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Biomedical Imaging and Instrumentation

Biomedical Imaging and Instrumentation - Poster Session E

U21 - Lab-in-your-palm chemiluminescence vertical flow assay for high-sensitivity troponin testing

Saturday, October 11, 2025
10:00 AM - 11:00 AM PDT
Location: Exhibit Hall F, G & H

Introduction: : Cardiac troponin I (cTnI) is the gold-standard biomarker for diagnosing myocardial infarction (MI). Clinical guidelines for MI recommend detecting cTnI at a few pg/mL level to distinguish MI patients from healthy individuals. However, high-sensitivity assays for cTnI are often inaccessible in underserved or decentralized settings due to their dependence on expensive instrumentation and trained personnel. Existing point-of-care testing (POCT), while more accessible, typically lacks the sensitivity, reproducibility, and robustness needed for early and accurate MI detection. To bridge this diagnostic gap, we developed a deep learning-enhanced chemiluminescence vertical flow assay (CL-VFA) platform designed for rapid, affordable, high-sensitivity cTnI testing. Our system integrates (i) a paper-based vertical flow sensor using polymerized enzyme labels for high-intensity CL signal (Fig. 1a), (ii) a portable Raspberry Pi-based reader that captures low-intensity CL signal with enhanced sensitivity (Fig. 1b), and (iii) a tray-based modular cartridge that enables stable, high-quality CL imaging by preventing reagent evaporation and mobility (Fig. 1c). Together, these lab-in-your-palm design and system optimization enable lab-grade assay in resource-limited environments, supporting broader access to high-quality cardiac diagnostics.

Materials and

Methods: : The CL-VFA consists of two modular cartridges. The first cartridge performs immunoassay using stacked paper layers loaded with 50 µL of serum mixed with polymerized enzyme-antibody conjugates. After washing away unbound reagents, the sensing membrane tray is transferred to the imaging cartridge, where a CL substrate is added and the signal captured by a Raspberry Pi-based portable reader system. This reader includes a CMOS camera, a lens, a touchscreen display, and a 3D-printed housing, utilizing the self-emitting nature of CL to simplify the imaging system by eliminating external excitation components, unlike colorimetric or fluorescence methods. The sensing membrane is positioned at a fixed distance ( < 2 cm) from the camera aperture, thereby enhancing light collection efficiency by minimizing photon loss. Adjustable exposure settings allow precise tuning of sensitivity and dynamic range. This design maintains mechanical simplicity while enabling optimal signal detection. CL intensities are extracted from the captured images using a computational code, then processed through a pipeline of four deep neural networks to accurately infer cTnI concentrations in the sample. The total assay time is 25 min, including immunoassay, washing, and imaging. To determine the limit of detection (LoD) of CL-VFA, cTnI was spiked into cTnI-free serum, and the assay was performed using serum samples diluted to different concentrations with the same serum matrix.

Results, Conclusions, and Discussions:: In this project, through engineering of the mobile reader and cartridge designs, we addressed two main issues: (i) the lack of a compact, high-quality CL readout system and (ii) the instability of CL signal generation and imaging, which hindered the successful implementation of CL modality in point-of-care biosensing. First, we developed a Raspberry Pi-based reader that provides a hand-held footprint (8×17.5×10.5 cm3 and 0.65 kg), with a total building cost of ~$222. Our portable reader achieved a detection limit of 6.1×10^(-5) optical density (OD) of conjugate concentration, outperforming both a CCD-based benchtop system (1.3×10^(-4) OD) and a smartphone-based reader (1.6×10^(-3) OD) by 2.1-fold and 26.2-fold, respectively (Fig. 1d). Improved signal-to-noise (S/N) ratios were attributed to enhanced thermal management, increased pixel size, and closer sensor proximity. In parallel, CL signal instability during imaging was addressed by engineering a tray-based cartridge that replaced the absorbent pad with a flat plastic support stage. As shown in Figs. 1c and 1e, the results indicate that the original absorbent-pad design led to evaporation and continuous downward flow of the CL reagent, even though the absorbent pads were fully wet. It resulted in signal decay and increased variability (average coefficient of variation [CV]: 22.9%). In contrast, the optimized imaging cartridge setup prevented reagent movement and evaporation, significantly reducing signal variability (average CV: 4.6%) and enabling the signal to reach saturation in 4 min. Using cTnI spiked serum samples, we demonstrated a strong correlation between the CL signal and cTnI concentration across six orders of magnitude (few pg/mL to 10^(5) pg/mL), an LoD of 0.16 pg/mL, and an average CV of 5.4% (Fig. 1f). With the combination of a paper-based sensor ($4.27 per test), a custom portable reader, and neural networks, our CL-VFA created a compact, rapid, affordable, simple, and highly sensitive cTnI POCT.

In conclusion, innovations in mobile reader and cartridge design have significantly enhanced CL signal capture and reproducibility. These improvements enable clinical lab-grade sensitivity on a low-cost, field-deployable platform for high-sensitivity cTnI testing, demonstrating how advances in reader engineering and VFA cartridge design can overcome the challenges in point-of-care diagnostics.

Acknowledgements and/or References (Optional): :