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Technologies for Blood Diagnostics

  • Author(s): Waldeisen, John Robert
  • Advisor(s): Lee, Luke P.
  • et al.
Abstract

For over three decades, the lateral flow assay (LFA) has remained the diagnostic gold standard for immunodetection. In the developed world, the diverse capabilities of these assays is relatively unknown. However in resource-limited settings, LFAs are the best diagnostic tool a clinician currently has other than a microscope to diagnose disease. Although most diseases are treatable and preventable, three diseases alone are responsible for killing more than 5 million people each year: HIV, malaria, and tuberculosis (TB). A devastating lack of diagnostic accessibility in the developing world prohibits accurate diagnosis leading to poor therapeutic administration of drugs on-hand and the increased incidence of drug resistance. Even here in the U.S., the dissemination of broad-spectrum antibiotics has led to the emergence of virulent strains of drug-resistant sepsis and mortality rates have spiked to greater than 35-50%.

This dissertation is part of an endeavor to alleviate the health disparities that are prevalent throughout our world. A recently realized mechanism for fluid actuation, degas-driven fluid flow, is applied for the development of point-of-care devices. The mechanism of degas-driven fluid flow is similar to capillary action, as the material properties of the device inherently determine fluidic actuation. However, larger fluid volumes can be controlled and advance fluidic logic can be programed, allowing the adaption of molecular diagnostic assays for pathogenic biomarker detection. Here, a 1D Fickian model of degas-driven fluid flow is present that is capable of predicting fluid movement. The understanding learned from this model is then applied for the development of polymeric point-of-care microfluidic diagnostic devices. Three specific diagnostic devices are discussed. The first is a blood sample preparation device capable of extracting nucleic acid and protein biomarkers for the simultaneous detection of HIV, malaria, and TB. Detection of TB is demonstrated on-chip with isothermal loop-mediated amplification. The second device is a hybrid LFA that employs sedimentation-based plasma separation, powered by degas-driven fluid flow, to assay for elevated levels of anti-phospholipid IgM antibodies in blood. This device is intended for the monitoring of patient response to TB drug therapy in resource-limited settings. Finally, the third device is intended for the self-monitoring of patients on anticoagulation prophylaxes. The device simultaneously measures hematocrit in addition to the INR value thus enabling the recognition of asymptomatic hemorrhage, an often-fatal side effect common to anticoagulant therapy. All devices are capable of naked-eye visual readout eliminating the need for external equipment.

Next, the disassembly of colloidal-based nanoparticle assemblies is optimized for the visual naked-eye detection of biomolecules on microfluidic devices. The cleavage of the nanoassemblies disengages the plasmon coupling between nanoparticles and shifts the observed dark field scattered light from orange to green. The integration of this detection scheme is incorporated for point-of-care biorecognition. Finally, the dissertation concludes with the presentation of the real-time PCR antibiogram for diagnosing drug-resistant sepsis. This method combines universal phenotypic susceptibility testing with the rapid diagnostic capabilities of PCR. Detection, susceptibility testing, minimum inhibitory concentration determination, and identification are achieved in less than 24 hours.

In summary, several technologies for performing blood diagnostics at the point-of-care and in the clinical diagnostic laboratory are introduced. The ideas presented in this dissertation are an effort to transform point-of-care testing by enabling the early detection of disease and mitigating health inequalities across the world.

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