(6w) Increasing Global Access to Diagnostic Testing Using Low-Cost, Non-Instrumented Paper-Based Microfluidics

INCREASING GLOBAL ACCESS TO DIAGNOSTIC TESTING USING LOW-COST, NON-INSTRUMENTED PAPER-BASED MICROFLUIDICS

Bhushan J. Toley

4^th year Postdoctoral Fellow

Bioengineering Department, University of Washington

Doctoral Thesis

Title: Microtechnologies for mimicking tumor-imposed transport limitations and developing targeted cancer therapies

Funding: NIH, NSF

Advisor: Prof. Neil Forbes

Postdoctoral Project

Title: Development of Multiplexable Autonomous Disposables for Nucleic Acid Amplification Tests (MAD NAAT)

Funding: DARPA, NIH

Advisor: Prof. Paul Yager

Research Interests

The primary aim of my research is to reduce the cost of, and increase access to state-of-the-art medical diagnostic testing by bringing it to the point-of-care. Through my research, I aim to develop portable and low-cost diagnostic tests that are deliverable to low resource settings and are operable by untrained users, and that emulate the abilities of a high-tech lab. The core technology I plan to use is paper-based microfluidics, which enables controlled transport of microliter volumes of fluids without the need of pumps or electricity.

Research Experience

The common theme of my research projects till date has been the design of devices that enable fluid manipulation at sub-millimeter length scales, the use of such (microfluidic) devices for conducting biological processes, and quantitatively analyzing these biological processes by applying core chemical engineering principles of transport, thermodynamics, and chemical kinetics. 

The aim of my doctoral research was to develop methods that would enable quantification of drug and nutrient gradients in tumor tissues in vitro. To this end, I developed a microfluidic in-vitro model of continuously perfused tumor tissue that mimics the region of a tumor adjacent to a blood vessel. I used this device for a) assessing the anti-cancer therapeutic potential of several bacterial strains, and b) estimating the transport parameters of two standard chemotherapeutic drugs – doxorubicin and Doxil. By applying chemical transport models to these systems, I estimated key transport parameters of the therapeutic agents and showed that a) motility governs the location of bacterial accumulation in tumor tissue, and b) ideal chemotherapeutic drugs must have an optimal combination of diffusion, binding, and cytotoxicity to be effective. In another study, I quantified the effect of surface charge of gold nanoparticles carrying doxorubicin on the rate and location of their tumor penetration. This work was published in Nature Nanotechnologyand has been cited over 150 times. 

The aim of my postdoctoral work is to develop a paper-based platform device that can conduct a sample-to-answer nucleic acid amplification test (NAAT). While paper-based microfluidic devices enable fluid flow without pumps, they have relatively limited flow control and valving capabilities compared to conventional microfluidic systems. To bridge this gap, I developed several new paper-microfluidic valving techniques. In my first publication, I demonstrated that relative differences in properties of porous materials can be used to shunt fluids from the main fluidic path to generate controlled delay lines. In my second publication, I presented a valving toolkit based on expandable elements that can conduct many types of fluidic operations, like on-switches, off-switches, and flow-diversion switches. A combination of these valves can be used to conduct arbitrarily complex, multi-step fluidic operations in paper-based devices. In addition to designing fluidic systems, I conducted extensive experiments to optimize the performance of isothermal strand displacement amplification (iSDA) – a new method of DNA and RNA amplification – in paper substrates. Simultaneously, I developed kinetic mathematical models of the iSDA reaction network that helped us gain key insights into the molecular mechanisms of iSDA. My research has led to the development of a prototype paper-based microfluidic device that can detect as low as 100 copies of Staph. aureusbacteria from a nasal swab by conducting an automatic multi-step molecular assay, making it the first device of its kind.

Mentoring and Teaching Experience

Over the course of my career, I have mentored 14 undergraduate students and 2 high school students; 7 out of these mentees have coauthored papers in peer-reviewed journals with me. I have served as teaching assistant for core chemical engineering undergraduate courses taken by juniors: i) Separation Processes – for 3 semesters and ii) Process Control and Dynamics – for 1 semester.

Future Research Directions

The aim of my future research is to develop patentable and commercializable technologies that will be used in devices that can provide the quality of testing available in fully equipped diagnostic labs at locations where access to such labs may be limited or non-existent. My initial projects will leverage my expertise in paper-based microfluidics in developing portable non-instrumented systems for conducting a) protein detection tests, and b) nucleic acid amplification tests. At the same time, I want to better understand how fluids flow in porous matrices like paper. I am interested in applying concepts developed in the field of geology to model fluid flow in paper-based materials. Ultimately, I would like to develop a paper microfluidic design tool that will guide researchers in the field to easily design paper-based microfluidic devices. 

Protein Detection: Enzyme-linked immunosorbent assay (ELISA) is a well-established analytical lab technique used to quantify the amount of a substance in a sample. The ELISA procedure requires several hours to complete and requires a trained technician and lab instruments to conduct the multiple reagent dispensing and washing steps. Automation of ELISAs has traditionally required an instrumented system for pumping fluids. I am interested in developing a paper-based microfluidic platform device that will store dried ELISA reagents for at least 6 months and conduct the ELISA at the push of a button after a sample is introduced into the device. Such a device will be completely electricity and battery-free and can be disposed-off after a single use. Because all ELISAs require the same fundamental fluidic operations, this platform can be adopted to conduct any ELISA, even in remote geographical locations. 

Molecular detection of DNA/RNA: I am also interested in developing a platform for conducting automatic sample-to-answer NAATs based on human sputum as the sample. Such a platform will have tremendous utility in diagnosis of TB. Recently, the Cepheid GeneXpert instrument revolutionized TB diagnostics; the GeneXpert automates the multiple steps in the molecular detection of TB from sputum, thus providing sophisticated diagnosis at the point-of-care. However, over the last few years, it has been realized that the cost of the instrument and disposable cartridges is prohibitive for the people who have the most need for it, i.e. those in the developing world. The paper-based sample-to-answer TB test that I will develop will operate on a single battery but will be instrument-free and significantly cheaper than the GeneXpert.

List of Peer-Reviewed Publications

1.Toley BJ, Wang JA, Gupta M, Buser JR, Lafleur LK, Lutz BR, Fu E, Yager P, “A versatile valving toolkit for automating fluidic operations in paper microfluidic devices”, Lab on a Chip, 2015, 15, 1432-44. (Featured on back cover)

2.Panpradist N, Toley BJ, Zhang X, Byrnes S, Buser JR, Englund JA, Lutz BR, “Swab sample transfer for point-of-care diagnostics: characterization of swab types and manual agitations methods”, PLoS ONE, 2014, 9(9), e105786

3.Toley BJ, McKenzie B, Yager P, Fu E et al, “Tunable delay shunts for paper microfluidic devices”, Analytical Chemistry, 2013, 85(23), 11545-11552.

4.Spicar-Mihalic P*, Toley BJ*, Houghtaling J, Liang T, Yager P, Fu E; “CO₂ laser cutting and ablative etching for the fabrication of paper-based devices”, Journal of Micromechanics and Microengineering, 2013, 23(6) (*equal contribution)

5. Toley BJ, Tropeano-Lovatt Z, Harrington J, McGarry MJ, Forbes NS; “Transport-reaction-coupled pharmacokinetic pharmacodynamics model captures key mechanisms of Doxorubicin and Doxil therapeutic efficacy”, Integrative Biology, 2013, 5, 1184-1196

6.Dai Y, Toley BJ, Swofford C, Forbes NS; “Construction of an inducible cell-communication system that amplifies Salmonella gene expression in tumor tissue”, Biotechnology and Bioengineering, 2013, 110(6), 1769-81

7.Toley BJ, Forbes NS; “Motility is critical for effective distribution and accumulation of bacteria in tumor tissue in vitro”, Integrative Biology, 2012, 4, 165-176

8. Toley BJ, Park J, Kim BJ, Venkatasubramanian R, Maharbiz MM, Forbes NS; “Micrometer-scale oxygen delivery rearranges cells and prevents necrosis in tumor tissue in vitro”, Biotechnology Progress, 2012, 28(2), 515-525

9.Toley BJ*, Ganz DE*, Walsh CL, Forbes NS; “Microfluidic device for recreating a tumor microenvironment in vitro”, Journal of Visualized Experiments, 2011, 57, DOI: 10.3791/2425 (* equal contribution)

10. Kim BJ, Han G, Toley BJ, Kim C, Rotello VM, Forbes NS; “Tuning payload delivery in tumor cylindroids using gold nanoparticles”, Nature Nanotechnology 2010 Jun, 5(6), 465-72