Tyler Sodia*, Lindsey Armstrong, Austin Haider, Marcos Maldonado*, Anika James, Marlea Kudlauskas, Lisa Fetter*, Anna Nguyen*, Derek Clark, Ilia Mazin, Nazar Dubchak, Jena Jacobs, Jessica Daniel*, Aviva Bulow, Susan Jett*, Ryan Warren, Tiffany Ashbaugh, Michael McCoy, Ebony Miller, Jonathan Richards*, Laura Roon*, Becky Addison, Jeremy O’Brien, Travis Ingraham, Sarai Graves, Kathryn Norquest*, Stephen Schaffner*, Kyra Brandt, Elina Baravik*, Yerelsy Reyna*, Josh Sowick, Jody Stephens*, Ryan Masterson*, Mason Preusser, Tonya Santaus, Amanda Faux, Matthew Stoddard, Morgan Miller.
(* denotes a researcher with a publication from the lab)
Dr. Andrew J. Bonham
Professor of Chemistry & Biochemistry
Dr. Bonham’s Curriculum Vitae
Dr. Bonham’s work focuses on understanding and investigating Transcription Factors, essential human proteins that regulate the bodies growth and response to disease. These transcription factors are essential components of gene regulation, and there is great interest in probing their presence and activity in both academic analysis and clinical diagnostics. Current methods to address these questions are often time-intensive or require specialized reagents, such as antibodies. At Metropolitan State University of Denver, Dr. Bonham is leading an innovative undergraduate research program focused on engineering new tools for sensitive and quick detection of TF:DNA interactions.
Lab Member 2019-
Project: Electrochemical DNA Biosensors for Detection of Mannose-capped Lipoarabinomannan
Mycobacterium Tuberculosis (TB) is one of the world’s most prevalent bacterial pathogens. It is estimated that almost 10 million cases of TB emerge every year, and roughly one-fifth of these cases are fatal. The current detection and diagnosis of TB is done primarily via two methods; the TB skin test and the TB blood tests. Neither of these tests can differentiate between latent TB infection and TB disease. In order to differentiate these states, time-consuming sputum tests are required, which rely on culturing the mycobacterium. Designing a sensitive serologic biosensor would dramatically decrease the time line of diagnosis and therefore improve patient outcomes. One possible avenue for improved detection lies in the cell wall of TB, which includes many complex glycolipids—many of which are believed to have immunopathogenic mechanisms in physiologic pathways. Mannose-capped lipoarabinomannan (ManLAM) is one of the most prevalent of these glycolipids, and presents a novel target as a bio-marker for the sensitive detection of TB and related Mycobacterium strains. Here, we have utilized an existing aptamer sequence that binds to ManLAM to generate a sensitive electrochemical, DNA-based biosensor for the detection of TB. This biosensor is able to adopt multiple different folded conformations, only one of which presents the core aptamer sequence in a state capable of binding ManLAM. An appended redox-active tag (methylene blue) generates a measurable difference in electrochemical current upon this conformational change, providing a sensitive and quantitative measurement of ManLAM concentration. Such biosensors may ultimately allow rapid, on site, diagnosis of TB infection within the time constraints of patient-doctor interaction.
Lab Member 2019-
Project: Design of electrochemical biosensors for detecting Carrion’s Disease
Carrion’s disease is a neglected tropical disease (NTD) caused by infection by the bacteria B. bacilliformis, endemic to northern Peru, and affects primarily rural, impoverished populations. In rural areas, diagnosis is currently made using Giemsa-stained blood smears, but this technique has a very low sensitivity for the disease (24-36%) and requires trained individuals, which are in short supply. The disease can be diagnosed via qPCR, IFA, and ELISA tests, and show high sensitivity, but are not feasible for diagnosis in rural areas, due to their impoverished nature. Here, we propose start-up activities that should allow the creation of a biosensor-based strategy for field detection of the bacteria responsible for CD. Our CD biosensor will be developing through a process known as SELEX: systematic evolution of ligands by exponential enrichment using the commercial x-aptamer selection system. This process will generate a specific probe against the Pap31 protein shed by the pathogenic bacteri responsible for CD which will be adapted into an electrochemical DNA-based biosensor. E-DNA sensors should provide a rapid means of testing for CD. These sensors have a long shelf-life and are reusable, as well as requiring only small blood samples. Electrochemical sensors provide easily read and interpreted output suitable for point-of-care conditions and can be analyzed using portable equipment on site. Thus, only a finger lancet-derived blood sample obtained in the field would be required. Ultimately, these characteristics offer the broader impact of better, timelier and less invasive diagnostics for those affected by this disease.
Whooping cough caused by Bordetella pertussis can cause serious and prolonged health effects, particularly in infants. Quick and accurate diagnosis is essential in treating the infection. Early treatment can significantly reduce the duration of illness in the patient and lead to a milder case of illness overall. The current standard for testing set forth by the CDC is invasive, requiring nasopharyngeal swabs. Most medical facilities are not equipped to then process the sample collected, and it is instead sent out for analysis adding to the length of time required to get a diagnosis for the patient. Common laboratory tests for the detection of B. pertussis are PCR and culture, but each present unique challenges. The purpose of our research is to design and develop a rapid electrochemical biosensor that can detect P.69 pertactin. P.69 pertactin is a surface protein used by B. pertussis to bind to mammalian cells. A biosensor that can detect P.69 pertactin would improve several major issues associated with the current testing standards including size of the patient sample needed, time to get results, cost of testing and shipping, and accuracy of tests. The B. pertussis biosensor will be developed through the SELEX process and will create a specific oligonucleotide probe for P.69 pertactin . This probe will then be adapted into an electrochemical DNA-based biosensor. This biosensor would require a much smaller sample from the patient and give almost immediate results to the physician overseeing the case.
The goal of this project is to develop an electrochemical biosensor capable of rapid detection of pathogenic Mycoplasma bacteria in academic, research, and clinic settings. This research is highly significant because this biosensor will be capable of detecting two of the three pathogenic Mycoplasma strains that cause human disease, namely Mycoplasma pneumoniae and Mycoplasma genitalium. Mycoplasma pneumoniae, the more prevalent of the two, infects almost two million people every year in the U.S. It is responsible for contagious upper respiratory infections and is also known as atypical or “walking pneumonia” that spreads rapidly in crowded areas2. The second most prevalent, Mycoplasma genitalium, is responsible for approximately 15%–20% of nongonococcal urethritis (NGU) cases, 20%–25% of nonchlamydial NGU, and approximately 30% of persistent or recurrent urethritis3. In addition to causing these human diseases, Mycoplasma also continues to be a major contaminant of laboratory cell cultures4. Current methods of detections for Mycoplasma bacteria, such as molecular-based assays, PCR and serological analysis are time consuming, expensive, and less suitable for working under stringent conditions such as extreme temperature or pH1. Moreover, current serological methods create possibilities of false negative results for most infected individuals because they do not measure the presence of the microorganism. They instead focus on measuring the host immune response. Therefore, our research will aim at detecting the presence of these Mycoplasma strains with a diagnostic electrochemical biosensor that is designed for rapid, reliable, and reagentless serological detection of several common Mycoplasma strains. Using this electrochemical biosensor we will be able to detect each listed pathogenic Mycoplasma, by identifying a commonly secreted protein, P48. Dr. Bonham and his past student researchers designed a successful aptamer (a DNA oligonucleotide designed to selectively bind to and detect the P48 protein) with which we will work to create a final product that is sensitive enough to detect P48 protein in a minimal amount of human serum. This will allow us to potentially improve both prevention and diagnosis of Mycoplasma in patients who present a proposed infection.
Human Ecto-NOX Disulfide-Thiol Exchanger 2 (ENOX2) is a cell-surface metalocatalyst which is expressed solely on malignancies of all major human cancer types. ENOX2, along with its ubiquitous parent protein ENOX1, serve as terminal oxidases in plasma membrane electron transport chains, suggesting that such proteins are essential for cell growth. ENOX2 is frequently shed from malignancies into the sera and demonstrates site-specific transcript variants, and thus presents an attractive target for both diagnostic blood testing and anti-cancer therapeutic treatments. To better facilitate detection and targeting of ENOX2 in patient sera, we utilized SELEX to generate aptamers against recombinantly expressed ENOX2. These aptamers were characterized via gel mobility shift assays to determine the candidate aptamer with highest affinity for human ENOX2. The candidate with highest affinity was incorporated into an electrochemical DNA-based biosensor, where changes in DNA confirmation upon target binding result in changed dynamics of an appended redox reporter molecule, ultimately generating a rapid and quantitative change in the current of the electrochemical response. This novel ENOX2-targeting electrochemical biosensor allows rapid, dose-responsive readout for the presence of ENOX2 in both buffer and blood serum.