Every year infectious diseases are among the top major causes of death worldwide. Infectious diseases disproportionately impact resource-poor areas. While most developed countries have the ability to screen and quickly detect infectious diseases in humans, animals, and plants, developing countries lack this ability. In such areas, access to medical treatment and testing facilities is severely limited. Additionally, developing countries are more densely populated, which makes infectious disease surveillance exceedingly more difficult. As a result of this, infectious diseases tend to spread more quickly and outbreaks tend to occur more often in developing countries. Additionally, the increasing rate of antimicrobial resistant infections and emergence of novel infectious diseases increase the likelihood of global pandemic occurrence.

It is evident that developing novel devices for detection of infectious diseases would be greatly beneficial to global health. It is important to note that developed countries would benefit equally from such a device. Pandemics tend to burden the economy and put strain on resources even in developed countries. The impact of such a device is determined by a set of criteria put forth by the World Health Organization (WHO). Based on the ASSURED criteria, an ideal device for point-of-care (POC) diagnostics must be affordable, sensitive, specific; and yet it must also be user-friendly, rapid and robust, equipment free, and deliverable to end-users. Additionally, this device must be easily adaptable for detection of novel pathogens. The nucleic acid amplification test (NAAT) meets many of these criteria. However, NAAT’s current workflow involves many user liquid handling steps as well as a thermocycler.

This thesis aims to address these issues by first automating isothermal nucleic acid amplification, followed by coupling the amplification step with the lateral-flow immunoassay (LFA), which is a paper-based diagnostic device. Thus, taking advantage of the affordability and POC features of the LFA. The aim of these efforts is to work toward the ultimate goal of developing a device that receives the patient sample, then carries out all the steps in NAAT in an automated manner and produces results with just a push of a single button.

Point-of-care (POC) diagnostics are easy-to-use, low-cost devices that can rapidly detect a biomarker of interest without the need for highly trained personnel or expensive equipment. In resource-limited settings, POC testing is often the only viable option for patients, which highlights the importance of developing sensitive technologies for early disease detection and monitoring of treatment methods. Hydrogels are flexible 3D structures made with crosslinked hydrophilic components that absorb large amounts of water. These systems are sensitive to different chemical or physical stimuli, and the magnitude of their response is usually directly proportional to the magnitude of the applied external stimuli. In the diagnostics field, hydrogels are mostly being used for enzyme detection and immunodiagnostics. For these applications, the biomarker acts as a signal that triggers a volume, color, or state change of the hydrogel, an example of the latter being a gel-sol transition or vice versa. This technology often requires the chemical modification of at least one of the main hydrogel components so that the system is specific towards the molecule of interest. Accordingly, these devices need to be manufactured in a laboratory setting with complex equipment and often expensive reagents, and they also require personnel with extensive knowledge and experience to conduct the assay and its analysis. Thus, it would be desirable to design a hydrogel system that is responsive to the presence of the biomarker of interest without the need for chemical modification of the reagents to design a robust, POC-friendly device that can be manufactured in all parts of the world. To develop this system, we focused on calcium alginate dextran hydrogels beads for the detection of abnormal concentrations of ions, such as bicarbonate and phosphate, in patient samples. This system can be synthesized by a simple ionic crosslinking method based solely on the complexation of the oppositely charged species, i.e., alginate and calcium, which comprise the gel matrix. For disease detection, we took advantage of a calcium depletion mechanism based on the reaction of the biomarkers of interest with the divalent calcium cation in the calcium alginate gel matrix. The timeframe for hydrogel degradation was therefore correlated with the sample’s ion concentration, making our POC device semi-quantitative. To our knowledge, this is the first demonstration of the use of nonionic polymers to improve the morphology of calcium alginate hydrogel beads using a simple processing method that involves minimal labor, equipment, and reagents. The simplified bead synthesis protocol combined with a user-friendly device that we developed allows for the rapid detection of high serum bicarbonate or urine phosphate levels at the POC.

Providing effective healthcare to underrepresented populations is a major challenge worldwide due to environmental, economic, and social disparities. Both developing countries and low income rural/urban areas in the US tend to have reduced healthcare access which limits the frequency and quality of medical screenings as well as treatment options. This has led to an increased effort in the development of point-of-care devices which emphasize, immediate and onsite results. One device that has the potential to fulfill these requirements is the lateral-flow immunoassay (LFA). While the LFA has been successful in the form of the over-the-counter pregnancy test, it still does have some disadvantages that limit its ability to fully replace sophisticated laboratory-based assays. Throughout this thesis, I will be discussing the development of cutting-edge technologies to address the two major disadvantages of the LFA which are its poor quantitative characteristics and low sensitivity.Cerebrospinal fluid (CSF) leaks are a common complication of numerous procedures in Otolaryngology. These leaks are relatively difficult to diagnose and the current laboratory-based tests to do so have a turn-around time that is unacceptable for rapid clinical decision making. We addressed this problem by developing the first rapid diagnostic test kit for the detection of CSF leaks. In Chapter 2, we describe the development of a barcode-style LFA for the semi-quantitative detection of beta-trace protein, which is present in CSF at high concentrations. This work was expanded upon in Chapter 3, where we created a complete, standalone rapid diagnostic test kit composed of our barcode-style LFA, a collection swab, dilution buffers, disposable pipettes, and instructions. Clinical studies demonstrated excellent predictive capabilities of this kit in distinguishing patient samples containing CSF from those that did not. In some cases, a more quantitative readout than what the barcode-style LFA can achieve is required. In Chapter 4, we developed a new technique to introduce an even more quantitative readout to the LFA without the need of electronic readers. This technology was applied to the therapeutic drug monitoring of the cardiac glycoside digoxin. Digoxin capture and binding occurs on a modified LFA test strip which utilizes platinum nanozyme probes that possess catalase-like activity. This test strip is combined with a reaction that controls the anisotropic etching of gold nanorods to produce a wide range of visible color hues dependent on the initial digoxin concentration in the sample which allowed for naked-eye quantification. We then shifted to the development of technologies to improve the detection limit and sensitivity of the LFA. Our lab previously discovered the phenomenon of an aqueous two-phase system (ATPS) separating on paper, which allowed for the seamless integration of biomarker preconcentration and detection on the LFA. In Chapter 5, we have extended the functionality of an ATPS separating on paper to automate the sequential delivery of signal enhancement reagents in addition to concentrating biomarkers. The timing of reagent delivery was controlled by changing the initial composition of the ATPS and a mathematical model was developed to help predict the ATPS flow behavior. We applied this technology to automate biomarker preconcentration and nanozyme signal enhancement on the LFA, resulting in a 30-fold improvement in detection limit over the conventional LFA when detecting Escherichia coli, all while maintaining a single application step. In late 2019, a new virus known as SARS-CoV-2 began to sweep across the globe, leading to the COVID-19 pandemic. The development of highly sensitive and easily deployable rapid diagnostic tests is a vital aspect of managing this pandemic. In Chapter 6, we discuss ongoing efforts to develop a highly sensitive, nanozyme signal enhanced LFA for the rapid detection of the SARS-CoV-2 nucleocapsid protein. A 3D-printed casing was designed to store the test strip and all reagents in a way that maintains user friendly operation by eliminating liquid handling steps. Besides developing highly sensitive diagnostics, the ability to deploy these tests quickly and in a highly scalable manner is crucial for being able to manage a pandemic. The work described in Chapter 7 highlights the integration of novel fibronectin-based antibody mimetics, called monobodies, into the LFA for the detection of the nucleocapsid protein of SARS-CoV-2. These monobodies are advantageous over monoclonal antibodies in that they are rapidly developed and produced recombinantly in Escherichia coli which is more affordable and scalable than monoclonal antibody production.

Infectious diseases are a significant problem, accounting for 1 in every 4 deaths worldwide. The field of bioengineering is constantly innovating and advancing diagnostic technology; however, more often than not, these innovations are accessible only to communities with privileged resources. This has led to a growing focus on point-of-care (POC) diagnostics. Due to their ease of use, speed, and low cost, POC diagnostics can effectively test patients in resource-poor settings. One of the most well known POC technologies is the lateral-flow immunoassay (LFA). Most easily recognized for its use in pregnancy tests, LFA is a paper-based diagnostic that produces visually interpreted results using a colorimetric indicator decorated with antibodies specific to the target. Recently, we have seen rapid advancement in the fields of paper fluidics and paper diagnostics, which can have a tremendous impact on the future of LFA technology. In light of this, we focused our work in 3 distinct directions, which involves development of quantitative experimental methodologies, mathematical modeling, and improving the ease-of-use of the advanced LFA technology.

Development of new paper-based devices would benefit significantly from being able to quantitatively assess the effects of engineering the device on important LFA parameters. For example, it would be useful to know the effects of manipulating the gold nanoprobes and test strips on the forward (kf,s) and reverse (kr,s) rate constants for the probe binding to and dissociating from the test line, respectively. We discuss our novel approach for determining these rate constants and the volumetric flow rate by using mathematical modeling and radioactive iodine-125 (125I). Moreover, we demonstrate how radioactivity and paper strips can also be used to determine the volume of fluid in and before the test line, the concentration of gold nanoprobes, and the number of antibodies per gold nanoprobe.

As the field of paper-based diagnostics continues to rapidly expand, it becomes more important to incorporate modeling into their design. A model can be used to determine the effects of LFA parameters on desired outputs, such as the amount of probe bound to the test line. Such predictions become increasingly important as systems become more complicated, and the effects of changing different operating conditions become less intuitive due to the many physical, chemical, and biological processes that are simultaneously occurring. Moreover, a mathematical model allows the engineer to quantitatively predict the influence of well-defined changes in certain parameters. We have derived a simple model that could be used in combination with our novel estimation methods for LFA parameters to predict the amount of probe binding to the test line, an important performance indicator of LFA. We will discuss the derivation of the model, and demonstrate the model’s ability to predict empirical results.

Our lab’s recent innovations have improved LFA sensitivity by utilizing aqueous two-phase systems (ATPSs) to thermodynamically concentrate the target molecule prior to detection. In the third direction, we describe a diagnostic design that dehydrates the ATPS components within the paper, creating a non-dilutive, one-step diagnostic process. We investigate the importance of ATPS component dehydration order and demonstrate an improvement in the limit of detection for Chlamydia trachomatis LFA using our dehydrated ATPS diagnostic design.

Hydrogels are multifunctional biomaterials popularly used in a wide range of applications. In this thesis, calcium alginate hydrogels were thoroughly studied with a special focus on synthesizing calcium alginate hydrogel beads using a simple set up involving only a syringe and a gelation bath. By introducing an additional nonionic polymer component to the alginate solution during synthesis, the hydrogel beads exhibited greatly improved morphology with regard to uniformity and sphericity. These hydrogel beads also demonstrated excellent dye retention properties and stability in water. Additionally, they exhibited a gel-sol transition process in response to external stimuli such as calcium chelators. Taking advantage of these properties, the novel hydrogel beads were integrated in diagnostics applications for point-of-care detection of metabolic alkalosis and biomarkers in bacterial infections, with the degradation of gel and release of dye as disease indicators. For metabolic alkalosis detection, the assay utilized the reaction of sodium bicarbonate and citric acid to produce citrate, a metal chelator capable of competitively binding to calcium cations in the gel matrix to trigger hydrogel degradation. This resulted in successful detection of elevated bicarbonate concentrations in less than one hour. For detection of biomarkers in bacterial infections, novel hydrogel beads were synthesized with an additional enzyme substrate component in their gel matrix, which allowed for substrate cleavage and gel degradation to occur in the presence of bacterial enzymes. Results from preliminary studies showed that the gel-sol response of the hydrogel beads towards the bacterial enzymes α-amylase and trypsin was successfully achieved.

Cervical cancer is the fourth most common cancer in women, with essentially all cases caused by long-lasting infection with high-risk types of the human papillomavirus (HPV). Unlike other cancers, the risk of developing cervical cancer can be greatly reduced with vaccines that protect against infection from these high-risk HPV types. Furthermore, widespread and routine gynecologic screening, such as the Papanicolaou (Pap) test and HPV testing, have significantly decreased the incidence of cervical cancer and deaths from the disease in industrialized countries.

However, despite being potentially preventable and curable, cervical cancer continues to be a leading cause of cancer-related death for women living in regions with limited health care resources. Nearly 85% of all cervical cancer cases occur in less developed regions of the world, and in 2012, almost nine out of ten (87%) cervical cancer related deaths affected women in resource-poor settings. This global disease burden is largely due to the lack of effective cervical cancer screening programs and access to prophylactic vaccines, creating a clear need for new point-of-care (POC) screening technologies for women in developing countries. As such, women in resource-poor communities could greatly benefit from a simple, low-cost, and equipment-free test that can be self-administered and can provide easily interpretable results in a reasonable amount of time.

One common paper-based diagnostic device that has the potential to satisfy the aforementioned criteria is the lateral-flow immunoassay (LFA), a rapid antibody-based test that is most recognized for its application as the over-the-counter pregnancy test. However, despite success in certain applications, the sensitivity of LFA remains inferior in comparison to that of gold-standard diagnostic laboratory assays, limiting its use in more widespread clinical applications. To address this problem, our group has previously utilized various aqueous two-phase systems (ATPSs) to preconcentrate target biomolecules, after which the phase containing the concentrated biomolecule was extracted and applied to LFA for improved detection. Our group has also discovered that by integrating ATPS with LFA, particular types of paper and 3D paper architectures can significantly accelerate the rate of phase separation of an ATPS, leading to shorter assay times and fewer sample preparation steps, in addition to sensitive results.

The focus of this thesis was to investigate various components required to create a new ATPS integrated LFA screening test that could detect for HPV16. This test serves as a precursor to a commercialized device that could determine if women are at risk for cervical cancer in health care limited settings. One main focus of this thesis was to identify and optimize two ATPS systems that can lyse cells and subsequently concentrate the desired biomarkers. The lysing ability of the nonionic Triton X-114 micellar system and polyethylene glycol (PEG) – potassium phosphate salt (PEG-salt) system with Triton X-100 were studied, and their abilities to phase separate as they flowed through paper were determined. Paper constructs were then optimized to ensure accelerated phase separation on paper such that the ATPS could concentrate desired biomarkers to the front of fluid flow for downstream detection.

After confirming that both ATPS solutions exhibited the desired cell lysis and phase separation properties, the partitioning of model hydrophilic and hydrophobic particles indicative of the size of HPV were studied. A mathematical model was then developed based on thermodynamic principles, and this model considered contributions from excluded-volume and hydrophobic interactions to predict partition coefficients of a particle in a Triton X-114 micellar ATPS. The model reasonably predicted the measured experimental partition coefficient, and can be further developed in the future to provide even more accurate predictions of partition coefficients.

Lastly, the limit of detection of HPV16 L1 was determined with LFA only. Studies suggest that the Triton X-114 micellar system has the potential to improve the detection of HPV16 upon integration with LFA. Additionally, to our knowledge, this is the first successful demonstration of detection of the HPV16 L1 major capsid protein using LFA. In the future, this paper-based device can be further improved to detect for desired cervical cancer biomarkers to provide cervical cancer risk determination results that are both sensitive and quick.

Aqueous two-phase systems (ATPSs), traditionally utilized in industrial bioseparations, are showing increasing potential as an approach for concentrating components in paper-based point-of-care bioassays. Specifically, our lab was the first to demonstrate that ATPSs can increase the sensitivity of the lateral-flow immunoassay (LFA) by concentrating biomarkers into one of the two phases, after which, the phase containing the concentrated biomarkers was extracted prior to their detection. While this method demonstrated consistent improvements to the LFA, its applicability in point-of-care settings was restricted by two main factors. First, the time to separate into two distinct phases varied among different classes of ATPSs, but usually required hours to achieve effective biomolecule concentration. Second, the method required several user steps in the form of sample mixing with the ATPS components, and the subsequent extraction and application of the phase containing the concentrated biomolecule to the LFA after phase separation occurred. More recently, our lab demonstrated that when the mixed, homogenous ATPS was applied to a paper membrane, phase separation was observed within the paper membrane itself as the solution wicked across the paper. This largely unexplored phenomenon reduced the phase separation time of a polyethylene glycol (PEG)-salt ATPS from hours in a tube to minutes on paper.

This thesis focuses on advancing the phenomenon of paper-based phase separation as a means of enhancing the phase separation rate of ATPSs and making them more suitable for point-of-care applications. First, we extended the paper-based phase separation phenomenon to a naturally slow phase separating system, the Triton X-114 ATPS. Next, we investigated the dehehydration of the phase forming components directly into the paper matrix, and the subsequent phase separation upon resolubilization of the components by a liquid sample. Within these two investigations, the ATPS was then integrated with the LFA to improve the detection of biomarkers of infectious diseases such as malaria and chlamydia. Furthermore, we investigated the use of the Washburn equation as a mathematical framework to describe the flow behavior of ATPS phases within porous media in order to better predict phase separation behavior within paper.

The Triton X-114 ATPS is a micellar ATPS that is comprised of the Triton X-114 nonionic surfactant. This particular ATPS, previously used by our lab to concentrate biomarkers for the LFA, is one of the slowest separating systems, partially due to the small interfacial tension and density difference between its two phases (the micelle-poor and micelle-rich phases). We applied the Triton X-114 ATPS to paper membranes and demonstrated, using a distinctively different design in which the solution flows vertically up a multilayered paper wick, that paper-based phase separation can also be achieved with the Triton X-114 system. In this case, we found that gravitational effects had no influence on the flow of the dense gold nanoparticles as the less dense micelle-poor phase containing the gold wicked ahead of the micelle-rich phase. This was the first time that a micellar ATPS was applied directly to a fiberglass paper membrane to significantly speed up its macroscopic separation from at least 8 hrs in a test tube to approximately 3 min on paper. The paper-based Triton X-114 ATPS was then integrated with the LFA to simultaneously concentrate a malaria protein biomarker into the leading micelle-poor phase, and then detect it without the need of a user-dependent phase extraction step. The single-step integration improved the LFA detection limit for the protein by 10-fold in buffered saline and complex serum media. This was also the first time within our lab that we concentrated an infectious disease biomarker in complex biological fluids.

The design used for the abovementioned Triton X-114 study is applicable when dealing with oral and vaginal swab samples as the swab would need to be mixed with a buffer solution to solubilize the target. However, in urine, saliva, or blood applications, one would prefer to just add the biological fluid to the device. The second focus of the thesis was therefore to further improve the user-friendliness of the ATPS-LFA integration by removing initial sample preparation steps. To achieve this, we investigated the novel concept of sequential rehydration of the two-phase components that were initially dehydrated into the paper matrix as a way to achieve paper-based ATPS phase separation. We used two different polymer-salt ATPSs: the PEG-salt system and the UCON-50-HB-5100 (UCON)-salt system, optimizing the component concentrations and rehydration order to yield the appropriate phase separation conditions for each system. Upon rehydration of the components, phase separation successfully occurred within the paper, leading to the formation of a leading polymer-poor phase and a lagging polymer-rich phase. As mentioned above, the benefit of this method is that a biological sample no longer needs to be manually mixed with the components and instead can be directly added to the device. These dehydrated systems were then integrated with the LFA to produce paper-based assays in which all the necessary components for target concentration and detection were stored within the paper matrix. The dehydrated PEG-salt ATPS and UCON-salt ATPS were integrated with the LFA to simultaneously concentrate and detect large Chlamydia trachomatis whole bacteria and smaller human IgM antibodies, respectively. Ultimately, our designs demonstrated 10-fold improvements to the detection limits for both Chlamydia trachomatis and IgM, and could therefore improve LFA sensitivity without adding steps to the user. These exciting developments solve the initial limitations of the ATPS-LFA integration, making their use as point-of-care diagnostic devices for infectious diseases one step closer to reality.

Although paper-based phase separation of several different aqueous two-phase systems have been experimentally demonstrated, there is a need for a mathematical model that can accurately predict two-phase system wicking behavior in paper-based devices, as it would benefit the process of device design. We decided to evaluate the Washburn model as a framework for fluid flow of the PEG-salt ATPS and Triton X-114 ATPS in porous media. Using a combination of imbibition studies and characterization of the Washburn fluid parameters for each individual phase, we determined that the viscosity difference between the two phases is a dominant factor in the ability of a given ATPS to phase separate in paper. More specifically, the Washburn model correctly predicts that the less viscous phase will constitute the leading phase, independent of flow direction in a horizontal or vertical orientation. In this validation of the model, we then applied it to predict the phase separation capabilities of two polymer-polymer systems, the PEG-Dextran system and the PEG-polyacrylic acid (PEG-PAA) system. We predicted and successfully demonstrated that the PEG-PAA system could phase separate due to its large enough difference in phase viscosities, while the PEG-Dextran system could not phase separate due to its small viscosity difference. Furthermore, this theoretical framework was extended to predict the phase separation of ATPSs in polyethylene glycol dimethacrylate-based microporous hydrogels. We were able to predict and show for the first time that phase separation of various ATPSs could be achieved within hydrogels, demonstrating that the phase separation enhancement phenomenon can occur in various types of porous media.

Nanomedicine focuses on the development and engineering of novel and unique therapeutic and diagnostic agents that can overcome the challenges associated with using traditional modalities. Nanoparticles (NPs) in the size range between 1 and 1000 nm have many advantages for use in these applications, such as, low polydispersity, established characterization methodologies, and the ability to be loaded with therapeutics for diseases, conjugated to targeting ligands to enhance specificity, and coated with polymers to improve stability and half-lives.

This thesis focused on developing nanotechnology to address both therapeutic and diagnostic applications due to the universal need to understand and maintain colloidal stability. Maintaining colloidal stability is crucial in therapeutic applications since aggregated NPs will have a reduction in the number of targeting ligands available for interacting with cell-surface receptors by reduction of the surface area to mass (or volume) ratio. The drug release from the NPs can also be significantly altered, and the aggregate sizes may no longer be appropriate for targeting tumors passively and for being actively internalized into cancer cells. The aggregation also has the possibility of severely reducing the shelf-life of the drug carriers. In the case of diagnostics, in addition to reducing the number of targeting ligands and shelf-life, aggregation can change the optical properties of NPs. Moreover, aggregated NPs in the well-established lateral-flow immunoassay (LFA) can experience difficulty flowing through the test strip and may give rise to invalid results. Accordingly, irrespective of the application, the studies in this thesis required an understanding and tuning of the electrostatic and steric, excluded-volume stabilizing interactions when designing the NPs.

With regard to cancer therapy, this thesis focused on further testing the limits of a transferrin (Tf) variant that was previously engineered by our laboratory for improving the delivery of cytotoxins in a Tf-cytotoxin molecular conjugate format. Specifically, our previously derived mathematical model was first extended to theoretically determine if conjugating a Tf variant to a NP would increase its association with a cancer cell, thereby increasing the probability of delivering a toxic payload to a cancer cell. Upon finding that the theoretical predictions supported such a construct, the Tf variant was conjugated to the NPs and then radiolabeled with iodine-125, and these NPs were found to associate with prostate cancer cells for a greater period of time relative to the native Tf counterpart, as predicted by our extended mathematical model. Subsequently, poly(lactide-co-glycolide) (PLGA) NPs encapsulating the chemotherapeutic doxorubicin (DOX) was conjugated to a Tf variant and was shown to have improved drug carrier efficacy both in vitro and in vivo relative to the native Tf counterpart. The in vivo results were especially exciting as a single intravenous injection was found to dramatically inhibit tumor growth in a mouse model for prostate cancer. This work corresponded to the first ever investigation of the drug carrier properties of NPs conjugated to a Tf variant. In contrast to our laboratory's previous studies with molecular drug conjugates, which are very promising for cancers treated locally, these Tf variant-conjugated NPs administered intravenously have potential for treating most types of cancers.

With regard to diagnostic applications, this thesis focused on revolutionizing LFA. One of the most common applications of LFA is the pregnancy test, where gold NPs are used as the colorimetric indicator. The small sizes of the gold NPs also allow the use of low sample volumes, and their large surface area to mass ratio enables efficient capture of target molecules. Their optical properties can also be exploited to directly visualize a result. Although LFA has many features that make it attractive for use as a point-of-care diagnostic, the use of LFA for detecting biomarkers at low concentrations is severely limited due to its low sensitivity. To increase LFA sensitivity, our laboratory is the only one to have used aqueous two-phase complex fluid systems for concentrating biomarkers prior to their detection. This pioneering work is described in the thesis, where we began by using an aqueous two-phase Triton X-114 micellar system to pre-concentrate the model virus M13 prior to its detection with LFA.

This first combination of an aqueous two-phase system (ATPS) and LFA improved the detection limit of LFA for M13 by 10-fold, and has led to many new exciting avenues of research. Subsequently, an aqueous two-phase polyethylene glycol (PEG)-salt system was investigated to concentrate the same virus but in a much shorter time period. The same PEG-salt system was then examined for its ability to concentrate the model protein Tf. In this case, due to Tf being significantly smaller than M13, gold NPs were used to bind and transport Tf into one of the two phases of the aqueous two-phase PEG-salt system. However, due to the high concentrations of salt present in one of the phases, the gold NPs were manipulated to ensure colloidal stability through the entire process from concentration to detection. These gold NPs were also manipulated to drive the gold NPs to the interface instead of one of the two bulk phases with the idea of further concentrating the target biomolecules, as the interface corresponds to a three-dimensional region that is only a few molecular diameters thick. Moreover, this thesis discusses the discovery of a very exciting phenomenon where the paper membranes used in LFA can enhance the phase separation process of an ATPS, which enables the concentration of the target molecules as they flow directly on the test strip. This ability to simultaneously concentrate and detect biomarkers directly on paper removes the need to extract a phase from a test tube and also reduces the time to result. Lastly, the combination of an ATPS and LFA was further extended to include magnetic fields and a new solid-liquid interface to enhance extraction. All of these technologies have the potential to dramatically improve the state of health care in resource-poor settings by providing rapid, accurate, and inexpensive diagnostics, leading to improved patient management, treatment, and outbreak prevention.

Delivery of naked drugs in the body faces many challenges, such as poor solubility in blood and interstitial fluids, enzymatic and proteolytic degradation in the liver, clearance by the kidneys, recognition by the immune system, inability to cross the cell membrane, and nonspecific toxicity to normal cells. Over the years, drug encapsulation and delivery through nanocarriers have been sought as a potential approach to overcome many of these obstacles. Traditionally, research in this area has been dominated by vesicles assembled from lipids. These lipid based vesicles, also known as liposomes, are currently the most widely used vesicle for drug delivery with commercial formulations (DOXIL® and MyocetTM) in the market. However, liposomes are intrinsically unstable in vivo unless they are coated with polymers such as polyethylene glycol (PEG), which decreases their clearance from the body. Even with the addition of PEG, there are limitations as the added PEG chain increases the hydrophilic head group of the lipids, increasing their tendency to form micelles instead of bilayers. Due to these limitations, researchers have investigated alternative systems, such as polymer-based vesicles or polymersomes. The disproportionality that exists between the hydrophilic PEG head group and the hydrophobic tail of a lipid can be overcome by synthesizing synthetic PEG amphiphilic polymers with longer hydrophobic segments. In addition, by synthesizing building blocks that are larger than lipids, membrane stability can be improved along with slower leakage of drugs. However, some polymersomes suffer from lacking the ability to incorporate biofunctionality without impairing their ability to self-assemble into vesicles.

In order to contribute to this exciting drug delivery field and find a material that could potentially improve upon current lipids and synthetic polymers, our lab investigated the use of novel amino acid-based polymers as the building blocks for forming vesicles. The use of amino acids offers many advantages. They have an intrinsic biocompatibility, which implies great potential for low immunogenicity and toxicity. The variety of amino acids and their many possible sequence combinations can be used to custom design polypeptides with different chemical properties and biofunctionalities. Moreover, recent advances in polymerization techniques allow the synthesis of long chains of monodisperse polypeptides. Vesicles formed from polypeptides can offer increased stability over conventional liposomes by providing thicker membranes that contribute to increased attractive interactions between the building blocks. We have investigated vesicles comprised of polypeptides that have been synthesized by Dr. Timothy Deming's group (UCLA), which employs a transition metal-mediated α-amino acid N-carboxyanhydride (NCA) polymerization technique to synthesize the amphiphilic block copolypeptides. The vesicles formed from these polypeptides can be prepared in different sizes in bulk quantities.

This thesis focused on investigating the potential for using these polypeptide vesicles as drug delivery vehicles. Initially, vesicles comprised of positively charged polypeptides were studied, specifically the lysine-leucine (K60L20) and arginine-leucine (R60L20) block copolypeptides. Due to their positive charge, these vesicles have a disadvantage of being toxic to cells through interactions with the net-negatively charged plasma membranes. Therefore, in order to identify a design criterion for producing positively charged vesicles with low cytotoxicity, we focused on optimizing the hydrophilic/hydrophobic ratio of the polypeptide by varying the length of the hydrophobic block while maintaining a constant length for the hydrophilic block. The K60L20 polypeptides were used for this study due to their relative ease of preparation compared to the R60L20 polypeptides. It was found that varying the hydrophilic/hydrophobic ratio in the lysine-leucine block copolypeptide affects its ability to form vesicles, where polypeptides with long hydrophobic segments formed less toxic vesicles with appropriate sizes for drug delivery. Among the copolypeptides investigated, the K60L20 copolypeptide composition showed the most potential for drug delivery applications, as this copolypeptide was able to form monodisperse nanoscale vesicles with the least amount of micelles and small aggregates that were more toxic than the vesicles themselves.

The optimized block copolypeptide composition was then applied to the arginine-leucine block copolypeptide, and the ability of these arginine-based vesicles to transfect mammalian cells was systematically investigated. The arginine-leucine R60L20 block copolypeptide was studied, since the arginine residues had previously been shown to enhance the delivery of cargo into cells. Plasmid DNA was used as our model therapeutic, since only low concentrations of plasmid DNA are required for an effect. This was an important point, since high concentrations of R60L20 vesicles will lead to significant cytotoxicity. Our transfection results with our R60L20 vesicles demonstrated that there is potential for using this novel material as a transfection agent, as they were able to achieve transfection with low cytotoxicity and immunogenicity.

In contrast to plasmid DNA, high concentrations of the vesicles are required when delivering small molecule chemotherapeutics due to their IC50 values being in the micromolar range. Since the R60L20 vesicles themselves become cytotoxic at high concentrations, we also investigated the negatively charged vesicle formed from the glutamate-leucine (E60L20) block copolypeptide. A main advantage of these vesicles, over the positively charged vesicles, is that they are less toxic to cells. However, this is also a disadvantage, since the electrostatic repulsive interactions between the vesicles and the net-negatively charged cell membranes can inhibit the vesicles from entering cells. We were able to overcome this limitation by conjugating the vesicle surface with transferrin (Tf), a ligand that has been commonly used for targeting many types of cancer cells. We investigated the intracellular trafficking behavior of these Tf-conjugated EL vesicles, and they were shown to recycle back to the cell surface once they were internalized. This behavior was similar to what has been observed for other nanoparticle systems conjugated with Tf.

Even with this recycling behavior, other Tf-conjugated nanoparticle systems were found to exhibit improved drug delivery efficacy. Accordingly, we investigated the ability of our Tf-E60L20 vesicles to deliver the chemotherapeutic doxorubicin (DOX). Before performing the in vitro efficacy study, the vesicle surface was conjugated with poly(ethylene glycol) (PEG) to improve its stability for future in vivo applications. The targeting Tf ligand was then conjugated to the resulting vesicle, and DOX was then encapsulated in the vesicle interior. A previously developed mathematical model was applied to our system to predict trends that helped guide experiments and allowed for reduced experimentation and faster optimization. Once the carrier was complete, in vitro cytotoxicity studies were performed to demonstrate proof-of-concept that the newly developed Tf-conjugated vesicles exhibit a significant improvement in drug delivery efficacy over the non-targeted version.

The objectives of this thesis were to investigate ionic liquid aqueous two-phase systems (ATPSs) as a concentration method for improving the lateral-flow immunoassay (LFA) detection of antigens, and demonstrate potential advantages of these ATPSs over conventional polymer-salt and micellar ATPSs used in the past. ATPSs have been widely utilized for liquid-liquid extraction and purification of biomolecules, with some studies also demonstrating their capacity as a biomarker concentration technique for use in diagnostic settings. As the limited polarity range of conventional ATPSs can restrict their use, ionic liquid (IL)-based ATPSs have been recently proposed as a promising alternative to polymer- and micellar-based ATPSs, since ILs are regarded as tunable solvents with excellent solvation capabilities for a variety of natural compounds and proteins. This study demonstrates the first application of IL ATPSs to point-of-care diagnostics. ATPSs consisting of 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF4]) and sodium phosphate salt were utilized to quickly concentrate biomarkers prior to detection using the LFA. We found the phase separation speed of the IL ATPS to be very rapid and a significant improvement upon the separation speed of both polymer-salt and micellar ATPSs. This system was successfully applied to both sandwich and competitive LFA formats and enhanced the detection of both Escherichia coli bacteria and the transferrin protein up to 8-fold and 20-fold, respectively. This system’s compatibility with a broad range of biomolecules, rapid phase separation speed, and tunability suggest wide applicability for a large range of different antigens and biomarkers.