Rapid and Highly Sensitive Detection of Ricin in Biological Fluids Using Optical Modulation Biosensing

Abstract

Ricin, a highly toxic glycoprotein derived from the seeds of Ricinus communis, poses significant risks in bioterrorism and toxicology due to its rapid absorption and ease of dissemination. Rapid, ultra-sensitive detection is crucial for timely medical intervention and implementing security measures. However, existing methods often lack sufficient sensitivity or require lengthy processing, limiting their utility for trigger-to-treat scenarios. Here, we present an optical modulation biosensing (OMB)-based ricin assay capable of detecting low concentrations of ricin in buffer, plasma, and biological samples. The assay combines magnetic-bead-based target capture with fluorescent signal enhancement, achieving a limit of detection (LoD) of 15 pg/mL in buffer and 62 pg/mL in plasma, with a 4-log dynamic range. Optimized protocols reduced the assay time to 60 min, maintaining an LoD of 114 pg/mL in plasma while preserving accuracy and reproducibility. The assay successfully detected ricin in bronchoalveolar lavage fluid and serum from mice that were intranasally exposed to ricin, with signals persisting up to 48 h post exposure. Its rapid, high-throughput capabilities and simplified workflow make the OMB-based assay a powerful tool for toxicology, forensic analysis, and counter-bioterrorism. This study highlights the OMB platform's potential as a sensitive and robust diagnostic tool for detecting hazardous biological agents.

Keywords: bioterrorism; fluorescence-based immunoassay; optical modulation biosensing; ricin; toxicology diagnostics.

Figure 1

Principles of optical modulation biosensing (OMB). (a) In an OMB-based ricin assay, magnetic beads are coated with anti-ricin capture antibodies to bind the target antigen, ricin. Detection is achieved using a biotinylated anti-ricin antibody linked to a fluorescent marker. (b) A small, sharp-tipped, permanent magnet is placed beneath the sample well, causing the magnetic beads in the solution to aggregate. To eliminate background noise from unbound fluorescent analyte in the solution, the laser beam is alternated between the background solution and the fixed beads, allowing for accurate subtraction of the noise from the fluorescent signal of the beads.

Figure 2

The analytical performance of the OMB-based ricin assays in buffer. For the MH1-MH75 configuration (solid blue line), the dynamic range was 4-log, the LoD was 15 pg/mL, and the CV was less than 15%. For the MH75-MH1 configuration (dashed orange line), the dynamic range was 4-log, the LoD was 100 pg/mL, and the CV was less than 53%. For the MH1-MH75 dose–response curve, the error bars represent the standard error of five experiments ($n=5$), with six repetitions at the blank concentration and three repetitions at each of the other concentrations in each experiment. For the MH75-MH1 dose–response curve, the error bars represent the standard deviation of 17 repetitions at the blank concentration ($n=17$) and 5 repetitions at each of the other concentrations ($n=5$).

Figure 3

The turnaround time optimization results for the OMB-based MH1-MH75 ricin assay in 50% plasma. In a series of experiments, the turnaround time was reduced from 110 min (solid blue line) to 80 min (dashed purple line), and then to 60 min (dashed-dotted orange line). The error bars represent the standard error of three to five experiments ($n=3–5$). The calculated LoDs of the 110, 80, and 60 min assays were 62 pg/mL, 128 pg/mL, and 114 pg/mL. For each experiment, the mean signal of the blank concentration was calculated based on six repetitions, while three to four repetitions were performed for all other concentrations.

Figure 4

The detection of ricin in the bronchoalveolar lavage fluid (BALF) and serum of mice following intranasal exposure. Normalized fluorescence signals in (a) BALF samples and (b) serum samples of mice after intranasal exposure to ricin. The highest detected ricin levels in murine BALF samples were observed at 6 h, and a detectable signal remained in samples even at 48 h. Serum samples from mice showed the highest signal at 24 h, with detectable levels persisting up to 48 h after exposure. The signal at 72 h was not significantly different from the control. The error bars of the BALF sample experiment represent the standard deviation of five mice ($n=5$) at each time point. The error bars of the serum sample experiment represent the standard deviation of four to seven repetitions for each time point $\left(n=4–7\right)$.