Below is the proposal for this project. The proposal was submitted to the Johns Hopkins University’s Hopkin’s Office of Undergraduate Research. I was awarded the Provost’s Undergraduate Research Award for my ideas and I am now completing the project along with NASA to use the recommended device to monitor astronout in long-time deep space missions.
Free-Flow Isotachophoresis Device to Separate Nucleic Acids and Proteins in Bodily Fluids:
The overall goal of this PURA project is to develop a robust electrokinetics-based micro-total analysis platform for extracting nucleic acids and proteins from human samples, including blood serum, urine and saliva. Typically, sample preparation and purification processes are performed manually in a laboratory and minimal techniques exist to automatically process and prepare complex fluid matrix in small microfluidic devices for use with downstream chemical analysis systems, such PCR. The novel platform that I propose to develop here will make significant impact in the field of portable diagnostics and disease detection. The enabling methods I intend to develop is based on a powerful, but often overlooked, electrokinetic phenomena known as isotachphoresis (ITP). ITP is an electrokinetics method with both sensing and diagnostic purposes, which has potential to be applied to a broad range of applications, including detection of disease, food/water contaminants and biowarfare agents. This project will focus its efforts on developing ITP into a robust tool for enabling disease detection in microfluidic systems. I will accomplish this by developing the ITP into a technology capable of performing continuous, free-flow separation of proteins and nucleic acids from human body fluids on small microfluidic devices. After developing the ability to separate, purify and extract proteins and nucleic acids continuously using ITP, I will then work towards developing the breakthrough capability to extract proteins and nucleic acids simultaneously from a single sample.
Very often, scientists are not capable of extracting low concentration targets present at parts- per-million levels from raw diagnostic samples. To combat this issue, new sample preparation methods need to be developed. Interest in micro-total analysis platforms that can aid in sample processing procedures has increased over the last decade due to their ability to rapidly process samples with minimal reagent requirements. The low residence time of these devices makes them ideal for rapidly and simply processing samples through concentrating and separating molecules.
Free-flow electrophoresis (FFE) methods are capable of performing preparative sample pre- concentration and separation on charged ions and molecules. The isotachophoresis method I propose to develop uses electrokinetics principles to prepare samples through electric field-mediated concentration and separations. Current ITP devices are bulky, unreliable, difficult to fabricate and allow only for separation and pre-concentration in small microliter batch volumes. Batch-wise separation is tedious and limits research time because it demands high involvement from scientists to load multiple batches of raw sample, collect the processed fluid, and repeat the process until the entire sample volume has been processed.
Our lab has developed an easy-to-use and lightweight ITP device that is capable of performing ITP separation and concentration of fluorescent molecules in a continuous manner. I propose to leverage this work and use the developed device to design, characterize and demonstrate its capability of separating and concentrating target molecules and in this way, revolutionize sample preparation by making it a single step, continuous process capable of processing large mesofluidic volumes of fluid quickly and easily.
Our lab has built a flow meso-fluidic device capable of performing ITP separation and concentration of fluorescent molecules shown in Figure 1. I propose further technical development of the device to make it an easy-to-use and lightweight ITP device to rapidly extract, concentrate andseparate proteins and nucleic acids from a liquid sample. This would in turn produce a continuous free-flow ITP method to pre-process raw samples. I will develop an understanding for how proteins and nucleic acids concentrate by ITP in a continuous flow environment. I will then perform experiments in electrolyte buffers with known concentrations of nucleic acids and proteins to further understand ITP concentration and separation behavior of simple solutions. Finally, I will optimize the ability to achieve targeted molecular purification by changing parameters such as electric field strength, channel length, fluid flow rate, and choice of buffer chemistries. This will reveal the viable use of ITP microfluidic platforms in preparative processes.
The project set-up will be the same for all experiments performed throughout the remainder of the scholastic year to maintain consistency in result presentations. A project schedule is shown on Figure 2, and a more detailed explanation is provided below it.
The proposed microfluidic ITP device is shown on Figure 2. The project set-up is as follows is based on the idea that biomolecular separation and concentration occurs at the interface between co-flowing positive and trailing electrolyte fluids. A positive (+) trailing electrolyte (TE) mixture will be fed from the top inlet (1) to the ITP continuous device and a (+) leading electrolyte (LE) solution will be fed through the bottom inlet (5) for all protein extraction and separation processes. For separating nucleic acids, the experiments will be performed with TE/LE electrolyte solutions with negative co-ions for isolating negatively charged nucleic acids. For both classes of biomolecules, mixed solutions of fluorescently labelled samples will be mixed with TE buffer, loaded on a small cryo tube and delivered to the device through the middle inlet (3). Fluid flow will be induced using an external pressure system. The microfluidic inlet will be pressured at a pressure of 0.25 psi using our labs custom-made microfluidic flow controller. An electric field will be applied across the channel by applying a voltage difference between embedded gallium electrodes on the sidewalls of the microfluidic channels using an small DC power supply.
To quantify the field-induced molecular electro-migration, I will track the concentration profiles of the fluorescent-labelled biomolecules using the laboratory’s inverted confocal microscope. More precisely, I will measure the dynamic changes in fluorescent intensity of each sample being as biomolecules accumulate between the trailing and leading electrolytes (positive or negative depending on the experiment) to track concentration changes along the device’s main channel.
My first set of experiments will be performed using a known initial concentration of fluorescently- labelled proteins (FITC-avidin) to assess and understand how ITP concentration is influenced by the strength of the electric field. A second set of experiments will be performed to determine the influence of other important physicochemical parameters, including inlet flow rate, applied voltage, and buffer chemistry in order to produce any observed ITP-driven molecular concentration.
I will successfully carry out the goals listed in this proposal using my extensive theoretical and practical experience in the microfluidics field. As a senior majoring in chemical and biomolecular engineering, I have taken several classes on transport phenomena where I have learned the theory behind fluid dynamics and electrophoresis. As a research and development intern at the Diabetes Research Institute during the summer of 1016 I co-designed and assembled a high perfusion device for integrated use with a microfluidic dispenser. I have also extended my understanding of microfluidic platforms working with Dr. Zachary Gagnon and have contributed to formulating a method for monitoring microfluidic flows using impedance spectroscopy. During my time at the microfluidics laboratory, I have gained hours of experience in microfabrication and clean room practices necessary to develop a state-of-the-art microfluidic device.
The proposed project will demonstrate the capability of using our continuous ITP device design to rapidly and simply separate and concentrate target molecules by changing ITP parameter values. Phase I of the proposal will further understanding of proteins and nucleic acids in a continuous ITP system. The second phase will allow us to determine if the determined relationships of phase one can really be used to pre-process samples through pre-concentration and separation of target-molecules.
The results could be used in the future to develop a free-flow bi-direction ITP system that allows for simultaneous nucleic acid and protein extraction. This kind of system would allow for the integration of our novel device with other common laboratory processes as a sample preparation step of a continuous system (Figure 4).