Victoria Dagostino

Bioengineering, '19


Development of a Point of Care Assay for Detecting High Risk HPV in Resource Limited Settings

Mentor: Cesar Martin Castro, MD (MGH)

More than 85% of women who die of cervical cancer live in low- and middle- income countries1. The main reason for this disparity is that methods of combating cervical cancer exist, but are often not accessible in resource limited areas. Over 90% of cervical cancer develops from a persistent infection with a high risk type of human papilloma virus (HPV)2, meaning early detection and treatment of high risk Human papilloma virus types can prevent cervical cancer fatalities. However, detection is expensive, time consuming, and requires specialized equipment. Therefore to prevent cervical cancer deaths, an inexpensive and easy-to-use assay must be developed that can detect high risk HPV at the point of care. This project is to develop a high-throughput screening assay for four of the most common high risk HPV types directly from patient samples using an integrated portable device based on lens-free imaging. The device can be created using a 10x10mm glass coverslip coated with PDMS which is etched to create multiple lanes. Surface functionalization chemistry will be used to attach DNA probes for four HPV types, one in each lane. DNA from patients’ cervical brushings will be captured by complementary DNA probes. Magnetic microbeads conjugated to DNA probes will be attracted towards the functionalized surface by use of a magnet, and then magnetically washed away. Target DNA will capture the magnetic beads with the corresponding probe, and these beads will remain specifically bound to HPV-specific DNA while other beads are washed away. Using the Digital Diffraction Diagnostic (D3) lens free imaging device previously developed and optimized at MGH3, the beads in each lane will be counted to accurately diagnose the patient. This device will be further optimized to diagnose multiple patients simultaneously. A new innovation from the precedent system is implementing an isothermal amplification known as Recombinase Polymerase Amplification (RPA). The isothermal DNA amplification will replace the conventional PCR requiring a thermocycler and be well suited for use in resource limited settings. Cervical cancer mortality can be prevented by the detection and treatment of high risk HPV, and the vision of this project is to make that possible across the world. 1. World Health Organization. (June 2016). Human Papillomavirus (HPV) and cervical cancer [Fact sheet]. Retrieved from 2. American Cancer Society. (5 December 2016). What are the risk factors for cervical cancer [Fact sheet]. Retrieved from 3. Hyungsoon Im, Cesar M. Castro, Huilin Shao, Monty Liong, Jun Song, Divya Pathania, Lioubov Fexon, Changwook Min, Maria Avila-Wallace, Omar Zurkiya, Junsung Rho, Brady Magaoay, Rosemary H. Tambouret, Misha Pivovarov, Ralph Weissleder, and Hakho Lee. Digital diffraction analysis enables low-cost molecular diagnostics on a smartphone. PNAS 2015 112 (18) 5613-5618; published ahead of print April 13, 2015, doi:10.1073/pnas.1501815112

A. The Digital Diffraction Diagnostic (D3) device. This device is a miniature, portable, lens free imaging device developed at MGH for cancer diagnosis in low- and middle- income countries. B. Holographic image taken by D3 of polystyrene beads and silica beads. These beads are conjugated with DNA probes. In the presence of high risk HPV DNA, two bead-probe conjugates will form a dimer. C. A reconstruction of the holographic image: brighter spots represent a polystyrene bead, darker spots represent a silica bead, and the red arrows point to polystyrene-silica bead dimers representing the presence of HPV DNA. This reconstruction can be done remotely by uploading the holographic image to a secure cloud. Source: