An Electrically Active Microneedle Electroporation Array for Intracellular Delivery of Biomolecules

The objective of this research is the development of an electrically active microneedle array that can deliver biomolecules such as DNA and drugs to epidermal cells by means of electroporation. Properly metallized microneedles could serve as microelectrodes essential for electroporation. Furthermore, the close needle-to-needle spacing of microneedle electrodes provides the advantage of utilizing reduced voltage, which is essential for safety as well as portable applications, while maintaining the large electric fields required for electroporation. Therefore, microneedle arrays can potentially be used as part of a minimally invasive, highly-localized electroporation system for cells in the epidermis layer of the skin. This research consists of three parts: development of the 3-D microfabrication technology to create the microneedle array, fabrication and characterization of the microneedle array, and the electroporation studies performed with the microneedle array. A 3-D fabrication process was developed to produce a microneedle array using an inclined UV exposure technique combined with micromolding technology, potentially enabling low cost mass-manufacture. The developed technology is also capable of fabricating 3-D microstructures of various heights using a single mask. The fabricated microneedle array was then tested to demonstrate its feasibility for through-skin electrical and mechanical functionality using a skin insertion test. It was found that the microneedles were able to penetrate skin without breakage. To study the electrical properties of the array, a finite element simulation was performed to examine the electric field distribution. From these simulation results, a predictive model was constructed to estimate the effective volume for electroporation. Finally, studies to determine hemoglobin release from bovine red blood cells (RBC) and the delivery of molecules such as calcein and bovine serum albumin (BSA) into human prostate cancer cells were used to verify the electrical functionality of this device. This work established that this device can be used to lyse RBC and to deliver molecules, e.g. calcein, into cells, thus supporting our contention that this metallized microneedle array can be used to perform electroporation at reduced voltage. Further studies to show efficacy in skin should now be performed.Ph.D.Committee Chair: Mark G. Allen; Committee Member: Mark R. Prausnitz; Committee Member: Oliver Brand; Committee Member: Pamela Bhatti; Committee Member: Shyh-Chiang She

ELECTROCHEMICAL PROCESSES IN MICROFLUIDICS SYSTEMS UNDER AC ELECTRIC FIELDS

Alternating current (AC) electric signal has been widely applied in microfluidic systems to induce AC electrokinetic behavior. AC electrokinetic phenomena including AC electrophoresis, AC dielectrophoresis, AC electroosmosis flow and AC electrothermal flow are widely applied in (bio)particle sorting, separation and concentration and micropumps. However, numbers of non-ideal AC electrokinetic behaviors have been reported: human erythrocyte deformation in AC dielectrophoresis system has been observed; flow reversal in AC electroosmosis flow pumps has also been reported. In this dissertation, a systematic study on human erythrocyte crenation in AC dielectrophoresis system was firstly conducted. Multiple possible physical mechanisms inducing cell crenation including temperature, temperature jump, pH, shear force and osmotic pressure was examined. pH change and osmotic pressure change induced by ionic concentration change was attributed as mechanisms induced cell crenation. Such pH change and ionic concentration gradient in AC non-uniform electric fields were then further studied. pH change was detected using Fluorescein sodium salt in sodium chloride solution at relative frequency ~5 to 25 times to electrode charging frequency. Time, spatial, frequency and peak-to-peak potential dependencies have been examined on such pH change and pH gradient. Ion concentration behavior was detected by using ionized FITC molecule as fluorescing ion dissolved in inert solvent methanol. Electrode surface is also coated with HfO2 dielectric layer to minimize effect from electrochemical reaction on FITC emission intensity. Time, spatial, frequency and peak-to-peak potential dependencies have also been examined on ion concentration change and ion concentration gradient. Both pH change and ion concentration change have been observed under high relative AC frequency

Fabrication and characterization of a mcro/nanofluidic platform for electroporation.

For traditional electroporation devices, there are a number of problems associated with these devices such as insufficient understanding of its theoretical mechanism, low cell viability, inadequate electroporation efficiency, excess voltage applied to generate required electric field due to the large size of these devices and sample contamination. Although newly developed microfluidic electroporation devices have solved most of the above existing problems in traditional bulk electroporation devices, they appear to lack the ability to control the precise dose of biomolecules or genes transfecting into cells and, from a manufacturing perspective, the fabrication methods do not enable repeatable production of such devices on the large scale. Here, we introduce a new, repeatable method for fabricating 3-D Micro/Nanofluidic electroporation platforms and characterize these platforms to demonstrate their ability to electroporate live cells. Some of the new methods developed in this work include a direct-write fiber technique via three-axis robotic dispensing system, dry film resist photolithography, film-to-film bonding and replica molding to create the desired electroporation platform. A robotic dispensing system was utilized to control the fiber diameter, which was determined vii by the: 1) prescribed dispense time; 2) pressure of the dispensing system valve; 3) rate at which the stage traversed; 4) diameter of the dispensing tip; 5) polymer solution viscosity and surface tension; and, 6) programmed drawing length. Thin dry film photoresist was utilized to replace liquid photoresist in order to achieve high-quality film-to-film bonding after drawing nanofibers onto one substrate containing the thin-film structure. Polydimethylsiloxane (PDMS) was employed as the bulk material to fabricate the target micro/nano electroporation substrate using replica molding and micro/nanofibers etching. Characterization of the direct-write fiber technique via robotic dispensing system to acquire suspended and complex fibers of the required dimension repeatedly under prescribed conditions were completed. Combining this fiber direct-write method and traditional clean room techniques, a total of 18 micro- to nano-scale electroporation devices (6 for each group of 1 ìm, 500 nm, and 300 nm diameter) were successfully developed and mass produced in two weeks with relatively high repeatability (within 20% of the design). Finally, metrology and characterization studies were performed on the electroporation platforms to validate the micro/nanochannel’s existence and its connectivity to two micro-chambers. Furthermore, biomolecules and other fluorescent particles were successfully transported through the micro/nanochannel and transferred (via electroporation) into the cells. Preliminary results of electroporation experiment performed on this micro/nano-electroporation platform illustrated that the duration of the entire electroporation process was significantly shorter than times reported previously by other investigator’s nano-electroporation platforms

A Theoretical Analysis of the Feasibility of a Singularity-Induced Micro-Electroporation System

Electroporation, the permeabilization of the cell membrane lipid bilayer due to a pulsed electric field, has important implications in the biotechnology, medicine, and food industries. Traditional macro and micro-electroporation devices have facing electrodes, and require significant potential differences to induce electroporation. The goal of this theoretical study is to investigate the feasibility of singularity-induced micro-electroporation; an electroporation configuration aimed at minimizing the potential differences required to induce electroporation by separating adjacent electrodes with a nanometer-scale insulator. In particular, this study aims to understand the effect of (1) insulator thickness and (2) electrode kinetics on electric field distributions in the singularity-induced micro-electroporation configuration. A non-dimensional primary current distribution model of the micro-electroporation channel shows that while increasing insulator thickness results in smaller electric field magnitudes, electroporation can still be performed with insulators thick enough to be made with microfabrication techniques. Furthermore, a secondary current distribution model of the singularity-induced micro-electroporation configuration with inert platinum electrodes and water electrolyte indicates that electrode kinetics do not inhibit charge transfer to the extent that prohibitively large potential differences are required to perform electroporation. These results indicate that singularity-induced micro-electroporation could be used to develop an electroporation system that consumes minimal power, making it suitable for remote applications such as the sterilization of water and other liquids

Development of Cell Lysis Techniques in Lab on a chip

The recent breakthroughs in genomics and molecular diagnostics will not be reflected in health-care systems unless the biogenetic or other nucleic acid-based tests are transferred from the laboratory to clinical market. Developments in microfabrication techniques brought lab-on-a-chip (LOC) into being the best candidate for conducting sample preparation for such clinical devices, or point-of-care testing set-ups. Sample preparation procedure consists of several stages including cell transportation, separation, cell lysis and nucleic acid purification and detection. LOC, as a subset of Microelectromechanical systems (MEMS), refers to a tiny, compact, portable, automated and easy-to-use microchip capable of performing the sample-preparation stages together. Complexity in micro-fabrications and inconsistency of the stages oppose integration of them into one chip. Among the variety of mechanisms utilized in LOC for cell lysis, electrical methods have the highest potential to be integrated with other microchip-based mechanisms. There are, however, major limitations in electrical cell lysis methods: the difficulty and high-cost fabrication of microfluidic chips and the high voltage requirements for cell lysis. Addressing these limitations, the focus of this thesis is on realization of cell lysis microchips suitable for LOC applications. We have developed a new methodology of fabricating microfluidic chips with electrical functionality. Traditional lithography of microchannel with electrode, needed for making electro-microfluidic chips, is considerably complicated. We have combined several easy-to-implement techniques to realize electro-microchannel with laser-ablated polyimide. The current techniques for etching polyimide are by excimer lasers in bulky set-ups and with involvement of toxic gas. We present a method of ablating microfluidic channels in polyimide using a 30W CO2 laser. Although this technique has poorer resolution, this approach is more cost effective, safer and easier to handle. We have verified the performance of the fabricated electro-microfluidic chips on electroporation of mammalian cells. Electrical cell lysis mechanisms need an operational voltage that is relatively high compared to other cell manipulation techniques, especially for lysing bacteria. Microelectro-devices have dealt with this limitation mostly by reducing the inter-distance of electrodes. The technique has been realized in tiny flow-through microchips with built-in electrodes in a distance of a few micrometers which is in the scale of cell size. In addition to the low throughput of such devices, high probability of blocking cells in such tiny channels is a serious challenge. We have developed a cell lysis device featured with aligned carbon nanotube (CNT) to reduce the high voltage requirement and to improve the throughput. The vertically aligned CNT on an electrode inside a MEMS device provides highly strengthened electric field near the tip. The concept of strengthened electric field by means of CNT has been applied in field electron emission but not in cell lysis. The results show that the incorporation of CNT in lysing bacteria reduces the required operational voltage and improves throughput. This achievement is a significant progress toward integration of cell lysis in a low-voltage, high-throughput LOC. We further developed the proposed fabrication methodology of micro-electro-fluidic chips, described earlier, to perform electroporation of single mammalian cell. We have advanced the method of embedding CNT in microchannel so that on-chip fluorescent microscopy is also feasible. The results verify the enhancement of electroporation by incorporating CNT into electrical cell lysis. In addition, a novel methodology of making CNT-embedded microfluidic devices has been presented. The embedding methodology is an opening toward fabrication of a CNT-featured LOC for other applications

Ablation of Cardiac Tissue with Nanosecond Pulsed Electric Fields: Experiments and Numerical Simulations

Cardiac ablation for the treatment of cardiac arrhythmia is usually performed by heating tissue with radio-frequency (RF) electrical currents to create conduction-blocking lesions in order to stop the propagation of electrical waves. Problems associated with RF ablation are recurrence of arrhythmias after successful treatments, tissue loss beyond the targeted tissue, long duration of the ablation procedure, and thermal side effects including thrombus formation that may lead to stroke. Here, we propose a new, non-thermal ablation method using nanosecond pulsed electric fields (nsPEFs) with better-controlled ablation volume, shorter procedure time, and no thermal side effects. We demonstrate that we can create non-conductive transmural lesions using different electrode configurations. We also develop a numerical model of nsPEF ablation, which allows us to estimate the critical electric field which leads in cardiac tissue and helps to provide a guideline for clinical tissue ablation. Our experimental model is a Langendorff-perfused rabbit heart. The heart is placed in a life-support system, and optical mapping is performed to study its electrical activity. We further developed the capability to apply short sequences of nanosecond pulses to tissue through pairs of customized electrodes. In order to characterize the 3D geometry of an ablated volume, we have adopted propidium iodide and TTC staining in conjunction with tissue sectioning. Our results obtained by optical mapping data and PI/TTC stained tissue show that fully transmural lesions can be obtained faster and with better control over the ablated volume than in conventional (RF) ablation, in the absence of thermal side effects. In order to aid nsPEF ablation planning, we used the COMSOL finite element software to create a model of the electric field distribution in cardiac tissue, which has a complex anisotropic architecture, for different electrode configurations. The experimental and numerical results are consistent and suggest a critical electric field strength of 3kV/cm for the death of cardiac tissue. This threshold obtained by the numerical model can function as a guideline for future clinical nsPEF treatment of atrial fibrillation. In summary, we have developed nsPEF ablation for the treatment of cardiac arrhythmia to provide better control over the ablated volume than conventional (RF) ablation, to reduce procedure time, and to avoid thermal side effects. Our ultimate goal is to bring this technology to the clinical practice

Single Cell Analysis

Cells are the most fundamental building block of all living organisms. The investigation of any type of disease mechanism and its progression still remains challenging due to cellular heterogeneity characteristics and physiological state of cells in a given population. The bulk measurement of millions of cells together can provide some general information on cells, but it cannot evolve the cellular heterogeneity and molecular dynamics in a certain cell population. Compared to this bulk or the average measurement of a large number of cells together, single-cell analysis can provide detailed information on each cell, which could assist in developing an understanding of the specific biological context of cells, such as tumor progression or issues around stem cells. Single-cell omics can provide valuable information about functional mutation and a copy number of variations of cells. Information from single-cell investigations can help to produce a better understanding of intracellular interactions and environmental responses of cellular organelles, which can be beneficial for therapeutics development and diagnostics purposes. This Special Issue is inviting articles related to single-cell analysis and its advantages, limitations, and future prospects regarding health benefits

High Throughput and Highly Controllable Methods for in Vitro Intracellular Delivery

In vitro and ex vivo intracellular delivery methods hold the key for releasing the full potential of tissue engineering, drug development, and many other applications. In recent years, there has been significant progress in the design and implementation of intracellular delivery systems capable of delivery at the same scale as viral transfection and bulk electroporation but offering fewer adverse outcomes. This review strives to examine a variety of methods for in vitro and ex vivo intracellular delivery such as flow-through microfluidics, engineered substrates, and automated probe-based systems from the perspective of throughput and control. Special attention is paid to a particularly promising method of electroporation using micro/nanochannel based porous substrates, which expose small patches of cell membrane to permeabilizing electric field. Porous substrate electroporation parameters discussed include system design, cells and cargos used, transfection efficiency and cell viability, and the electric field and its effects on molecular transport. The review concludes with discussion of potential new innovations which can arise from specific aspects of porous substrate-based electroporation platforms and high throughput, high control methods in general

Micro Electromechanical Systems (MEMS) Based Microfluidic Devices for Biomedical Applications

Micro Electromechanical Systems (MEMS) based microfluidic devices have gained popularity in biomedicine field over the last few years. In this paper, a comprehensive overview of microfluidic devices such as micropumps and microneedles has been presented for biomedical applications. The aim of this paper is to present the major features and issues related to micropumps and microneedles, e.g., working principles, actuation methods, fabrication techniques, construction, performance parameters, failure analysis, testing, safety issues, applications, commercialization issues and future prospects. Based on the actuation mechanisms, the micropumps are classified into two main types, i.e., mechanical and non-mechanical micropumps. Microneedles can be categorized according to their structure, fabrication process, material, overall shape, tip shape, size, array density and application. The presented literature review on micropumps and microneedles will provide comprehensive information for researchers working on design and development of microfluidic devices for biomedical applications

Intracellular delivery by membrane disruption: Mechanisms, strategies, and concepts

© 2018 American Chemical Society. Intracellular delivery is a key step in biological research and has enabled decades of biomedical discoveries. It is also becoming increasingly important in industrial and medical applications ranging from biomanufacture to cell-based therapies. Here, we review techniques for membrane disruption-based intracellular delivery from 1911 until the present. These methods achieve rapid, direct, and universal delivery of almost any cargo molecule or material that can be dispersed in solution. We start by covering the motivations for intracellular delivery and the challenges associated with the different cargo typesñYsmall molecules, proteins/peptides, nucleic acids, synthetic nanomaterials, and large cargo. The review then presents a broad comparison of delivery strategies followed by an analysis of membrane disruption mechanisms and the biology of the cell response. We cover mechanical, electrical, thermal, optical, and chemical strategies of membrane disruption with a particular emphasis on their applications and challenges to implementation. Throughout, we highlight specific mechanisms of membrane disruption and suggest areas in need of further experimentation. We hope the concepts discussed in our review inspire scientists and engineers with further ideas to improve intracellular delivery