Lehigh University Annual Progress Report: 2009 Formula Grant

Transcription

1 Lehigh University Annual Progress Report: 2009 Formula Grant Reporting Period July 1, 2012 June 30, 2013 Formula Grant Overview Lehigh University received $119,007 in formula funds for the grant award period January 1, 2010 through December 31, Accomplishments for the reporting period are described below. Research Project 1: Project Title and Purpose Development of a Point-of-Care Opto-Fluidic HIV Viral Load Detector - The intended result of this work is the design and construction of a hybrid platform that uses a combination of filtration and concentration methods in order to quantify the number of circulating HIV virons directly from patient blood samples. The purpose of this project is to perform fundamental studies to gauge the feasibility and performance of the device. After primary feasibility studies, we expect that the device will address the challenges associated with a point of care diagnostic platform that is both portable and easy to use in both resource-limited and general settings. Anticipated Duration of Project 1/1/ /31/2013 Project Overview The purpose of this project is to create a novel, low-cost, globally deployable diagnostic device capable of detecting viral loads in HIV patients. HIV infection affects more than 33 million people worldwide, most of whom live in areas without even the most basic laboratory facilities. Although state-of-the-art technologies based on nucleic acid amplification are sensitive and quantitative for viral load analysis, they are widely accepted to be technically too demanding and too costly, thus severely limiting their applicability at the point of care (POC). The goal of this project is to furnish the device in a usably portable high-performance platform. Since the device will quantify whole viral particles from blood using a combination of filtration and detection, we must first verify the individual components of the technique. There are two main factors in the operation of the device; one being the straining/concentration element and the other being the labeling and detection of the filtered viral particles. In order to detect the viruses, they must first be isolated from the other blood-borne debris and concentrated significantly in order to be optically detected. The clinically relevant level of circulating viruses can be as low as nine orders of magnitude below even the most sensitive Lehigh University 2009 Formula Grant Page 1

2 optical detection techniques. In order to accomplish the necessary concentration enhancement, we will combine nanoporous filtration membranes with an optical concentrator. The filtration membranes will strain and concentrate the viruses by six orders of magnitude. After this step, an optical concentrator will be used to further enhance the concentration. Additionally, a systematic fluorescent labeling of the virions will occur in tandem with the membrane element to allow optical detection. Thus the isolation of the viruses requires a combination of mechanical and optical techniques, both of which require fundamental research to move forward. In addition to system development, we will also evaluate the system performance using clinical samples obtained from Lehigh Valley Hospital. The funding of this project will support both the fundamental research and clinical evaluation. Principal Investigator Xuanhong Cheng, PhD Assistant Professor Lehigh University 5 E. Packer Ave, Whitaker Lab Bethlehem, PA, Other Participating Researchers H. Daniel Ou-Yang, PhD employed by Lehigh University Timothy Friel, MD employed by Lehigh Valley Hospital Expected Research Outcomes and Benefits The end result of this project will be a compact device capable of quantifying HIV viral loads directly from whole blood. We expect that at the completion of this study, we will have explored the fundamental questions that surround the device; including the performance of the filtration membranes, the effectiveness of the fluorescent labeling and the concentrating power of the optical elements. Once the device has been tested with model systems and clinical trials, we expect that in the future a compact version of the platform will improve the public health by providing a cheaper, faster and more accessible diagnostic method for viral infections. Although the focus in this project is HIV viral load quantification, the concentrating and detection platform could easily be applied to the detection of other pathogens for clinical diagnosis, homeland security and epidemiological monitoring purposes. Summary of Research Completed While previous periods focused on optical sensor development to count nanoparticles in a suspension, in the current funding period we attempted to fabricate nanoporous membranes for the processing of viral particles. Combining aluminum anodization with nanotemplating, we demonstrated that large area, long-range ordered membranes with pore size comparable to HIV Lehigh University 2009 Formula Grant Page 2

3 viruses could be fabricated without the access of sophisticated clean room facilities. More specifically, we used monolayers of hexagonally packed silica nanobeads to indent aluminum. After electrochemical anodization of the aluminum, the resulting oxide contains straight, high aspect ratio nanopores with the periods and pore arrangements comparable to the imprinting mold. Not only does this method significantly improve the ordering of the nanopores over those formed through self-ordering, it also allows continuous tuning of the pore geometry. Furthermore, tunable cell sizes can be readily achieved for cell sizes significantly greater than 500 nm, which was considered an upper limit of porous anodic aluminum oxide. The preindentation approach also allows us to quantitatively characterize the relationships among various physical parameters and reaction conditions, such as the cell size, intrinsic pore size, anodization voltage, electrolyte composition and oxide film growth rate. This information is important for fundamental understanding of the anodic aluminum oxide (AAO) formation. The resulting membranes with submicron pore size are under evaluation as a nanofiltration medium for virus separation and concentration in the lab. Methods To create the the nanoimprinting mold, monodispersed silica nanobeads with sizes ranging from 280nm to 760nm were synthesized by hydrolysis of tetraethyl orthosilicate (TEOS) (Sigma- Aldrich, Cat# ) following a recipe modified from the literature. Close-packed silicananobead monolayers were prepared through convective deposition on plain glass slides following the method reported by Kumnorkaew. To prepare nanoimprinted aluminum thin films, five micrometers of aluminum were evaporated onto clean glass slides using an electron-beam evaporator (Indel Systems-2602). The silica monolayer was then pressed onto the aluminum surface at a pressure of 30 kn/cm 2 for 30 seconds using an Instron instrument to create a pattern of nano-dimple arrays. To create nanoimprinted aluminum substrates, aluminum was anodized in phosphoric acid (Alfa Aesar, Cat# A18067) under vigorous stirring for 1 hour. The concentrations of phosphoric acid were varied from 0.6 M to 3 M for 280-nm samples, from 0.2 to 0.6 M for 410-nm samples, from 0.04 to 0.2 M for 500-nm samples, from 4 to 15 mm for 645-nm samples, and from 1 to 5 mm for 760-nm samples. The anodization voltage was held constant at 112, 164, 195, 255, and 296 V to form AAO films with indentation periods of 280, 410, 500, 645, and 760 nm respectively. For comparison, conventional two-step anodization was carried out at 195 V in 0.1 M H 3 PO 4 for 16 h for the 1 st anodization and 1 h for 2 nd anodization. Nanofeatures obtained in this work were characterized using a field-emission SEM (Hitachi 4300). The nanobead, cell and pore sizes, and AAO film thickness were analyzed by ImageJ. The FFT analysis was performed by Gwyddion. Results of Data Generated and Analyzed While anodic aluminum oxide is well known to contain hexagonally packed submicron pores, there are various practical limitations to its wide application as the nano-filtration medium. First, only limited pore geometries are available through the self-ordering process. Second, the ordered Lehigh University 2009 Formula Grant Page 3

4 pore arrangements only exist in small domains on the order of microns. Thirdly, intrinsic spatial ordering behavior during anodization requires several days of processing and multiple steps of anodization, which limits the fabrication throughput. Here, we tested a pre-indentation method to control the order of the pores during aluminum anodization as well as improve the anodization throughput. The procedure to create long-range ordered AAO takes three steps. First, monolayers of tightly packed silica nanobeads are deposited on flat glass slides as the imprinting molds. Polystyrene nano-fillers are co-deposited and melted afterwards to stabilize the silica monolayer in the indentation process. Next, the silica monolayers are pressed onto an aluminum surface at30 kn/cm 2 to create nanodimples arrays, which guide pore nucleation sites in the subsequent anodization process. Afterwards, the indented aluminum is anodized in phosphoric acid at a constant voltage. The resulting AAO is analyzed by scanning electron microscopy (SEM). The nanopores in the pre-indented AAO films have a long range order over a millimeter length scale. The pores are hexagonally packed and the pore periods closely match the indentation molds. The regular ordered cell arrangement is not limited to the top surface, but is also maintained through the film thickness. Additionally, AAO films from the pre-patterned surface give rise to an obvious 6-fold symmetry FFT pattern with high intensity and narrow peak distribution. This is in contrast to the conventional two-step anodization without surface imprinting, which yields many smaller domains and a ring-like FFT pattern with low intensity and board distribution. Thus, pre-patterning greatly improves the pore size uniformity and long range pore order over structures formed by conventional self-organization. The other disadvantage of conventional two-step anodization is the limited pore geometries. The self-ordering phenomena occur only in narrow process windows determined by the type of acid used: sulfuric acid at 25V, oxalic acid at 40V and phosphoric acid at 195V, given 63 nm, 100 nm and 500 nm pore intervals, respectively. When the anodization process is accomplished outside the self-ordering regimes, the degree of spatial ordering decreases drastically. On the other hand, the pre-patterning procedure enables tunable cell sizes through selection of imprinting molds of different periods. Here, cell sizes in the range of nm are obtained in phosphoric acid, while 500 nm has been considered as the upper limit of the pore periods of anodic aluminum oxide. When the pore geometries are compared to the anodization voltages, it is found that the cell size increases linearly with the anodization voltage, governed through the relationship D c V a 2.60 nm/volt. The pore sizes (D p ) also increase linearly with the voltage with a relationship of D p V 0.6 nm/volt. The porosity of the template guided anodization films, calculated as P = (π/2 3)(D p /D c ) 2, is between ~6% and 9%, slightly lower than the 10% porosity reported in the literature through self-organization. This is likely a result of residual stress in the aluminum layer from the imprinting process. Since the pores can be widened in a post-anodization process, the lower porosity should not be a limitation of AAO formed through nanoindentation. In addition to the anodization voltage, we also found the composition and concentration of the electrolytes important in porous AAO growth. In this study, phosphoric acid is used in all of the Lehigh University 2009 Formula Grant Page 4

5 anodization and the acid concentration is varied, so the influence of pore formation by acidity can be compared. While acid concentration does not affect the cell and pore sizes to an observable level, the film growth rate is heavily dependent on the acid concentration. Under all indentation conditions, the film growth rates increase exponentially with the acid concentrations. The best fittings for the growth rate (GR) versus acid concentration (C) are: GR = e 0.36*C for interpore distance of 280 nm; GR = e 2.19*C for interpore distance of 410 nm; GR = e 8.11*C for interpore distance of 500 nm; GR = e *C for interpore distance of 645 nm; and GR = e *C for interpore distance of 760 nm. The growth rate cannot increase infinitely: when the acid concentration exceeds a critical value, the anodization current density surges within 30 seconds and the film is burnt. This critical concentration of phosphoric acid is found to decrease with the anodization voltage: for anodization voltage of 164, 195, 255, and 296 V, the critical concentrations are 0.65, 0.24, 0.016, and M, respectively. The critical concentration for creating 280 nm cell size at 112V is beyond the most concentrated phosphoric acid used in this study of 3M. The study of critical acid concentration for stable anodization provides a practical guidance for preindentation guided AAO growth. The maximum growth rate of ~ 1-2 µm/h is slightly lower than that reported in the literature of ~2-6 µm/h for self-ordered mild anodization. This is likely a result of low anodization temperature used in this study. In this work, the anodization temperature is maintained at -7 C using a cooling batch of ice/ethanol/dry ice. The acid solution is also stirred through the anodization process. The temperature used in the literature is usually ~0 C. As expected from reaction kinetics, lower temperature used in this study leads to a lower film growth rate. Lehigh University 2009 Formula Grant Page 5