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The prevalence of malaria across the globe creates the need for the development of diagnostic devices, as many deaths caused by malaria could be prevented by early diagnosis and treatment. Since typical symptoms of malaria, such as fever and chills, are challenging to distinguish from other diseases, diagnostic tests are crucial for allowing patients to receive the appropriate medical care. While current gold-standard methods such as microscopy are able to identify the presence and species of malaria parasites, the sensitivity achieved with these methods depends on the microscopist’s training, as well as access to resources such as lab equipment and power. Disparities in supplies and training lead to late or incorrect diagnosis in resource-limited areas across the globe. This has led to the development of point-of-care devices, which provide more rapid results without the need for expensive lab equipment or training. One example of such a device is the lateral-flow immunoassay (LFA), which has commonly found use as at-home pregnancy tests and recently as COVID-19 tests. Although the LFA is simple to use and provides easy-to-read results, currently available LFAs for malaria detection suffer from lower sensitivity compared to laboratory tests. In this thesis, I investigate methods for improving the detection of the malaria antigen Plasmodium falciparum lactate dehydrogenase (pLDH). Methods to improve the detection of the LFA include biomarker preconcentration and signal enhancement. Biomarker preconcentration can be achieved using aqueous two-phase systems (ATPSs), which are liquid-liquid extraction systems composed mostly of water. Molecules that are added into an ATPS can be concentrated into the smaller of the two phases. In addition, the sensitivity of the LFA can be improved through the use of signal enhancement, which aims to increase the intensity at the detection zone with the use of additional or modified colorimetric indicators. In Chapter 2, we focus on us

The prevalence of malaria across the globe creates the need for the development of diagnostic devices, as many deaths caused by malaria could be prevented by early diagnosis and treatment. Since typical symptoms of malaria, such as fever and chills, are challenging to distinguish from other diseases, diagnostic tests are crucial for allowing patients to receive the appropriate medical care. While current gold-standard methods such as microscopy are able to identify the presence and species of malaria parasites, the sensitivity achieved with these methods depends on the microscopist’s training, as well as access to resources such as lab equipment and power. Disparities in supplies and training lead to late or incorrect diagnosis in resource-limited areas across the globe. This has led to the development of point-of-care devices, which provide more rapid results without the need for expensive lab equipment or training. One example of such a device is the lateral-flow immunoassay (LFA), which has commonly found use as at-home pregnancy tests and recently as COVID-19 tests. Although the LFA is simple to use and provides easy-to-read results, currently available LFAs for malaria detection suffer from lower sensitivity compared to laboratory tests. In this thesis, I investigate methods for improving the detection of the malaria antigen Plasmodium falciparum lactate dehydrogenase (pLDH). Methods to improve the detection of the LFA include biomarker preconcentration and signal enhancement. Biomarker preconcentration can be achieved using aqueous two-phase systems (ATPSs), which are liquid-liquid extraction systems composed mostly of water. Molecules that are added into an ATPS can be concentrated into the smaller of the two phases. In addition, the sensitivity of the LFA can be improved through the use of signal enhancement, which aims to increase the intensity at the detection zone with the use of additional or modified colorimetric indicators. In Chapter 2, we focus on us

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 advant

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 advant

Chemotherapy treatments involving the delivery of naked drugs to the body must overcome many complications such as poor solubility, enzymatic degradation, and clearance from the body, all of which can result in the drug having a short circulation half-life, low efficacy, and undesirable side effects. One solution to overcome these problems is to encapsulate the drug within a nano-sized drug delivery vehicle. Nano-sized drug delivery vehicles are advantageous since they can protect the drug from degradation during its circulation in the body, release the drug in a controlled manner, and provide passive targeting to the tumor tissue. Many materials for drug delivery vehicles have been investigated. Liposomes, which are vesicles composed of natural or synthetic phospholipids, have been thoroughly investigated, and many liposomal formulations have been successful in the market. However, one limitation of liposomes is that they are less stable due to being comprised of relatively smaller molecules that exhibit weaker attractive interactions in a self-assembled vesicle. This disadvantage has motivated the development of other materials for drug delivery such as synthetic polymers, which are longer molecules that can exhibit stronger attractive interactions in a self-assembled vesicle. Synthetic control of the polymers also allows for fine tuning of the hydrophilic and hydrophobic chain lengths. Another material that has been recently gaining popularity for use in drug delivery is the polypeptide. These amino acid-based building blocks provide further advantages. Similar to polymers, monodisperse polypeptides can be synthesized with precise control due to recent advances in polymerization techniques, and the longer chains provide stability for the self-assembled vesicles. They are also naturally occurring and have the potential to be biocompatible. These polypeptides can also adopt secondary structures to further improve the stability of the vesicles. Our laboratory previo

Chemotherapy treatments involving the delivery of naked drugs to the body must overcome many complications such as poor solubility, enzymatic degradation, and clearance from the body, all of which can result in the drug having a short circulation half-life, low efficacy, and undesirable side effects. One solution to overcome these problems is to encapsulate the drug within a nano-sized drug delivery vehicle. Nano-sized drug delivery vehicles are advantageous since they can protect the drug from degradation during its circulation in the body, release the drug in a controlled manner, and provide passive targeting to the tumor tissue. Many materials for drug delivery vehicles have been investigated. Liposomes, which are vesicles composed of natural or synthetic phospholipids, have been thoroughly investigated, and many liposomal formulations have been successful in the market. However, one limitation of liposomes is that they are less stable due to being comprised of relatively smaller molecules that exhibit weaker attractive interactions in a self-assembled vesicle. This disadvantage has motivated the development of other materials for drug delivery such as synthetic polymers, which are longer molecules that can exhibit stronger attractive interactions in a self-assembled vesicle. Synthetic control of the polymers also allows for fine tuning of the hydrophilic and hydrophobic chain lengths. Another material that has been recently gaining popularity for use in drug delivery is the polypeptide. These amino acid-based building blocks provide further advantages. Similar to polymers, monodisperse polypeptides can be synthesized with precise control due to recent advances in polymerization techniques, and the longer chains provide stability for the self-assembled vesicles. They are also naturally occurring and have the potential to be biocompatible. These polypeptides can also adopt secondary structures to further improve the stability of the vesicles. Our laboratory previo

The objective of this thesis was to investigate a concentration method using aqueous two-phase systems (ATPS) for improving the detection of proteins and viruses at the point-of-care. In the first part of the thesis, aqueous two-phase micellar systems were generated using Triton X-114 surfactant to concentrate a model protein, namely transferrin (Tf). In the second part of the thesis, aqueous two-phase polymer-salt systems were generated using polyethylene glycol (PEG) and potassium phosphate salt to concentrate a model virus, namely bacteriophage M13 (M13). In both studies, the concentration step was combined with a detection assay, namely the lateral-flow immunoassay (LFA), to enhance the detection of protein and viral targets.The detection of proteins at the point-of-need has several applications such as detecting food allergens in a food sample and protein toxins used as biowarfare agents in-field. For such applications, a sensitive, yet rapid, inexpensive, and portable detection assay that requires minimal training and power is desired. Due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements, the LFA is an appropriate assay for such applications. However, the LFA detection limit for proteins is inferior to lab-based assays, such as the enzyme-linked immunosorbent assay (ELISA), and needs to be improved. Aiming to improve the protein sensitivity of the LFA, we employed an aqueous two-phase micellar system composed of Triton X-114 surfactant to concentrate Tf prior to the detection step. However, one challenge with concentrating small biomolecules, such as proteins, is that they partition evenly between the two phases due to experiencing fewer excluded-volume interactions compared to larger biomolecules. To address this issue, we developed a novel approach involving larger colloidal gold nanoparticles decorated with anti-Tf antibodies in the concentration step to bind Tf and aid its transport to the micelle-poor phase. By

The objective of this thesis was to investigate a concentration method using aqueous two-phase systems (ATPS) for improving the detection of proteins and viruses at the point-of-care. In the first part of the thesis, aqueous two-phase micellar systems were generated using Triton X-114 surfactant to concentrate a model protein, namely transferrin (Tf). In the second part of the thesis, aqueous two-phase polymer-salt systems were generated using polyethylene glycol (PEG) and potassium phosphate salt to concentrate a model virus, namely bacteriophage M13 (M13). In both studies, the concentration step was combined with a detection assay, namely the lateral-flow immunoassay (LFA), to enhance the detection of protein and viral targets.The detection of proteins at the point-of-need has several applications such as detecting food allergens in a food sample and protein toxins used as biowarfare agents in-field. For such applications, a sensitive, yet rapid, inexpensive, and portable detection assay that requires minimal training and power is desired. Due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements, the LFA is an appropriate assay for such applications. However, the LFA detection limit for proteins is inferior to lab-based assays, such as the enzyme-linked immunosorbent assay (ELISA), and needs to be improved. Aiming to improve the protein sensitivity of the LFA, we employed an aqueous two-phase micellar system composed of Triton X-114 surfactant to concentrate Tf prior to the detection step. However, one challenge with concentrating small biomolecules, such as proteins, is that they partition evenly between the two phases due to experiencing fewer excluded-volume interactions compared to larger biomolecules. To address this issue, we developed a novel approach involving larger colloidal gold nanoparticles decorated with anti-Tf antibodies in the concentration step to bind Tf and aid its transport to the micelle-poor phase. By