A Study on Development of Paper-Based Lateral Flow Biosensing Platforms for High-Performance Point-of-Care Testing

Abstract

As the global prevalence of high burden diseases, such as cardiovascular diseases, shifts towards low- and middle-income countries, the importance of medical diagnostics based on point-of-care testing (POCT) is rapidly increasing. However, the majority of current diagnostic tests that meet clinical standards rely on high-end biochemical analyzers in central hospitals, thereby restricting easy access to high-performance testing for many in developing countries, as well as in suburban/rural areas of developed countries. Paper-based lateral flow assays (LFAs) are among the most widely used and ideal biosensing platforms for POCT owing to their simplicity, ease of manufacture, low-cost, and long shelf life, which makes it highly deliverable to end-users at various resource-setting levels. Conventionally, colloidal gold nanoparticles (AuNPs) have been used as an exclusive label for LFAs. However, owing to the low extinction coefficient of AuNPs, the typical LFAs are incapable of high- performance biomarker testing, i.e., (i) highly sensitive detection of target at sub-ng/mL and pg/mL level and (ii) highly quantitative and precise analysis within coefficient of variation of 10%. Alternatively, signal amplification-based LFAs generating enhanced optical signals via bio/chemical reactions have enhanced the analytical capacity. However, current bioconjugation schemes for signal amplification have limited labeling of enzymes to very few reactive moieties in the antibody or restricted surface of AuNPs, thereby preventing trace level biomarker detection. Moreover, the signal amplification-based LFAs commonly require manual and multi-step reactions that increases complexity, imprecision, testing time, and cost, as trade-offs, compared to conventional LFAs. Recently, alternative paper-based assays were developed to automate the multi-step reactions, but the structures and materials used in the assays are not optimized for the current framework of mass-production, thereby limiting the practical use of the signal amplification-based LFAs. This dissertation reports on the development of advanced paper-based LFA biosensing platforms for high-performance POCT, and demonstrates their clinical/practical utility by testing cardiac troponin I (cTnI), a clinical standard biomarker for the diagnosis of myocardial infarction (MI). The strategies of this research consist of two-tier approach based on chemistry, biotechnology, materials science, and engineering, namely (i) developing novel conjugation schemes that facilitate efficient incorporation of enzymes to AuNP conjugate by employing aldehyde activated horseradish peroxidase, (ald)HRP, and polymerized HRP (polyHRP), respectively, and (ii) constructing simple, programmable, and manufacturable LFA architectures that automate immunoreaction and signal amplification via fluid switching polymers (i.e., water- swellable polymer and water-soluble polymer). In Chapter 2, the process of design, synthesis, and evaluation of AuNP conjugates that include both Ab and HRP is reported. To increase the labeling efficiency of HRP on the AuNP-based conjugates while maintaining process simplicity, two synthesis methods were suggested and evaluated. Particularly, AuNP conjugate labeled with physical adsorption of (ald)HRP and subsequent covalent coupling of Ab, AuNP-(ald)HRP-Ab, is investigated as a new candidate for conjugating AuNP, HRP and Ab in a single framework. The conjugates were characterized via spectroscopy and dynamic light scattering methods. The sensitivity of the conjugates was evaluated by analyzing the activity of HRP and comparing the analytical sensitivity provided by the chemiluminescence (CL)-based enzyme-linked immunosorbent assay for the detection of cTnI as a model target. The results of this chapter demonstrated that the use of (ald)HRP successfully enhanced the sensitivity of the immunoassay compared with the conventional conjugates used for enzyme-based immunoassay. In Chapter 3, the application of the AuNP-(ald)HRP-Ab conjugate for the development of highly sensitive CL-based LFA is reported. When paired with the CL-based signal readout modality, the AuNP-(ald)HRP-Ab conjugate resulted in 110-fold enhanced sensitivity over the colorimetric response of a typical AuNP-Ab conjugate. The performance of the CL-based LFA using the conjugate was tested by assaying cTnI in standard and clinical serum samples. Testing the standard samples revealed a detection limit of 5.6 pg mL-1 and acceptably reliable precision (coefficient of variation, 2.3–8.4%), according to clinical guidelines. Moreover, testing the clinical samples revealed a high correlation (r = 0.97) with a standard biochemical analyzer, demonstrating the potential clinical utility of the CL-based LFA for high-performance cTnI testing. In Chapter 4, the development of a highly sensitive, automated, universal, and manufacturable LFA biosensing platform is described. This project is based on by (i) incorporating AuNPs into a polymeric network of HRP, polyHRP, with an Ab as a new scheme for enhanced enzyme conjugation and (ii) integrating a mass-producible and time-programmable amplification part based on a water-swellable polymer for automating the sequential reactions. Coupled with an HRP-catalyzed CL method, this platform enables automated, cost-effective (0.66 USD per test), and high-performance testing of human cTnI in serum samples within 20 min with a detection range of 6 orders of magnitude, detection limit of 0.84 pg mL-1 (595-fold higher than conventional cTnI-LFA), and coefficient of variation of 2.9–8.5%, which are comparable to the standard equipment and acceptable for clinical use. Moreover, cTnI analysis results using clinical serum/plasma samples revealed a strong correlation (R2 = 0.991) with contemporary standard equipment, demonstrating the practical application of this platform for high-performance POCT. In Chapter 5, the development of a paper/soluble polymer hybrid-based LFA platform, paired with a smartphone-based reader, is reported. The testing architecture incorporates a polymeric barrier that programs/automates sequential reactions via a polymer dissolving mechanism. The smartphone-based reader with simple opto-mechanical parts offers a stable framework for accurate quantification. Coupled with catalytic/colorimetric gold-ion amplification, this platform produced results within 20 min, with a detection limit of 0.92 pg mL-1 and a coefficient of variation <10%, which is equivalent to the performance of a high-sensitivity standard analyzer, and operated within acceptable levels stipulated by clinical guidelines. Moreover, cTnI clinical sample tests indicate a high correlation (Pearson’s r = 0.981) with the contemporary analyzers, demonstrating the clinical utility of this platform in high-performance POCT.