Point of Care Molecular Diagnostics for Humanity

Citation

Malik, Imran Raouf (2014) Point of Care Molecular Diagnostics for Humanity. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9HH6H2D. https://resolver.caltech.edu/CaltechTHESIS:06062014-102704108

Abstract

Diagnostics of disease at POC (point of care) has been declared one of the Grand Challenge by the Bill and Melina Gates Foundation (BMGF). Infectious diseases constitute a major cause of disease burden and cause more than half a billion Disability-Adjusted Life Years (DALYs) and millions of deaths each year. They have an especially large effect on children under 5 years of age. We have analyzed data from the GBD 2010 (Global Burden of Disease) project to emphasize the damage caused by infectious diseases, and highlight the opportunity of using diagnostic tools to rapidly identify and treat diseases. To motivate the work of this thesis, we quantify the expected impact of appropriate diagnostic technologies.

We have also analyzed the requirements that a diagnostic tool should meet to generate the maximal global impact. We present various existing TPPs (Target Product Profiles) from different organizations and suggest some additions to these existing TPPs. We explain the particular molecular pathology technologies which have the potential to allow deployment of functional products in the developing world for point-of-care pathogen detection, especially in low-resource settings.

We perform a detailed analysis on existing polymerase chain reaction (PCR) systems and describe the problems caused with thermal performance and optical interrogation. We list the requirements that disposable cartridges for such instruments should meet and suggest a metal base design with polymer top. After detailed FEA simulations, we demonstrate that the thermal response can be modeled using a one-dimensional (1D) lumped element system. We show improvements in thermal response due to using a metal base and the effect of fluid height. We also performed thermal-structural simulations to quantify the stresses on the adhesive bonds of metal/polymer cartridges. Next, we explain fabrication of these cartridges. We show methods to dispense adhesive using a robot and a custom made jig to spread the adhesive during curing. The cartridge was tested with different PCR reagents and we obtained reaction efficiencies approaching those of the commercial real time PCR machines. Our fabrication technique is useful to join dissimilar materials and is production friendly. By developing custom software, we observed the cartridge performance in a continuous manner. We could see the thermal response of cartridges by continuous fluorescence monitoring, and used reflective aluminum which increase light collection efficiency.

We then present a simple and robust new way for thermal cycling. Robust thermal cycling has been a major challenge conducting PCR, especially in point of care situations. Here, we suggest a contact cooling approach, in which the cartridge rests on a thin metal plate with an integrated thin heater constructed from flexible printed circuit board (PCB) material. We use a solenoid to move a metal plate to cool down the sample cartridge during cycling. The metal plate then rests on a larger heat sink to disperse the shuttled heat. Our design is dust and water proof and was verified on a bench-top prototype.

A novel optical design for fluorescence detection during qPCR is also described. We suggest a lateral illumination waveguide geometry with prism coupling that eliminates lenses and is integrated into an injection molded cartridge. The light is homogenized using a light guide, and we quantify the sources of scattered stray light from the chamber edge by performing ray tracing simulations to optimize the precise geometry. The design is tolerant to misalignments and enables easy coupling of LED light into the chamber. As the light collection efficiency is high, the size of the chamber can be very small. We tested real PCR reactions using this concept and observed a rapid integration time, enabling very fast reading.

Sample preparation has been another challenge for all point-of-care (POC) lab-on-chip devices for many years. Here, we propose a new design which is robust, fast, flexible and simple, and uses a sliding seal to move the collected sample between various reservoir chambers. The sample moves on a slider sandwiched between seals that shuttles a DNA binding membrane between different reactions. Thus, size and volumes of reagents can be increased without increasing dead volumes. This design is easily automated, and positive displacement of fluids can work with many reagents without worrying about their characteristics such as foaming. The speed of the sample preparation protocols is high and complex protocols can be ported on this design concept, which we tested on real clinical samples and obtained impressive results. We designed and injection molded devices to test and verify this concept.

Finally, we focus on instrumentation and software required to allow our technology to be used at the POC. We describe our embedded electronics and describe the powerful micro-controller and various high performance ICs that are used to construct a fully functional for sample to answer instrument. We developed various versions of software. The developer software allows us to control our system and bench top setup. Our end user product includes a tablet and cell phone software interface. Software was developed for a windows 8 tablet, windows 8 phone and an Android based devices.

To conclude, we very briefly describe the POC systems that are under development: A portable qPCR system with a separate cartridge design, and a universal sample to answer system that performs qPCR, sample preparation and sample to answer protocols in one box depending on the cartridge.

As per best of our knowledge the cost of this technology is much lower than any other option in its class. The sample to answer instrument is expected to cost less than $500. The test cost is expected to be less than $5. The performance is not compromised. We hope that this work can help bring a transformative change in the practice of pathology especially in the developing world.

Thesis Files