sulfo-LC-SPDP

The effects of the hindered and non-hindered water soluble long-chain disulfide bonds on the stability and cytotoxicity of the ricin A chain (RTA) immunotoxin were examined. The RTA immunotoxins were prepared with the Fab fragments of anti-common acute lymphoblastic leukemia antigen (CALLA) monoclonal antibody (Fab-RTA) using sulfosuccinimidyl-6-[(-methyl-(-(2-pyridyldithio)toluamido]hexanoate (S-LC-SMPT) and sulfosuccinimidyl-6-[3-(2-pyridyldithio)-propionamido]hexanoate (S-LC-SPDP). The prepared Fab-RTA immunotoxins were evaluated for their conjugation yield, immunoreactivity, thermal and disulfide bond stability and cytotoxicity. The conjugation yield of the Fab-RTA immunotoxin from the water soluble long chain crosslinking agents, S-LC-SMPT and S-LC-SPDP, were comparable. Both Fab-RTA immunotoxins exhibited a similar immunoreactivity and thermal stability in aqueous solution. However, S-LC-SMPT -mediated Fab-RTA, sterically hindered, showed an enhanced disulfide bond stability in vitro over S-LC-SPDP mediated one. In the cytotoxicity against antigenic cell Daudi, the S-LC-SMPT -mediated RTA immunotoxin maintained a comparable cytotoxicity, compared with S-LC-SPDP mediated Fab-RTA immunotoxin.

Whole peptide-displayed phage particles are promising alternatives to antibodies in sensor development; however, greater control and functionalization of these particles are required. In this study, we aimed to identify and create highly sensitive and selective phage-based electrochemical biosensors for detecting ovomucoid, a known food allergen. Phage display was performed using two different phage libraries (cyclic and linear form of peptides), which displayed affinity peptides capable of binding specifically to ovomucoid. Throughout the biopanning, two phage clones that displayed both peptides (CTDKASSSC and WWQPYSSAPRWL) were selected. After the characterization of their binding affinities, both whole phage particles were covalently attached to a gold electrode using crosslinking chemistry (MUA-EDC/NHS and Sulfo-LC/SPDP); the developed phage sensor was characterized using cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS). The cyclic peptide-displayed phage sensor modified using EDC/NHS chemistry exhibited significantly better binding affinity (Kd= 2.36 ± 0.44 μg/mL) and limit of detection (LOD, 0.12 μg/mL) for ovomucoid than the linear phage sensor, resulting in good reproducibility and recovery, even in an actual egg and white wine samples. This approach may provide an alternative and more efficient way of sensing food allergens with desirable sensitivity, selectivity, and feasibility in food diagnostic applications.

Mesoporous silica nanoparticles (MSN) have emerged as an attractive class of drug delivery carriers for therapeutic agents. Herein, we explored the covalent immobilization of proteins into MSN to generate a stimulus-responsive controlled release system. First, MSN were functionalized with thiol groups using (mercaptopropyl)-trimethoxysilane (MPTMS). Functionalization was verified by X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared (FTIR) spectroscopy, and dynamic light scattering. The model enzyme carbonic anhydrase (CA) was coupled to sulfosuccinimidyl 6-[3'(2-pyridyldithio)-propionamido]hexanoate (Sulfo-LC-SPDP) at a low ratio of 1:1 to prevent enzyme inactivation and subsequently covalently immobilized into MSN via thiol-disulfide interchange. The enzyme could be released from MSN with 10 mM glutathione, which represents intracellular redox conditions, while it remained bound to the MSN at extracellular redox conditions represented by 1 μM glutathione. The activity of the released enzyme was >80% demonstrating that the enzyme was still largely functional and active after immobilization and release. Human cervical cancer (HeLa) cells were incubated with the MSN-CA bioconjugates at various concentrations for 24 h and the data show good biocompatibility. In summary, we demonstrate the potential of MSN as drug delivery systems for proteins.