Advancing Troponin Point-of-Care Testing through Silicon Photonic Biosensors: Enhancing the Accuracy of Heart Attack Diagnosis
Creators: name: Beh, Siew Joo
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 320602
subject: Medical biotechnology diagnostics (incl. biosensors)

description: The adoption of cardiac troponin point-of-care testing in clinical settings remains challenging despite the rapid advancement of biosensing technology in the last decades. Point-of-care testing provides a faster troponin result at a fraction of the time, but its accuracy must correlate well with laboratory testing. The major drawback of point-of-care testing stems from its lower diagnostic sensitivity compared to central laboratory-based assays, such as benchtop chemiluminescent analyser, which can detect multiple cardiac biomarkers at a higher level of sensitivity within a single assay. However, this standard testing has its flaws. Even with the generation of the so called ‘high-sensitivity’ troponin assays, these benchmark assays still show discordant in the diagnoses of myocardial injury with different troponin I or T assays as shown by various comparative clinical studies published in the last 20 years. 

Since early 1990s, the measurement of cardiac troponin assay has been based on the specific detection of either cardiac troponin I or troponin T subunit proteins, which are released into the bloodstream during myocardial infarction. However, these commercial troponin assays do not capture the complete extent of the infarction, as cardiac troponin is a complexed protein that metabolises into different isomeric forms of troponin molecules. The chosen antibodies, usually in pair for capturing and detection purposes, only detect selected few isomer forms, resulting in serious underestimation of cardiac troponin in patient samples and increases the potential for misdiagnosis. Recent studies published by Katrukha et al emphasised the differences of troponin composition in blood over time following acute myocardial infarction. These findings highlight the importance of using multiple antibodies to capture different isomeric forms of troponin for a more comprehensive diagnosis of the severity and extent of myocardiac injury.

To that end, in order to achieve a highly sensitive and accurate point-of-care testing that can rival the laboratory benchmark, it is first necessary to develop an assay that has the capability of discerning and quantifying troponin isomers in the blood. These studies provide insight and motivation for this doctoral thesis to detect the various troponin isomers during acute myocardial infarction progression to enhance the accuracy of troponin point-of-care testing. Ultimately, the next generation of troponin assays should provide clinical profiling of patients with acute myocardial infarction based on the composition of troponin in blood. This would enable clinicians to identify early to late acute myocardial infarction patients, potentially impacting timely treatment for these patients. 

Photonic biosensors offer high sensitivity, multiplexing and label-free sensing capabilities, making them an emerging field with significant potential for advancing point-of-care diagnostics. First, they require no additional labelling that complicates the detection process. This increases the speed of analysis and lowers the overall cost, both of which are important for point-of-care diagnostic testing. Second, they can be easily integrated into a compact platform which is crucial for medical testing in emergency situations especially in remote areas. Medical professionals require access to devices that are easy to use and quick to deliver result to save lives. Third, photonic biosensors have the capability to be multiplexed, allowing the testing of all isomeric forms of troponin simultaneously, which increases the overall assay specificity. In future, this can be extended into the addition of other cardiac biomarkers to further enhance the specificity. Lastly, they can achieve high sensitivities, with some studies reporting detection limits in the attomolar to femtomolar range which can ensure troponin to be detected even in samples with ultra-low concentration.

Initially, a commercial surface plasmon resonance biosensor was utilised with a two-step approach to detect all troponin I forms. While the approach could differentiate between these isomers, it fell short of fully quantifying them. The insights gained, including the limitations found with surface plasmon resonance, led to the consideration of silicon photonic biosensor as a more suitable platform. An integrated Mach-Zehnder-based silicon photonic biosensor, coupled with an automated microfluidic system, was then employed, enabling precise sample manipulation necessary for biosensing. The performance of this biosensor was analysed in terms of sensitivity, reproducibility, and selectivity for troponin complex. The biosensor exhibited selective troponin detection within 10 minutes, operating across the ng/mL-µg/mL range with high reproducibility, and achieving a low limit of detection of 3 ng/mL, highlighting its potential for rapid and precise troponin detection. Nevertheless, further enhancements in sensitivity, reproducibility and selectivity were imperative to advance this system towards clinical point-of-care applications. A one-step silicon surface functionalisation using Silane-PEG molecules, based on the research of Cosa et al research on functionalising glass surfaces for fluorescence studies, was then introduced. This method was carefully adapted and optimised to be applied on silicon surfaces, with the purpose of creating a uniform monolayer of antibodies across the sensor surface and generating reproducible biosensors for cardiac troponin detection. This surface chemistry method was tested on the silicon photonic biosensor and the biosensor response was compared to the previous physiosorbed method. Despite the inherent limitations of this new approach, a significant improvement of at least 76% was observed in the biosensor response. The findings of this thesis demonstrated the capability of integrated silicon photonic biosensors for troponin detection. Further refinement of surface functionalisation and testing of the two-step methodology are necessary to enable the technology’s use with blood samples. The ultimate objective is to advance this technology for clinical readiness, with the goal of developing a point-of-care device that can improve patient outcomes and alleviate long-term burdens on the healthcare system.