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Title:Point-of-care microfluidic assays for measuring expression level of antigen on blood cells
Author(s):Ghonge, Tanmay
Director of Research:Bashir, Rashid
Doctoral Committee Chair(s):Bashir, Rashid
Doctoral Committee Member(s):Kenis, Paul; Pan, Dipanjan; Cunningham, Brian
Department / Program:Bioengineering
Discipline:Bioengineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Microfluidics, Point-of-Care, BioMEMS, Smartphone, Sepsis
Abstract:Elevated expression of a membrane protein CD64 on neutrophils is linked with the onset of sepsis, a life-threatening syndrome that contributes to millions of deaths annually worldwide. The survival rate of septic patients falls rapidly every hour the appropriate medication is delayed. Therefore, automated and periodic measurements of CD64 expression (nCD64) at the patient’s bedside could lead to timely medical intervention, thereby saving lives and billions of dollars. Gold standard assays for measuring nCD64, such as flow cytometry, require manual sample preparation and long incubation times. For point-of-care applications, however, an assay should be able to measure nCD64 with little to no sample preparation. This dissertation addresses the need by investigating portable, point-of-care platforms for measuring nCD64 from whole blood without any off-chip sample preparation. Our first platform is an electrical biosensor that measures nCD64 by measuring the fraction of immunologically captured cells expressing CD64. It consists of a capture chamber, for immunologically capturing cells and microfluidic coulter counters at its entrance and exit. In our study, we found that the fraction of cells expressing CD64 correlates linearly with nCD64. For our study, we optimized the geometry of the capture chamber and the coulter counters from the first principles of fluid mechanics and electrostatics respectively. This biosensor can produce a readout of nCD64 starting from just 10 μL of blood in 10 mins. Although this technique does not require off-chip sample preparation, red blood cells have to be lysed on-chip so that white blood cells can be counted by the electrical counters. Lysing red blood cells on-chip adds a step which requires multiple pumps running in parallel. Ideally, the technique should be able to measure nCD64 without red blood cell lysis. Towards that, we have developed an optical, microfluidic cell capture assay that works from whole blood. We demonstrate the proof-of-concept of this assay by measuring the density of biotin molecules on beads. We injected beads in a capture chamber that is functionalized with neutravidin. Beads that have a higher density of travel, on average, shorter distances in the chamber before they get captured compared to the beads with a lower density of biotin. We developed a statistical model to extract the probability of capture (ε) per interaction with a pillar from the spatial distribution of beads in the channel. ε is found to be linearly proportional to the surface density of biotin. We expanded this optical technique to measure the nCD64 on neutrophils. One μL blood whole blood is injected in a microfluidic channel consisting of a capture chamber functionalized with anti-CD64 antibodies. As was the case with beads, the immunologically captured have a distinct spatial signature of capture depending on the CD64 expression level. Samples with higher CD64 expression travel, on average, a shorter distance in the channel. Using the same statistical model used to quantify biotin density on beads, we quantified CD64 expression on neutrophils. To make this technique easily translatable to a point-of-care device, we assembled a smartphone-imaging set-up to replace bulky microscopes. Our smartphone microscope can measure CD64 from whole blood without the need for any sample preparation in about 20 mins. We believe that deploying this technology in hospitals could save millions of lives worldwide.
Issue Date:2019-02-28
Type:Text
URI:http://hdl.handle.net/2142/105135
Rights Information:Copyright 2019 Tanmay Ghonge
Date Available in IDEALS:2019-08-23
Date Deposited:2019-05


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