Feasibility study for the rapid screening of target molecules using translational diffusion coefficients: diffusion-ordered NMR spectroscopy of biological toxins

A panel of 15 biological toxins ranging between ~60–28,000 g/mol was used to evaluate the feasibility of screening aqueous samples for toxin analytes based on their translational diffusion coefficients, D _t. Toxin D _t values were measured by pulsed-field gradient ¹H NMR spectroscopy using a bipolar pulse pair, longitudinal eddy current delay pulse sequence incorporating water suppression to achieve the maximum dynamic range for toxin signals. To collect data for an effective screening protocol, reference D _t values were determined from five independent measurements at both 25 and 37 °C for all toxins in the panel. In the protocol, D _t values are measured at both temperatures for a suspected toxin target in a sample, and for assignment as a potential toxin analyte, the measurements are required to fall within ±0.25 × 10⁻⁶ cm²/s of both reference D _t values for at least one toxin in the panel. Only solution viscosity was found to influence sample D _t measurements appreciably; however, the measurements are easily corrected for viscosity effects by calculating the D _t value of the suspected toxin at infinite dilution. In conclusion, the protocol provides a rapid and effective means for screening aqueous samples for all toxins in the panel, narrowing toxin identification to ≤2 possibilities in virtually all cases.