Immunoassays are the gold standard for detecting biomarkers in disease diagnosis and treatment. While fluorescent immunoassays are popular, conventional methods using 96-well plates are cumbersome, time-consuming, and require bulky equipment, hindering point-of-care testing. Lab-on-a-chip technology offers miniaturized alternatives, with various microfluidic chips developed for faster and more efficient assays. However, sensitivity remains a crucial parameter to improve. Nanostructures, particularly plasmonic nanostructures, offer significant potential for enhancing the sensitivity of immunoassays due to the metal-enhanced fluorescence (MEF) effect. While various nanostructures (e.g., gold films, nanoparticles, nanopillars, nanorods, nanowells) have been explored, their fabrication often involves complex and expensive techniques like e-beam lithography or nanoimprinting. This research aims to overcome these limitations by utilizing a simple and cost-effective fabrication method – oblique angle deposition (OAD) – to create Au nanorod arrays integrated into microfluidic chips for highly sensitive and rapid immunoassays. The authors previously demonstrated the effectiveness of OAD in creating nanorod arrays for other applications. This study focuses on integrating these arrays into a microfluidic device for the detection of cardiac troponin I (cTnI), a key biomarker for heart attacks, along with other clinically relevant biomarkers such as prostate-specific antigen (PSA) and carcinoembryonic antigen (CEA).

The paper reviews existing literature on immunoassays, highlighting the limitations of traditional methods and the advancements in lab-on-a-chip technology to address these limitations. It discusses different microfluidic platforms such as plastic microfluidic chips for one-step immunoassays (Gervais and Delamarche), multiplexed volumetric bar-chart chips (Song et al.), and other lab-on-a-chip approaches. The review also explores the use of nanostructures for enhancing the sensitivity of immunoassays, mentioning examples like plasmonic gold films, nanoparticles, nanopillars, nanorods, and nanowells. It critically evaluates the existing nanofabrication techniques, such as e-beam lithography (EBL), nanoimprinting, and colloidal lithography, pointing out their drawbacks in terms of cost, complexity, and throughput. The literature review sets the stage for the proposed method, emphasizing the need for a simple, cost-effective, and high-throughput fabrication method for creating sensitive and rapid immunoassay devices.

The study employed oblique angle deposition (OAD) to fabricate dense arrays of gold (Au) nanorods directly onto glass substrates. A shadow mask was used to define the deposition area, eliminating the need for advanced lithography techniques. The morphology of the nanorods was characterized using scanning electron microscopy (SEM), revealing their average diameter and length. COMSOL Multiphysics simulations were used to optimize the nanorod dimensions (diameter and pitch) to maximize the metal-enhanced fluorescence (MEF) effect, targeting the excitation and emission wavelengths of the Alexa 488 fluorophore. The Au nanorod arrays were functionalized with a self-assembled monolayer (SAM) of 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) (DSP) to covalently immobilize capture antibodies (cAb). For the immunoassay, the target protein (e.g., cTnI) was incubated with detection antibodies (dAb) conjugated with Alexa 488. The sandwich immunoassay was performed on both Au nanorod arrays and reference glass slides for comparison. The fluorescence intensity was measured to determine the detection limit and dynamic range. For the flow-through assay, the Au nanorod arrays were integrated into a microfluidic chip with a serpentine microchannel design for efficient mixing and a capillary pump to control the flow rate. The dAb was pre-patterned in a deposition zone, and the sample was introduced at the inlet, flowing through the channels, binding with the dAb and then the cAb on the nanorods. The fluorescence intensity was then measured in the reaction chamber.

The key findings demonstrate a significant improvement in the sensitivity of immunoassays using the integrated Au nanorod arrays. The detection limit for cTnI was remarkably reduced to 33.9 fg mL⁻¹ (~1.4 fM) on the nanorod substrates compared to 22.9 ng mL⁻¹ (~1.0 nM) on glass slides, representing a more than 6 × 10⁵-fold improvement. Similarly, significant sensitivity enhancements were observed for PSA and CEA assays. The flow-through microfluidic device enabled rapid and sensitive detection of cTnI in human serum with a detection limit of 6.9 pg mL⁻¹ (~0.3 pM) within 6 minutes. The dynamic range of the assays on the nanorod arrays was also significantly broader than on glass slides, spanning several orders of magnitude. The simulations confirmed that the optimized nanorod geometry effectively enhanced the electric fields around the nanorods, leading to the observed MEF effect. The SEM images showed the successful fabrication of dense, irregular nanorod arrays using OAD, which contributed to the enhanced assay performance.

The results demonstrate the high sensitivity and speed of the developed immunoassay platform. The significant enhancement in sensitivity is attributed to the combined effects of the large surface area provided by the nanorod arrays and the MEF effect, which amplifies the fluorescence signal. The use of OAD offers a simple, scalable, and cost-effective fabrication process compared to conventional nanofabrication techniques. The rapid flow-through assay format minimizes the assay time and sample volume, making it suitable for point-of-care applications. The ability to multiplex the assay by simultaneously detecting multiple biomarkers further enhances the clinical utility of this platform. The findings have significant implications for early disease detection and diagnostics, potentially leading to improved patient care and management.

This research successfully demonstrated a highly sensitive and rapid microfluidic immunoassay platform using Au nanorod arrays fabricated via OAD. The simple, cost-effective fabrication method and the superior performance, including ultra-low detection limits and fast assay times, make this technology promising for point-of-care diagnostics. Future work could explore the integration of other functionalities into the microfluidic chip, such as sample preparation and signal processing modules, to create a fully integrated diagnostic device. Further optimization of nanorod geometry and exploration of other plasmonic materials could potentially lead to even higher sensitivity and broader applicability.

While the study demonstrated significant improvements in sensitivity and speed, potential limitations include the need for further validation in larger clinical studies. The long-term stability of the Au nanorod arrays and the potential for non-specific binding should also be investigated. The current design might need modifications for certain sample types or complex matrices that could interfere with the assay. Future work should address these aspects to enhance the robustness and reliability of the platform.