The invention of pronuclear microinjection initiated the field of transgenic research. Over 30 years later microinjection remains the most straight-forward and most commonly used transgene delivery option. In this work we address the current progress of microelectromechanical systems (MEMS) used as transgenic delivery mechanisms. The nanoinjector is a specially designed MEMS device which uses electrostatic charge to manipulate transgene molecules. The process of nanoinjection was designed as an alternative to microinjection which causes less damage to developing embryos, improves embryo survival, birth rates, and overall efficiency of injections. In vivo testing of nanoinjection demonstrates it is both safe and effective. Additionally nanoinjection has the potential to make transgenesis via yeast artificial chromosomes more practical as the nanoinjector may prevent shearing of the YAC molecules. A second nanoinjection protocol termed intracellular electroporetic nanoinjcetion (IEN) was designed to allow for cytoplasmic injections. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In IEN injections the nanoinjector is used to place transgenic molecules in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronucleus using electrical pulses. Electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm, giving transgene access to the pronucleus before degradation can occur. In this report we describe the principles which allow for localized electroporation of the pronuclei including: the location of the pronuclei between 21-28 hours post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on the movement of transgenic DNA based on the voltages delivered by IEN. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.

Delivering foreign molecules into living cells is a broad and ongoing area of research. Gene therapy, or delivering nucleic acids into cells via non-viral or viral pathways, is an especially promising area for pharmaceutics. All gene therapy methods have their respective advantages and disadvantages, including limited delivery efficiency and low viability. Nanoinjection, or delivering molecules into cells using a solid lance, has proven to be highly efficient while maintaining high viability levels. In this thesis, an array of solid silicon lances was tested by nanoinjecting tens of thousands of HeLa cancer cells simultaneously. Several molecule types were injected in different tests to understand cell uptake efficiency and cell viability. Voltage was used to determine the impact of an electric field on molecule delivery. Propidium iodide, a dye that fluoresces when bound to nucleic acids and does not fluoresce when unbound, was delivered into cells using the lance array. Results show that the lance array delivers propidium iodide into up to 78% of a nanoinjected HeLa cell culture, while maintaining 78%-91% viability. Using similar protocol as in propidium iodide experiments, plasmid DNA containing the code for a fluorescent protein was nanoinjected into HeLa cells, resulting in an average expression rate of up to 0.21%. Since gene expression only occurs in cells which have integrated DNA into the genome in the nucleus, a different DNA detection method was developed to determine total DNA count in cells following nanoinjection. DNA strands tagged with a radioactive isotope were nanoinjected into HeLa cells. Liquid scintillation was employed to quantify and discriminate between DNA delivered to cells and DNA that remained in solution around cells following nanoinjection. The largest average amount of DNA delivered to cells was 20.0 x 10^3 DNA molecules per cell. Further development of the radioactive nanoinjection process is needed to more fully understand the parameters that affect DNA delivery efficiency. In all experiments with propidium iodide and DNA molecules, low accumulation voltage, coupled with a short pulsed release voltage, resulted in the greatest molecule delivery efficiencies when compared to tests without voltage or with a constant voltage only. Lastly, an automated nanoinjection system was developed to eliminate variability in user applied nanoinjection force. The automated system was found to reduce variability in average propidium iodide uptake values by 56%. In conclusion, experimental testing of the multi-cell nanoinjection process has shown promising molecule delivery results into human cells, suggesting that further optimization of the process would have positive implications in the field of academic and clinical gene therapy.

The ability to inject DNA and other foreign particles into cells, both germ cells (e.g. to produce transgenic animals) and somatic cells (e.g. for gene therapy), is a powerful tool in genetic research. Nanoinjection is a method of DNA delivery that combines mechanical and electrical methods. It has proven to have higher cell viability than traditional microinjection, resulting in higher integration per injected embryo. The nanoinjection process can be performed on thousands of cells simultaneously using an array of microneedles that is inserted into a monolayer of cells. This thesis describes the needle array design requirements and the fabrication process used to meet them. The process uses unpassivated and passivated deep reactive ion etching (DRIE) to create needles with a constant diameter shaft and a pointed tip. The needle diameter and height are about 1 µm and 8 µm, respectively. A buckling analysis and physical testing show that the needles can withstand the force required to penetrate the cells. The chip is attached to a plastic suspension with a counter electrode and electrical connections to a voltage source. The suspension's motion is defined by two compliant orthoplanar springs that have been vertically and rotationally offset for added stability. The base of the suspension is designed to exactly fit in the bottom of a cell culture dish, where the needle array can be pushed into the cell monolayer. Injection protocol was created and followed to perform tests with needle insertion only, voltage application only, and the full nanoinjection process. The average cell viability for the full injection process was 98.2% compared to an average control viability of 99.5%. Zero volt injections with a high concentration of propidium iodide, a cell impermeable dye with two positive charges, resulted in dye uptake from diffusion, proving that the needles are penetrating the cells. Tests comparing injections with and without voltage had high variability in dye uptake. Therefore, glass cover slips were placed in the culture dishes to provide more consistent injection conditions. This reduced variation in zero voltage tests. It is recommended that this procedure be followed for performing injections with voltage.

The Nanoinjector Lance Array has been developed to inject foreign material into thousands of cells at once using electrophoresis to attract and repel particles to and from the electrically-charged lances. A mathematical computer model simulating the motion of attracted or repelled proteins informs the design of the nanoinjection lance array system. The model is validated by accurately predicting protein velocity in electrophoresis experiments. A complete analysis of parameters is conducted via simulations and specific research questions regarding the counter electrode of the nanoinjector lance array system are explored using the model. A novel technique for fabricating lance arrays from collapsed carbon nanotube forests is explored and detailed. Experiments are conducted using the Nanoinjector Lance Array, attempting to inject three different kinds of protein molecules into a culture of HeLa cells. The experimental results are encouraging and suggest possibilities for future success. Other recommendations are made for future research regarding the model, carbon nanotube fabrication, and experimental testing.

A novel method for charged macromolecule delivery, called nanoinjection, has been developed at Brigham Young University. Nanoinjection combines micro-fabrication technology, mechanism design, and nano-scale electrical phenomenon to transport exogenous DNA across cell membranes on a nano-featured lance. DNA is electrically accumulated on the lance, precision movements of microelectromechanical systems (MEMS) physically insert the lance into cell, and DNA is electrically released from the lance into the cell. Penetration into the cell is achieved through a two-phase, self-reconfiguring metamorphic mechanism. The surface-micromachined, metamorphic nanoinjector mechanism elevates the lance above the fabrication substrate, then translates in-plane at a constant height as the lance penetrates the cell membranes. In-vitro studies indicate no statistical difference in viability between nanoinjected and untreated mouse zygotes. Pronuclear nanoinjection experiments on mouse zygotes, using microinjection as a control, demonstrate integration and expression of a nanoinjected transgene, and higher rates of zygote survival and pup births than the microinjection control. A new compliant mechanism analysis method, the minimization of potential energy method (MinPE method) is presented to model the equilibrium position of compliant mechanisms with more degrees of freedom (DOF) than inputs, such as a fully-compliant nanoinjector. The MinPE method position and force predictions agree with the method of virtual work and non-linear finite element analyses of under-actuated and underconstrained compliant mechanisms. Additionally, a performance-based comparison is made between quadratic shell finite elements elements and 3-D quadratic solid elements for modeling geometrically non-linear spacial deflection of thin-film compliant mechanisms. The comparison's results suggest the more computationally efficient quadratic shell elements can be used to model spatially deforming thin-film compliant mechanisms. Finally, this dissertation presents preliminary results for a proposed method of DNA transfer called cytoplasm-to-pronucleus nanoinjection. By placing a DNA coated lance into the cytoplasm of a mouse zygote and applying a voltage pulse of sufficient magnitude and duration, pores may open in the pronuclear membranes and DNA may be electrophoretically repelled from the lance. If effective, this process could result in transgenes without having to visualize and physically penetrate into the pronucleus. While embryo survival has been demonstrated under a variety of injection conditions, further study is needed to increase the process' consistency, and to determine if cytoplasm-to-pronucleus nanoinjection can generate transgenic animals.

Arrays of silicon lances were made using photolithographic and STS DRIE Bosch techniques. Arrays consist of a 10 mm square grid pattern of lances measuring 100 m tall and having a 3 mm diameter, each lance being spaced 30 mm apart. The tips of lances are pointed, allowing easier penetration through plant cell walls. A nanoinjector device was also made to accept the silicon lance arrays and perform nanoinjections. A nanoinjection consisted of 2 silicon lance arrays, with lances oriented towards each other, being moved into and out of a plant cotyledon placed between them. Prior to the nanoinjection, polar molecules in solution can be attracted to the lances through a process utilizing the nanoinjector device’s ability to control the electrical current between the 2 lance arrays. During the nanoinjection the displacement between the lances, the force exerted on the plant cotyledon and the electrical current between the lance arrays are controlled. Once the lances are inserted into the cells, the electrical current between the lance arrays is reversed, repelling the molecular load from the lance array. Propidium iodide (PI) and Cotton Leaf Crumple Virus (CLCrV) were used as molecular loads in nanoinjections. The nanoinjector also records and outputs data from the nanoinjection for analysis. Nanoinjections were performed on Arabidopsis and Cotton cotyledons. Changes in the force applied during a nanoinjection and varying the number of repeated nanoinjections on the same cotyledon were observed. Too much force or too many repeated injections causes physical damage to the cotyledon. An optimal force and number of repeated injections can be performed without causing physical damage to the cotyledon. Successful transfection of PI and CLCrV was not observed in a relatively small number of performed nanoinjection procedures on either Arabidopsis or Cotton cotyledons. Possible interacting variables and recommendations for further work are discussed.

The ability to deliver sequences of DNA and other molecular loads across the membrane of a cell and into its nucleus is an area of interest in the medical community. One of its many applications is that of gene therapy. In contrast to other forms of treatment, gene therapy seeks to treat diseases at the cellular level. The success of these treatments depends on the technologies for cell transfection that are available. Physical methods are sometimes able to overcome poor efficiencies of chemical methods and the safety concerns of viral methods, but are usually impractical due to the limited number of cells that are able to be transfected at a time, isolation, and immobilization of the cells. Nanoinjection is capable of using millions of small lances in an array to inject hundreds of thousands of cells simultaneously with relatively high efficiencies and viabilities. The solid nature of the lances also allows them to be smaller than their hollow-needle counterparts, which results in higher cell viability. Propidium Iodide (PI), a dye whose fluorescence increases greatly when bound to nucleic acids, was used as an injection molecule for testing the efficacy of the nanoinjection process on HeLa 229 cancer cells in a portion of the experiments, with a GFP plasmid of DNA being used in the rest. After injection, flow cytometry was used to detect the concentration of PI or the expression of the GFP in the injected cells. Since PI cannot normally penetrate the membrane of living cells, those found with high concentrations of PI were either successfully injected or dead, which can be determined by the flow cytometry. Investigation of the parameters that affect the efficiency of the nanoinjection process will help improve it for further research. Some of these parameters that were investigated include the force of injection, the material used for the lances (silicon versus carbon nanotubes), and the injection speed of the lance arrays. An injection device capable of small changes in deflection was designed to ensure accurate increments in force for testing, as well as a pulsed current control injection system. Results for injections of varying forces indicate a slow rise in PI uptake from 0 to 1.8 Newtons where it reaches a maximum uptake of 4.11 when normalized to the PI uptake of the positive controls. The PI uptake then remains relatively level as the force continues to increase, averaging an uptake of approximately 3.1. The slow rise is likely due to more of the cells being punctured as the force increases until most have been punctured and the PI uptake levels off. The viability of the injected cells was close to that of the controls with no clear trend. A comparison of lance arrays made from silicon and carbon nanotubes using DNA as the molecular load shows little difference between materials. Different injection speeds tested show that only 1-5% of the cells in the injection process are lost for speeds in the range of 0.08-0.16 mm/sec, whereas 49-69% of the cells are lost using speeds between 0.6-3 mm/sec.

<p>This thesis reports the results of biomarker measurements in three environmental monitoring projects. In the first project, which was part of the Swedish national environmental monitoring, biomarkers were measured annually in female perch (<i>Perca fluviatilis</i>) in two background areas in the Baltic Sea during 1988–2000, resulting in a unique 13-year series of measurements. The most important results were a strong decreasing temporal trend in the gonadosomatic index (GSI) and a strong increasing temporal trend in the hepatic ethoxyresorufin O-deethylase (EROD) activity in the Baltic Proper. In the second project, biomarkers and concentrations of classic pollutants were measured in female perch in the Stockholm recipient 1999–2001. This was the first time a large city was investigated as a point source of pollution, and the gradient was longer and included more stations than customary. Severe pollution conditions in central Stockholm were indicated by the poor health status of the perch: retarded growth, decreased frequency of sexually mature females, low GSI, disturbed visceral fat metabolism, increased hepatic EROD activity, decreased muscle acetylcholinesterase activity, increased frequency of hepatic DNA adducts, and a high concentration of biliary 1-pyrenol. Muscle ΣDDT and ΣPCB were measured as pollution indicators and were 10–28 respectively 12–35 times higher than the background levels in perch from the Baltic Proper. In the Stockholm archipelago two superimposed gradients were found. Whereas the response of several biomarkers consistently decreased with increasing distance from central Stockholm, the response of others first decreased from Stockholm to the middle archipelago and then increased to the open Baltic Sea. The latter biomarkers included the frequency of sexually mature females, GSI, hepatic EROD activity, and hepatic DNA adducts. In the third project, potential toxicity from munitions on the seafloor, at a dumpsite in the Stockholm archipelago, was analysed by the nanoinjection of sediment extracts into newly fertilised rainbow trout (<i>Oncorhynchus mykiss</i>) eggs, followed by the measurement of biomarkers in the developing larvae. No biological effects of the dumped munitions were found. The same stations in the Stockholm archipelago as in the second project were investigated as a positive control. The results of the three projects agreed well, which demonstrated the continuous pollution of the Baltic Sea and the severe pollution conditions and adverse biological effects in central Stockholm. Further investigations are urgently needed to identify which pollutants or other factors are causing the observed biological effects, both in the background areas in the Baltic Sea and in the Stockholm recipient.</p>

This thesis reports the results of biomarker measurements in three environmental monitoring projects. In the first project, which was part of the Swedish national environmental monitoring, biomarkers were measured annually in female perch (Perca fluviatilis) in two background areas in the Baltic Sea during 1988–2000, resulting in a unique 13-year series of measurements. The most important results were a strong decreasing temporal trend in the gonadosomatic index (GSI) and a strong increasing temporal trend in the hepatic ethoxyresorufin O-deethylase (EROD) activity in the Baltic Proper. In the second project, biomarkers and concentrations of classic pollutants were measured in female perch in the Stockholm recipient 1999–2001. This was the first time a large city was investigated as a point source of pollution, and the gradient was longer and included more stations than customary. Severe pollution conditions in central Stockholm were indicated by the poor health status of the perch: retarded growth, decreased frequency of sexually mature females, low GSI, disturbed visceral fat metabolism, increased hepatic EROD activity, decreased muscle acetylcholinesterase activity, increased frequency of hepatic DNA adducts, and a high concentration of biliary 1-pyrenol. Muscle ΣDDT and ΣPCB were measured as pollution indicators and were 10–28 respectively 12–35 times higher than the background levels in perch from the Baltic Proper. In the Stockholm archipelago two superimposed gradients were found. Whereas the response of several biomarkers consistently decreased with increasing distance from central Stockholm, the response of others first decreased from Stockholm to the middle archipelago and then increased to the open Baltic Sea. The latter biomarkers included the frequency of sexually mature females, GSI, hepatic EROD activity, and hepatic DNA adducts. In the third project, potential toxicity from munitions on the seafloor, at a dumpsite in the Stockholm archipelago, was analysed by the nanoinjection of sediment extracts into newly fertilised rainbow trout (Oncorhynchus mykiss) eggs, followed by the measurement of biomarkers in the developing larvae. No biological effects of the dumped munitions were found. The same stations in the Stockholm archipelago as in the second project were investigated as a positive control. The results of the three projects agreed well, which demonstrated the continuous pollution of the Baltic Sea and the severe pollution conditions and adverse biological effects in central Stockholm. Further investigations are urgently needed to identify which pollutants or other factors are causing the observed biological effects, both in the background areas in the Baltic Sea and in the Stockholm recipient.

Fundamentally altering cellular function at a genetic level is a major area of interest in the biologic sciences and the medical community. By engineering transfectable constructs that can be inserted to dysfunctional cellular systems, scientists can mitigate aberrant genetic behavior to produce proper molecular function. While viral vectors have been a mainstay in the past, there are many limitations, particularly related to safety, that have changed the focus of genome editing to incorporate alternative methods for gene delivery. Lance Array Nanoinjection (LAN), a second-generation microfabricated transfection biotechnology, is one of these alternative technologies. LAN works by utilizing both simultaneous electrostatic interaction with molecular loads and physical lancing of hundreds of thousands of target cell membranes. The purpose of this work is to demonstrate LAN in the context of in vitro transfection of immortalized culture cells and primary cells. As part of that exploration, three distinct areas of investigation are considered, which include: characterizing environmental factors that impact LAN transfection, demonstrating LAN genetic modification of immortalized HeLa 229 culture cells using an indicator marker, and lastly, investigating the effects of LAN on human primary, neonatal fibroblasts.