A lateral electrophoretic flow diagnostic assay

Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a “lateral e-flow assay” and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings

Global Health Diagnostics Utilizing Consumer Electronics: Development and Field Implementation

The global health implications of improper disease screening and diagnosis are tremendous, contributing to unnecessary disease burden and ongoing transmission of infectious diseases. While tools have been developed to help trained physicians and scientists diagnose disease in centralized healthcare facilities, many patients present with symptoms in resource-constrained settings. The limitations in infrastructure and skill present are often incompatible with existing diagnostic tools. The work in this dissertation centers on the engineering design, validation, and deployment of optical diagnostics targeted to these resource constraints. The approaches presented here take advantage of advances in mobile consumer electronics that have put high quality optics, automation, and data transmission capabilities into a compact and widely available package. In Chapter 1, I describe the limitations of current diagnostic tools for two diseases, oral cancer and tuberculosis, and outline the potential for development of novel tools around mobile devices. In Chapter 2, I present work that is a prerequisite for the rational design of reproducible and quantitative imaging with a mobile phone. Mobile phones and their cameras have seen rapid changes in specifications and performance, but they remain consumer devices with several corresponding limitations for medical use. I characterize the imaging capabilities of mobile phones across time as part of a custom portable microscope, detailing sampling limitations, aberrations, and the consequences of unwanted image processing. I conclude that mobile phones, through appropriate optical design and workflow management, can perform sufficiently for diagnostic imaging applications. In Chapter 3, I demonstrate a multi-color fluorescence imaging device based on a mobile phone for read-out of a sandwich immunoassay on a microfluidic chip. Microfluidic devices, systems that manipulate small volumes of liquid, have the potential to enable rapid and appropriate diagnostics at the point of care through reducing volume for patient samples and lowering cost. One of the limitations of microfluidic systems is that measuring the output of the device traditionally requires a large conventional microscope and scientific camera for visualization and quantification. Multi-color fluorescence on a mobile phone offers a powerful and portable alternative. Current diagnostics for tuberculosis, a disease that kills over 1 million people a year globally, have advanced dramatically in the recent decade, but the devices that detect the DNA of tuberculosis have not succeeded in extending testing capabilities to peripheral healthcare settings where they are most needed. New nucleic acid amplification tests are promising for this setting; they eliminate the need for cycling temperature and provide a more rapid time to answer. In Chapter 4, I demonstrate that it is possible to control and measure the amplification of DNA in real-time with this new class of assay using a mobile phone camera. Demonstration of this capability required development of an optical system, custom software, sample handling geometry, and thermal management. I also include initial validation of a primer set contributed by collaborators that is sensitive across a range of strains of tuberculosis. Ongoing work is focused on combining these capabilities into an integrated tuberculosis diagnostic. In Chapter 5, I present a field deployment of a diagnostic microscope for oral cancer built around a tablet computer. Oral cancer is the single largest cause of cancer mortality for men in India and other high burden countries. Cultural habits that increase the risk of developing oral cancer combine with delays in diagnosis leading to poor prognoses for patients. To improve the diagnostic process, it is important to screen patients early in the disease progression and refer them for a full diagnosis and care. In this work, I adapt both a manual microscope and automated slide-scanning device to the needs of oral cancer screening by brush biopsy. Through collaboration with clinical and corporate partners, these devices have been deployed and data collection has begun. I present initial images generated from patients and classified by pathologists that demonstrate the diagnostic workflow in practice. Affecting care in a global health setting is a complex challenge, but through work presented in this dissertation I contribute a combination of novel diagnostic method development, detailed device characterization, and field data collection that demonstrate the potential of global health diagnostics based on consumer electronics

Quantitative imaging with a mobile phone microscope.

Use of optical imaging for medical and scientific applications requires accurate quantification of features such as object size, color, and brightness. High pixel density cameras available on modern mobile phones have made photography simple and convenient for consumer applications; however, the camera hardware and software that enables this simplicity can present a barrier to accurate quantification of image data. This issue is exacerbated by automated settings, proprietary image processing algorithms, rapid phone evolution, and the diversity of manufacturers. If mobile phone cameras are to live up to their potential to increase access to healthcare in low-resource settings, limitations of mobile phone-based imaging must be fully understood and addressed with procedures that minimize their effects on image quantification. Here we focus on microscopic optical imaging using a custom mobile phone microscope that is compatible with phones from multiple manufacturers. We demonstrate that quantitative microscopy with micron-scale spatial resolution can be carried out with multiple phones and that image linearity, distortion, and color can be corrected as needed. Using all versions of the iPhone and a selection of Android phones released between 2007 and 2012, we show that phones with greater than 5 MP are capable of nearly diffraction-limited resolution over a broad range of magnifications, including those relevant for single cell imaging. We find that automatic focus, exposure, and color gain standard on mobile phones can degrade image resolution and reduce accuracy of color capture if uncorrected, and we devise procedures to avoid these barriers to quantitative imaging. By accommodating the differences between mobile phone cameras and the scientific cameras, mobile phone microscopes can be reliably used to increase access to quantitative imaging for a variety of medical and scientific applications

Cytoplasmic volume modulates spindle size during embryogenesis.

Rapid and reductive cell divisions during embryogenesis require that intracellular structures adapt to a wide range of cell sizes. The mitotic spindle presents a central example of this flexibility, scaling with the dimensions of the cell to mediate accurate chromosome segregation. To determine whether spindle size regulation is achieved through a developmental program or is intrinsically specified by cell size or shape, we developed a system to encapsulate cytoplasm from Xenopus eggs and embryos inside cell-like compartments of defined sizes. Spindle size was observed to shrink with decreasing compartment size, similar to what occurs during early embryogenesis, and this scaling trend depended on compartment volume rather than shape. Thus, the amount of cytoplasmic material provides a mechanism for regulating the size of intracellular structures

A lateral electrophoretic flow diagnostic assay.

Immunochromatographic assays are a cornerstone tool in disease screening. To complement existing lateral flow assays (based on wicking flow) we introduce a lateral flow format that employs directed electrophoretic transport. The format is termed a "lateral e-flow assay" and is designed to support multiplexed detection using immobilized reaction volumes of capture antigen. To fabricate the lateral e-flow device, we employ mask-based UV photopatterning to selectively immobilize unmodified capture antigen along the microchannel in a barcode-like pattern. The channel-filling polyacrylamide hydrogel incorporates a photoactive moiety (benzophenone) to immobilize capture antigen to the hydrogel without a priori antigen modification. We report a heterogeneous sandwich assay using low-power electrophoresis to drive biospecimen through the capture antigen barcode. Fluorescence barcode readout is collected via a low-resource appropriate imaging system (CellScope). We characterize lateral e-flow assay performance and demonstrate a serum assay for antibodies to the hepatitis C virus (HCV). In a pilot study, the lateral e-flow assay positively identifies HCV+ human sera in 60 min. The lateral e-flow assay provides a flexible format for conducting multiplexed immunoassays relevant to confirmatory diagnosis in near-patient settings

A multi-phone mobile microscope.

<p><b>A</b> Diagram of the magnifying optics and illumination added to a mobile phone to create a transmission light microscope. <b>B</b> Prototype of a field-ready mobile microscope – the CellScope – that has a folded optical path for compactness and is equipped with a multi-phone holder and iPhone 4. Phone-specific variants have been evaluated on five continents for various applications. <b>C</b> A Wright stained blood smear taken on the mobile microscope with an iPhone 4 and 20×/0.4 NA objective showing the inscribed field of view captured by the device. <b>D</b> Enlarged images of the small region of interest in <b>C</b> containing a granulocyte and red blood cells taken with four different mobile phones. The images demonstrate resolution, color, and brightness differences among phones.</p

Mobile phones differ from scientific cameras in selection of image capture and processing parameters.

<p><b>A</b> Common core hardware components underlie the capture process of both mobile phone cameras and scientific cameras. <b>B</b> The capture and processing parameters are set directly through the user interface of a scientific camera. <b>C</b> On mobile phones, an intermediate layer assesses the view of the camera in real-time and modifies image acquisition. This simplifies the user interface for traditional point-and-shoot photography but sacrifices the control desired by a scientific user.</p

Spatial resolution of mobile phone microscopy is dependent on microscope optics.

<p><b>A</b> The resolution that can be captured with a mobile phone microscope approaches that of a scientific camera coupled to the same optics across a range of numerical apertures. Inset shows the measured intensity profile across bars of non-transmitting chrome spaced at 512 line pairs per millimeter and taken with a 10×/0.25 NA objective, as well as the ideal target profile. The Michelson contrast calculated for this example group is 41%, indicating that features with this spacing are resolved. <b>B</b> Wright stained blood smear with an inset of a granulocyte and red blood cells taken with a 10×/0.25 NA objective and iPhone 4. <b>C</b> Image of the same sample and region of interest taken with a 40×/0.65 NA objective and iPhone 4 showing improved resolution.</p