-?, redirected polyclonal CD8 T cells against pathogenic CD8 T cells in a peptide-specific manner in the non-obese diabetic (NOD) mouse. Here, we describe mRNA electroporation for delivering peptide/?2m/CD3-? genes to a reporter T cell line and purified primary mouse CD8 T cells. The peptide/?2m/CD3-? products paired with endogenous MHC-I chains and transmitted strong activation signals upon MHC-I cross-linking. The reporter T cell line transfected with InsB15-23/?2m/CD3-? mRNA was activated by an InsB15-23-H-2Kd-specific CD8 T cell hybrid only when the transfected T cells expressed H-2Kd. Primary NOD CD8 T cells expressing either InsB15-23/?2m/CD3-? or islet-specific glucose-6-phosphatase catalytic subunit-related protein, amino acids 206-214 (IGRP206-214)/?2m/CD3-? killed their respective autoreactive CD8 T cell targets in vitro. Furthermore, transfer of primary CD8 T cells transfected with InsB15-23/?2m/CD3-? mRNA significantly reduced insulitis and protected NOD mice from diabetes. Our results demonstrate that mRNA encoding chimeric MHC-I receptors can redirect effector CD8 against diabetogenic CD8 T cells, offering a new approach for the treatment of type 1 diabetes.
Project description:The motion of polarizable particles in a nonuniform electric field (i.e., dielectrophoresis) has been extensively used for concentration, separation, sorting, and transport of biological particles from cancer cells and viruses to biomolecules such as DNAs and proteins. However, current approaches to dielectrophoretic manipulation are not sensitive enough to selectively target individual molecular species. Here, we describe the application of the dielectrophoretic principle for selective detection of DNA and RNA molecules using an engineered biological nanopore. The key element of our approach is a synthetic polycationic nanocarrier that selectively binds to the target biomolecules, dramatically increasing their dielectrophoretic response to the electric field gradient generated by the nanopore. The dielectrophoretic capture of the nanocarrier-target complexes is detected as a transient blockade of the nanopore ionic current, while any nontarget nucleic acids are repelled from the nanopore by electrophoresis and thus do not interfere with the signal produced by the target's capture. Strikingly, we show that even modestly charged nanocarriers can be used to capture DNA or RNA molecules of any length or secondary structure and simultaneously detect several molecular targets. Such selective, multiplex molecular detection technology would be highly desirable for real-time analysis of complex clinical samples.
Project description:We have formulated the dielectrophoretic force exerted on micro/nanoparticles upon the application of frequency-modulated (FM) electric fields. By adjusting the frequency range of an FM wave to cover the crossover frequency f X in the real part of the Clausius-Mossotti factor, our theory predicts the reversal of the dielectrophoretic force each time the instantaneous frequency periodically traverses f X . In fact, we observed periodic U-turns of vesicles, leukemia cells, and red blood cells that undergo FM wave dielectrophoresis (FM-DEP). It is also suggested by our theory that the video tracking of the U-turns due to FM-DEP is available for the agile and accurate measurement of f X . The FM-DEP method requires a short duration, less than 30 s, while applying the FM wave to observe several U-turns, and the agility in measuring f X is of much use for not only salty cell suspensions but also nanoparticles because the electric-field-induced solvent flow is suppressed as much as possible. The accuracy of f X has been verified using two types of experiment. First, we measured the attractive force exerted on a single vesicle experiencing alternating-current dielectrophoresis (AC-DEP) at various frequencies of sinusoidal electric fields. The frequency dependence of the dielectrophoretic force yields f X as a characteristic frequency at which the force vanishes. Comparing the AC-DEP result of f X with that obtained from the FM-DEP method, both results of f X were found to coincide with each other. Second, we investigated the conductivity dependencies of f X for three kinds of cell by changing the surrounding electrolytes. From the experimental results, we evaluated simultaneously both of the cytoplasmic conductivities and the membrane capacitances using an elaborate theory on the single-shell model of biological cells. While the cytoplasmic conductivities, similar for these cells, were slightly lower than the range of previous reports, the membrane capacitances obtained were in good agreement with those previously reported in the literature.
Project description:Characterization of cellular dielectrophoretic (DEP) behaviors, when cells are exposed to an alternating current (AC) electric field of varying frequency, is fundamentally important to many applications using dielectrophoresis. However, to date, that characterization has been performed with monotonically increasing or decreasing frequency, not with successive increases and decreases, even though cells might behave differently with those frequency modulations due to the nonlinear cellular electrodynamic responses reported in previous works. In this report, we present a method to trace the behaviors of numerous cells simultaneously at the single-cell level in a simple, robust manner using dielectrophoretic tweezers-based force spectroscopy. Using this method, the behaviors of more than 150 cells were traced in a single environment at the same time, while a modulated DEP force acted upon them, resulting in characterization of nonlinear DEP cellular behaviors and generation of different cross-over frequencies in living cells by modulating the DEP force. This study demonstrated that living cells can have non-linear di-polarized responses depending on the modulation direction of the applied frequency as well as providing a simple and reliable platform from which to measure a cellular cross-over frequency and characterize its nonlinear property.
Project description:This work determines the dielectrophoretic response of surface modified polystyrene and silica colloidal particles by experimentally measuring their Clausius-Mossotti factors. Commercial charged particles, fabricated ones coated with fibronectin, and Janus particles that have been grafted with fibronectin on one side only were investigated. We show that the dielectrophoretic response of such particles can be controlled by the modification of the chemistry or the anisotropy of their surface. Moreover, by modelling the polarizabilities of those particles, the dielectric parameters of the particles and the grafted layer of protein can be measured.