Immunological and Molecular Techniques for Point-of-Care Diagnostics

The development of technologies that enable early and accurate diagnosis is critical to the management of infectious diseases, especially with highly transmissible pathogens or in cases where the early administration of the right antibiotics could be life-saving. Demonstrating the presence of the relevant pathogen, rather than looking for serological markers, is the surest and fastest way of confirming an infection and diagnosing a disease. In this biosensing approach, pathogenic bacteria, viruses and protozoa, along with their secreted proteins and constituent nucleic acids are the principal analytical targets. Rapid screening tests, suitable for use as point-of-care (POC) devices by minimally trained personnel in decentralized laboratories and in the field, can be useful in diagnosing patients presenting with non-specific symptoms and in expediting control measures to manage emerging infectious diseases or in the unfortunate event of a bioterror attack. In this work, two POC-friendly technologies have been developed and evaluated analytically and/or clinically. The first assay used embedded, microfabricated linear retroreflectors as bio-sensing surfaces and micron-sized magnetic particles as light-blocking labels in a semi-homogeneous format, resulting in a highly sensitive diagnostic immunoassay. The magnetic properties of the particle labels were useful in sample pre-concentration to increase sensitivity, while fluidic force discrimination was used to increase specificity of the assay. This assay, which was readout by an automated sample capture and imaging approach, was determined to have a limit of detection of less than 4000 R. conorii per mL from buffer. Also, a panel of rapid assays based on Recombinase Polymerase Amplification, an isothermal nucleic acid amplification technique, were developed for the detection of urinary tract infection (UTI) causing bacteria. The five assays developed as part of this panel detected 100 genomes per reaction or less in around ten minutes, and showed no cross-reactivity with high concentrations of non-specific gDNA. The assay panel’s overall clinical sensitivity, as determined from a 25-sample cohort of culture-positive urine samples, was 84%, and the clinical specificity, determined using a 10-sample cohort of culture-negative urine samples, was 100%.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Chemical Engineering

Grantor

University of Houston

Year dc:date.issued

2014

Author dc:creator

• Raja Venkatasubramaniam, Balakrishnan

Advisor dc:contributor.advisor

• Willson, Richard C.

Committee members dc:contributor.committeemember

• Conrad, Jacinta C.
• Varadarajan, Navin
• Shih, Wei-Chuan
• Ruchhoeft, Paul

• Diagnostics
• Biosensors
• Pcr
• Recombinase polymerase amplification (RPA)
• Recombinase polymerase amplification
• Microfluidics
• Retroreflectors
• QNano
• Izon
• Filmarray
• Rickettsia
• Urinary tract infections

Statement dc:rights

• The author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/10657/2807

OAI identifier oai:identifier

oai:uh-ir.tdl.org:10657/2807