UCLA

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.