Smartphone-Enabled Plasmonic ELISA for Ultra‑Sensitive Myoglobin Detection at Point of Care

Abstract

Serum myoglobin rises within 1–2 h after an acute myocardial infarction (AMI), making it a critical early biomarker. We present a plasmonic immunoassay that exploits enzyme‑mediated localized surface plasmon resonance (LSPR) changes of gold nanorods (AuNRs) to quantify myoglobin with a linear range of 0.1–1000 ng mL⁻¹ and a limit of detection (LOD) of 0.057 ng mL⁻¹. A novel reader, built from a smartphone’s ambient light sensor (ALS) and a 3‑D printed accessory, translates LSPR shifts into a simple, low‑cost readout (~$2) suitable for resource‑limited settings. Myoglobin concentrations measured by this method correlated strongly with conventional ELISA results, underscoring its clinical relevance for rapid AMI triage.

Introduction

AMI remains a leading cause of mortality worldwide, with 1.5 million US and 500,000 Chinese cases annually. Early detection hinges on biomarkers that rise swiftly; serum myoglobin peaks 6–9 h post‑infarction and is detectable as early as 1–2 h [3–5]. Conventional ELISA, while reliable, requires laboratory infrastructure and skilled personnel, limiting its utility in point‑of‑care (POC) scenarios.

Plasmonic immunoassays combine ELISA’s specificity with the optical amplification of metal nanostructures, enabling naked‑eye readout and high sensitivity. Gold nanorods, with their tunable LSPR, are particularly attractive for signal transduction in biosensing. However, quantitative interpretation traditionally demands spectrometers or microplate readers, restricting field deployment. Here, we develop an AuNR‑based plasmonic ELISA for myoglobin, paired with a smartphone‑driven reader that harnesses the phone’s ALS to capture transmitted light intensity, offering a portable, inexpensive diagnostic tool.

Materials and Methods

Materials

Human myoglobin (Abcam, UK), anti‑myoglobin monoclonal antibodies (mAb1, mAb2, in‑house production), gold nanorods (AuNRs), glucose oxidase (GOx), horseradish peroxidase (HRP), and other reagents were sourced from standard suppliers. Serum samples were collected from Guangzhou Overseas Chinese Hospital.

Apparatus

LSPR spectra were recorded with a Synergy H1 microplate reader; ELISA absorbance was measured at 450 nm on a MK3 reader. AuNRs were characterized by a PHILIPS TECNAI‑10 TEM. The smartphone reader was assembled on a 3‑D printed chassis (part 1: 100 × 40 × 40 mm, part 2: 76 × 13 × 12 mm) and powered by two 1.5 V batteries. A 850 nm LED aligned with the ALS enabled transmitted‑light measurement via the Light Meter app (Android; compatible with iOS).

Synthesis of AuNRs

Seed‑mediated growth produced AuNRs with an aspect ratio tuned by AgNO₃ and CTAB concentrations. Final rods were incubated 12 h under stirring before use.

Plasmonic Immunoassay Protocol

96‑well plates were coated overnight with mAb1, blocked with BSA, and stored at –20 °C. GOx‑labeled mAb2 (GOx‑Ab2) was added to wells containing varying myoglobin concentrations, followed by glucose and AuNRs in the presence of HRP and CTAB. Etching of AuNRs by H₂O₂ generated a blue shift in the LSPR peak and reduced absorbance at 850 nm, proportional to myoglobin amount. The transmitted‑light intensity was recorded by the smartphone reader, generating a calibration curve. Serum samples were diluted tenfold and processed identically.

Data Analysis

Linear regression was performed using Origin 9.0; all assays were triplicated to ensure reproducibility.

Results and Discussion

Assay Principle

GOx on mAb2 converts glucose to gluconic acid and H₂O₂. H₂O₂, in the presence of HRP and Br⁻, etches AuNRs, shifting the LSPR peak to shorter wavelengths and lowering 850 nm absorbance. The extent of shift or intensity loss directly reflects myoglobin concentration.

Optimization

Optimal HRP (1.5 µM) and CTAB (6.25 µM) concentrations maximized LSPR shift. A 30‑min etching time with 100 µM H₂O₂ produced stable signals. Higher H₂O₂ levels induced a pronounced blue shift and morphological change from rectangular to elliptical rods, confirming the etching mechanism.

Smartphone Reader Performance

The ALS‑based reader measured transmitted‑light intensity at 850 nm with <1 % variance relative to a commercial microplate reader, achieving a 99.1 % correlation (R² = 0.991). The entire accessory cost is approximately $2.

Analytical Sensitivity

Using LSPR shift, the assay quantified myoglobin from 0.1 to 1000 ng mL⁻¹ with an LOD of 57.8 pg mL⁻¹. When read by the smartphone, the linear range remained identical, with an LOD of 64.1 pg mL⁻¹. Clinical serum measurements showed strong agreement with ELISA (R² = 0.98), validating the method’s diagnostic potential.

Comparison with ELISA

Traditional ELISA yielded a linear range of 25–1000 ng mL⁻¹ and an LOD of 22.7 ng mL⁻¹. The plasmonic assay therefore offers a ~400‑fold lower LOD and a broader dynamic range, making it superior for early AMI detection.

Conclusions

We have demonstrated a gold‑nanorod plasmonic ELISA that detects serum myoglobin with unprecedented sensitivity (LOD = 0.057 ng mL⁻¹) and a straightforward smartphone reader. The platform bypasses complex instrumentation, offering a viable, low‑cost solution for POC AMI diagnosis in resource‑constrained environments.

Abbreviations

• Ab1: Anti‑myoglobin antibody
• Ab2: Anti‑myoglobin antibody 2
• ALS: Ambient light sensor
• AMI: Acute myocardial infarction
• AuNRs: Gold nanorods
• ELISA: Enzyme‑linked immunosorbent assay
• GOx: Glucose oxidase
• HRP: Horseradish peroxidase
• H₂O₂: Hydrogen peroxide
• LSPR: Localized surface plasmon resonance
• Myo: Serum myoglobin
• POCT: Point‑of‑care testing