RESUMEN
Botulinum neurotoxins (BoNTs) are classified at a highest degree of threat in biodefense, due largely to their high lethality. With the growing risk of biowarfare, the shortcomings of the gold standard test for these neurotoxins, the mouse bioassay, have underscored the need to develop alternative diagnostic testing strategies. This paper reports on the detection of inactivated Clostridium botulinum neurotoxin serotype A (BoNT-A) and serotype B (BoNT-B), the two most important markers of botulism infection, by using a sandwich immunoassay, gold nanoparticle labels, and surface-enhanced Raman scattering (SERS) within the context of two threat scenarios. The first scenario mimics part of the analysis needed in response to a "white powder" threat by measuring both neurotoxins in phosphate-buffered saline (PBS), a biocompatible solvent often used to recover markers dispersed in a powdered matrix. The second scenario detects the two neurotoxins in spiked human serum to assess the clinical potential of the platform. The overall goal is to develop a test applicable to both scenarios in terms of projections of required levels of detection. We demonstrate the ability to measure BoNT-A and BoNT-B in PBS at a limit of detection (LoD) of 700â¯pg/mL (5 pM) and 84â¯pg/mL (0.6 pM), respectively, and in human serum at 1200â¯pg/mL (8 pM) and 91â¯pg/mL (0.6 pM), respectively, with a time to result under 24â¯h. The steps required to transform this platform into an onsite biodefense screening tool that can simultaneously and rapidly detect (<1â¯h) these and other agents are briefly discussed.
RESUMEN
N-Hydroxysuccinimide (NHS) ester terminal groups are commonly used to covalently couple amine-containing biomolecules (e.g., proteins and peptides) to surfaces via amide linkages. This one-step aminolysis is often performed in buffered aqueous solutions near physiological pH (pH 6 to pH 9). Under these conditions, the hydrolysis of the ester group competes with the amidization process, potentially degrading the efficiency of the coupling chemistry. The work herein examines the efficiency of covalent protein immobilization in borate buffer (50 mM, pH 8.50) using the thiolate monolayer formed by the chemisorption of dithiobis (succinimidyl propionate) (DSP) on gold films. The structure and reactivity of these adlayers are assessed via infrared spectroscopy (IR), X-ray photoelectron spectroscopy (XPS), electrochemical reductive desorption, and contact angle measurements. The hydrolysis of the DSP-based monolayer is proposed to follow a reaction mechanism with an initial nucleation step, in contrast to a simple pseudo first-order reaction rate law for the entire reaction, indicating a strong dependence of the interfacial reaction on the packing and presence of defects in the adlayer. This interpretation is used in the subsequent analysis of IR-ERS kinetic plots which give a heterogeneous aminolysis rate constant, ka, that is over 3 orders of magnitude lower than that of the heterogeneous hydrolysis rate constant, kh. More importantly, a projection of these heterogeneous kinetic rates to protein immobilization suggests that under coupling conditions in which low protein concentrations and buffers of near physiological pH are used, proteins are more likely physically adsorbed rather than covalently linked. This result is paramount for biosensors that use NHS chemistry for protein immobilization due to effects that may arise from noncovalently linked proteins.