Browsing by Subject "Immunoassay"

Detection and quantification of biomarkers directly from complex clinical specimens is desired and often required by healthcare professionals for the effective diagnosis and screening of disease, and for general patient care. Current methodologies to accomplish this task have critical shortcomings. Laboratory immunoassays, most notably enzyme-linked immunosorbent assay (ELISA) require extensive clinical infrastructure and complex user intervention steps to generate results and often are accompanied by a lengthy time-to-result. Conversely, available point-of-care (POC) diagnostic solutions, most notably available lateral flow immunoassays (LFIAs), often struggle with sensitivity and specificity in complex fluids, lack quantitative output and are not easily multiplexed. In this dissertation I will discuss the design, fabrication, testing, and refinement of an all-in-one fluorescence microarray integrated into a passive microfluidic fluid handling system to create a versatile and automated POC platform that can detect biomarkers from complex samples for disease management with the relative ease-of-use of an LFIA and the performance of a laboratory-grade test. The platform is driven by capillary and gravitational forces and automates all intervention steps after the addition of the sample and running buffer at the start of testing. The microfluidic cassette is built on a (poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) polymer brush which imparts two key functionalities, (1) it eliminates cellular and protein binding, and when combined with the vertical orientation of the microfluidic cassette prevents settling of debris during all assay steps. This allows for impressive sensitivities and specificities to be obtained from samples as complex as undiluted whole blood even when relying on gentle capillary and hydrostatic pressures for cassette operation. (2) Paradoxically, printed biorecognition elements can be stably and non-covalently immobilized into the POEGMA allowing for all reagents needed to conduct a sandwich immunoassay in a single step to be easily inkjet printed as spatially discrete spots into the POEGMA brush, which also stabilizes them at room temperature. Additionally, the microfluidic cassette is compatible with the “D4Scope” a handheld fluorescence detector that can quantify the output of the microfluidic cassette in seconds at the POC and is the only piece of auxiliary equipment required to operate the test.

This dissertation discusses early cassette prototypes and characterizes the performance of major device iterations (Chapter 2) before moving into three clinical applications of the cassette. First, a multiplexed serological test to detect antibodies against different proteins of the SARS-CoV-2 virus was developed (Chapter 3). Second, a multiplexed COVID-19 diagnostic test that simultaneously differentiates which variant you are infected with was developed (Chapter 4). Third, a sensitive fungal infection test for the diagnosis of talaromycosis was developed (Chapter 5). Finally, a rapidly iterative yet highly scalable injection molding fabrication process flow was created and characterized to improve performance and translatability of the cassette (Chapter 6).

Bioanalytical techniques such as immunoassays are ubiquitous in clinical and basic research laboratories and have transformed how we diagnose patients, monitor health, and study disease. Immunoassays typically use capture reagents, such as antibodies or antigens, to detect and quantify a biomarker of interest from a clinical specimen based on highly sensitive and specific binding interactions. While laboratory-based assays, such as enzyme-linked immunosorbent assay (ELISA), are the workhorses of clinical laboratories, they have several shortcomings that limit their overall utility, especially in low resource settings. Of note, ELISA requires multiple timed incubation steps, trained personnel, expensive equipment, and suffers from long times to result. To democratize access to clinical-grade tests, researchers have sought out different methods for point-of-care (POC) testing that are easy to perform and maintain high sensitivity and specificity. This dissertation describes the use of a “zero-background” polymer coating—poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA)—as a substrate for highly sensitive and specific POC diagnostic tests. The POEGMA coating eliminates nearly all non-specific protein adsorption and cellular adhesion, thus leading to high signal-to-noise ratios, even from complex biological samples, such as whole blood. In addition, the POEGMA brush contains all biomolecules necessary to complete an assay after addition of a liquid sample, thus allowing assays to be conducted in a single step. Further, the POEGMA coating stabilizes biomolecules on the surface, which allows tests to be stored at ambient conditions without refrigeration. Assays are read out using a fluorescence detector which quantifies the concentration for a given biomarker of interest. By inkjet printing capture biomolecules at discrete spatial addresses on the POEGMA-slides, multiplexing can be accomplished using a single fluorophore which greatly reduces the complexity and costs for assay readout. This dissertation focuses on adapting and applying this platform to several clinically relevant applications. First, we developed a test for molecular and cellular credentialing of breast cancer tissue at the POC (Chapter 2). With the onset of the coronavirus 2019 (COVID-19) pandemic, we adapted the platform to detect several different relevant biomarkers for COVID-19, including total antibody concentration against several viral proteins (Chapter 3), neutralizing antibodies (Chapter 4), and viral proteins (Chapter 5). All the tests developed for COVID-19 use multiplexed sensing strategies and can be conducted with minimal/no user intervention or clinical infrastructure. Taken together, these studies highlight the great potential for bioanalytical assays built upon POEGMA-coated substrates to be used for clinical applications in disease diagnosis, surveillance, and management.

Fluorescence-based methodologies have been used extensively for biosensing and to analyze molecular dynamics and interactions. An emerging, promising diagnostic tool are fluorescence-based microarrays due to their high throughput, small sample volume and multiplexing capabilities. However, their low fluorescence output has limited their implementation for in vitro diagnostics applications in a point-of-care (POC) setting. Here, by integration of a sandwich immunoassay microarray within a plasmonic nanogap metasurface, we demonstrate strongly enhanced fluorescence enabling readout by a fluorescence microarray even at low sensitivities. We have named this plasmonic architecture the plasmonically enhanced D4 (PED4) assay. The immunoassay consists of inkjet-printed capture and fluorescently labeled detection antibodies on a polymer brush which is grown on a gold film. Colloidally synthesized silver nanocubes (SNCs) are placed on top of the brush through a polyelectrolyte layer and interacts with the underlying gold film creating a nanogap plasmonic structure supporting local electromagnetic field enhancements of ~100-fold. By varying the thickness of the brush between 5 and 20 nm, a 151-fold increase in fluorescence and a 14-fold improvement in the limit-of-detection (LOD) is observed for the cardiac biomarker B-type natriuretic peptide (BNP) compared to the unenhanced assay, paving the way for a new generation of point-of-care clinical diagnostics.

To move the PED4 towards a single step point of care test (POCT), SNCs are conjugated with a secondary antibody that attaches specifically to the detection antibody. This allows SNCs to deposit on the surface without the need of a charged polyelectrolyte layer. In addition, multiplexing capabilities are demonstrated in this plasmonic platform where NT-proBNP, Galectin-3, and NGAL are simultaneously detected and fluorescently enhanced. Microfluidics integration and use of a low-cost detector is also demonstrated.

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