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- All Subjects: Bioengineering
- Creators: Caplan, Michael
- Resource Type: Text
Description
Monitoring complex diseases and their comorbidities requires accurate and convenient measurements of multiple biomarkers. However, many state-of-the-art bioassays not only require complicated and time-consuming procedures, but also measure only one biomarker at a time. This noncomprehensive single-biomarker monitoring, as well as the cost and complexity of these bioassays advocate for a simple, rapid multi-marker sensing platform suitable for point-of-care or self-monitoring settings. To address this need, diabetes mellitus was selected as the example complex disease, with dry eye disease and cardiovascular disease as the example comorbidities. Seven vital biomarkers from these diseases were selected to investigate the platform technology: lactoferrin (Lfn), immunoglobulin E (IgE), insulin, glucose, lactate, low density lipoprotein (LDL), and high density lipoprotein (HDL). Using electrochemical techniques such as amperometry and electrochemical impedance spectroscopy (EIS), various single- and dual-marker sensing prototypes were studied. First, by focusing on the imaginary impedance of EIS, an analytical algorithm for the determination of optimal frequency and signal deconvolution was first developed. This algorithm helped overcome the challenge of signal overlapping in EIS multi-marker sensors, while providing a means to study the optimal frequency of a biomarker. The algorithm was then applied to develop various single- and dual-marker prototypes by exploring different kinds of molecular recognition elements (MRE) while studying the optimal frequencies of various biomarkers with respect to their biological properties. Throughout the exploration, 5 single-marker biosensors (glucose, lactate, insulin, IgE, and Lfn) and one dual-marker (LDL and HDL) biosensor were successfully developed. With the aid of nanoparticles and the engineering design of experiments, the zeta potential, conductivity, and molecular weight of a biomarker were found to be three example factors that contribute to a biomarker’s optimal frequency. The study platforms used in the study did not achieve dual-enzymatic marker biosensors (glucose and lactate) due to signal contamination from localized accumulation of reduced electron mediators on self-assembled monolayer. However, amperometric biosensors for glucose and lactate with disposable test strips and integrated samplers were successfully developed as a back-up solution to the multi-marker sensing platform. This work has resulted in twelve publications, five patents, and one submitted manuscripts at the time of submission.
ContributorsLin, Chi En (Author) / La Belle, Jeffrey T (Thesis advisor) / Caplan, Michael (Committee member) / Cook, Curtiss B (Committee member) / Stabenfeldt, Sarah (Committee member) / Spano, Mark (Committee member) / Arizona State University (Publisher)
Created2018
Description
Synthetic manipulation of chromatin dynamics has applications for medicine, agriculture, and biotechnology. However, progress in this area requires the identification of design rules for engineering chromatin systems. In this thesis, I discuss research that has elucidated the intrinsic properties of histone binding proteins (HBP), and apply this knowledge to engineer novel chromatin binding effectors. Results from the experiments described herein demonstrate that the histone binding domain from chromobox protein homolog 8 (CBX8) is portable and can be customized to alter its endogenous function. First, I developed an assay to identify engineered fusion proteins that bind histone post translational modifications (PTMs) in vitro and regulate genes near the same histone PTMs in living cells. This assay will be useful for assaying the function of synthetic histone PTM-binding actuators and probes. Next, I investigated the activity of a novel, dual histone PTM binding domain regulator called Pc2TF. I characterized Pc2TF in vitro and in cells and show it has enhanced binding and transcriptional activation compared to a single binding domain fusion called Polycomb Transcription Factor (PcTF). These results indicate that valency can be used to tune the activity of synthetic histone-binding transcriptional regulators. Then, I report the delivery of PcTF fused to a cell penetrating peptide (CPP) TAT, called CP-PcTF. I treated 2D U-2 OS bone cancer cells with CP-PcTF, followed by RNA sequencing to identify genes regulated by CP-PcTF. I also showed that 3D spheroids treated with CP-PcTF show delayed growth. This preliminary work demonstrated that an epigenetic effector fused to a CPP can enable entry and regulation of genes in U-2 OS cells through DNA independent interactions. Finally, I described and validated a new screening method that combines the versatility of in vitro transcription and translation (IVTT) expressed protein coupled with the histone tail microarrays. Using Pc2TF as an example, I demonstrated that this assay is capable of determining binding and specificity of a synthetic HBP. I conclude by outlining future work toward engineering HBPs using techniques such as directed evolution and rational design. In conclusion, this work outlines a foundation to engineer and deliver synthetic chromatin effectors.
ContributorsTekel, Stefan (Author) / Haynes, Karmella (Thesis advisor) / Mills, Jeremy (Committee member) / Caplan, Michael (Committee member) / Brafman, David (Committee member) / Arizona State University (Publisher)
Created2019
Description
Sickle cell disease is a genetic disorder that can cause substantial helath problems. It is the result of a mutation in the DNA coding for hemoglobin. As a result of changes in two important amino acids, a person suffering from sickle cell disease will have erythrocytes that do not maintain the typical biconcave shape and instead for a crescent shape. Individuals with sickle cell disease may have many health problems tied to their irregular hemoglobin. The unusual shape of the erythrocytes leads to a much shorter cell life, which means that even though bone marrow remains active long past childhood to try to keep up with the loss of erythrocytes, the body is still unable to accommodate the rapid death of erythrocytes. The malformed erythrocytes can also cause vascular occlusion, blocking blood vessels and slowing blood flow. While sickle cell disease has the potential to spread worldwide, it is particularly common in Africa. This may be because people with the sickle cell trait have a high resistance to malaria, making them more likely to survive that ubiquitous disease and pass on their traits to their offspring. However, the mortality rate in young children with sickle cell disease is very high, in part because the spleen, already stressed by filtering out dead erythrocytes, has difficulties filtering out bacteria. One of the keys to stopping the spread of the disease is neonatal screening, but this requires specialized equipment that is fairly uncommon in rural areas, as can be seen in Kenya. Therefore, it would be highly beneficial to develop a more cost-effective and widely available method for testing for sickle cell disease.
ContributorsWold, John (Author) / Caplan, Michael (Thesis director) / LaBelle, Jeffrey (Committee member) / Snyder, Jan (Committee member) / Barrett, The Honors College (Contributor)
Created2012-05
Description
Microvillus Inclusion disease is a fatal disease found in the Navajo population caused by a single nucleotide polymorphism. It is characterized by intractable diarrhea and is often fatal early in life.1 The current method of diagnosis is sending duodenal biopsies for histopathological examination and confirmatory testing through genomic sequencing. The purpose of this experiment was to create a more simple and cost-effective diagnostic method for detecting Microvillus Inclusion disease. Three methods were explored (RFLP2, ARMS3,4, and Tentacle Probes5,6) and two methods were tested to determine their ability and their efficiency in detecting the SNP that causes the disease.2 Tests using the RFLP2 method and synthetic DNA resulted in 9% false-positive rate and 11% false-negative rate in a blind trial for detecting both target (mutation present) and non-target (mutation absent) DNA when gel analyzing software was used to compare Rf values after gel electrophoresis. Using the ARMS method3, a nine-sample randomized test was run that ended up with 22% false-positive rate and 19% false-negative rate from a blind trial when using a gel analyzing software to determine presence of the SNP by band intensity. Disclaimer: No DNA from human patients was used in this study. Only synthetic DNA used.
ContributorsHelmbrecht, Hawley Elizabeth (Author) / Caplan, Michael (Thesis director) / Carpentieri, David (Committee member) / Dubois, Courtney (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Sickle Cell Disease (SCD) is a prevalent genetic disease in Africa, and specifically in Kenya. The lack of available relevant disease education and screening mean that most don't understand the importance of getting testing and many children die before they can get prophylactic care. This project was designed to address the lack of knowledge with supplemental educational materials to be partnered with an engineering capstone project that provides a low cost diagnostic test.
ContributorsShawver, Jamie Christine (Author) / Caplan, Michael (Thesis director) / Snyder, Jan (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / Harrington Bioengineering Program (Contributor)
Created2014-05